Obestatin Plays Beneficial Role in Cardiomyocyte Injury Induced by Ischemia-Reperfusion In Vivo and In Vitro.

PubMed

Zhang, Qin; Dong, Xin-Wei; Xia, Jia-Ying; Xu, Ke-Ying; Xu, Zhe-Rong

2017-05-04

BACKGROUND Obestatin, primarily recognized as a peptide within the gastrointestinal system, has been shown to benefit the cardiovascular system. We designed this experiment to study the protective role and underlying mechanism of obestatin against ischemia-reperfusion(I/R) injury in myocardial cells. MATERIAL AND METHODS In an In vivo experiment, LAD was ligated for 0.5 h and then opened for reperfusion with obestatin for 24 h. Then, the infarction area was shown with TTC staining, and inflammation factors in serum were analyzed by qRT-PCR. In primary cultured cardiomyocytes, we measured the level of LDH, MDA, GSH, and SOD. Finally, we assessed cells apoptosis using flow cytometry and detected the concentrations of caspase-3, Bax, and Bcl-2 using Western blot analysis. RESULTS TTC staining showed that in the 3 obestatin groups, the infarct area became smaller with the increase of obestatin concentration. Obestatin also inhibited LDH expression in rat serum and decreased mRNA levels of TNF-α, IL-6, ICAM-1, and iNOS in rat cardiomyocytes after reperfusion. In primary cultured cardiomyocytes, obestatin decreased LDH content and increased GSH level after I/R injury. Obestatin was also found to antagonize the apoptosis of cardiomyocytes in a dose-dependent manner. Western blot analysis showed that obestatin downregulated the expression of caspase-3 and Bax and upregulated the expression of Bcl-2. CONCLUSIONS Obestatin can protect cardiomyocyte from I/R-induced injury in vitro and in vivo. This beneficial effect is closely related with its properties of anti-inflammation, anti-cytotoxicity, and anti-apoptosis. The protective effect of obestatin might be associated with activation of Bcl-2 and inhibition of caspase-3 and Bax.

FACS-based isolation, propagation and characterization of mouse embryonic cardiomyocytes based on VCAM-1 surface marker expression.

PubMed

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes.

FACS-Based Isolation, Propagation and Characterization of Mouse Embryonic Cardiomyocytes Based on VCAM-1 Surface Marker Expression

PubMed Central

Pontén, Annica; Walsh, Stuart; Malan, Daniela; Xian, Xiaojie; Schéele, Susanne; Tarnawski, Laura; Fleischmann, Bernd K.; Jovinge, Stefan

2013-01-01

Purification of cardiomyocytes from the embryonic mouse heart, embryonic stem (ES) or induced pluripotent stem cells (iPS) is a challenging task and will require specific isolation procedures. Lately the significance of surface markers for the isolation of cardiac cell populations with fluorescence activated cell sorting (FACS) has been acknowledged, and the hunt for cardiac specific markers has intensified. As cardiomyocytes have traditionally been characterized by their expression of specific transcription factors and structural proteins, and not by specific surface markers, this constitutes a significant bottleneck. Lately, Flk-1, c-kit and the cellular prion protein have been reported to specify cardiac progenitors, however, no surface markers have so far been reported to specify a committed cardiomyocyte. Herein show for the first time, that embryonic cardiomyocytes can be isolated with 98% purity, based on their expression of vascular cell adhesion molecule-1 (VCAM-1). The FACS-isolated cells express phenotypic markers for embryonic committed cardiomyocytes but not cardiac progenitors. An important aspect of FACS is to provide viable cells with retention of functionality. We show that VCAM-1 positive cardiomyocytes can be isolated with 95% viability suitable for in vitro culture, functional assays or expression analysis. In patch-clamp experiments we provide evidence of functionally intact cardiomyocytes of both atrial and ventricular subtypes. This work establishes that cardiomyocytes can be isolated with a high degree of purity and viability through FACS, based on specific surface marker expression as has been done in the hematopoietic field for decades. Our FACS protocol represents a significant advance in which purified populations of cardiomyocytes may be isolated and utilized for downstream applications, such as purification of ES-cell derived cardiomyocytes. PMID:24386094

Purification of cardiomyocytes from differentiating pluripotent stem cells using molecular beacons that target cardiomyocyte-specific mRNA.

PubMed

Ban, Kiwon; Wile, Brian; Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-10-22

Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Hyperoside protects against hypoxia/reoxygenation induced injury in cardiomyocytes by suppressing the Bnip3 expression.

PubMed

Xiao, Rui; Xiang, An-Li; Pang, Hong-Bo; Liu, Ke-Qiang

2017-09-20

Role of hyperoside in protecting cardiomyocytes from ischemia/reperfusion induced injury has been proved. However, possible protecting mechanisms remain unclear. To fix the problem, an essential pro-apoptotic protein Bnip3 was studied in our experiments. Neonatal rat cardiomyocytes were used and submitted to hypoxia for 8h followed by reoxygenation for 2h to simulate the ischemia/reperfusion injury. Hypoxia/reoxygenation(H/R) induced damage to cardiomyocytes and the protective effect of hyperoside were examined by means of MTT assay. H/R-induced apoptosis was assessed by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling(TUNEL) and DNA Ladder assay. mRNA expression of Bnip3 was determined by use of quantitative real-time reverse transcription polymerase chain reaction assay. Protein levels of Bnip3, Bax, Bcl-2 and cleaved caspase-3 were examined using western-blot assay. Our results showed that H/R caused great damage to cardiomyocytes, upregulated the protein expressions of Bnip3, Bax, cleaved caspase3, and decreased the expression of the anti-apoptotic protein of Bcl-2. Whereas, compared with the H/R group, a decrease in activities of Bnip3, Bax, cleaved caspase3, and a promoting expression of Bcl-2 were detected in the H/R goup pretreated with hyperoside. It was concluded in our study that H/R-induced apoptotic effect in cardiomyocytes could be attenuated by hyperoside, and the protective role of hyperoside, if not completely, could be partly through the suppression of the pro-apoptotic gene Bnip3. Copyright © 2017 Elsevier B.V. All rights reserved.

RhoA Regulation of Cardiomyocyte Differentiation

PubMed Central

Kaarbø, Mari; Crane, Denis I.; Murrell, Wayne G.

2013-01-01

Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac α-actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes. PMID:23935420

Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

PubMed Central

Park, Chi-Yeon; Choi, Seung-Cheol; Kim, Jong-Ho; Choi, Ji-Hyun; Joo, Hyung Joon; Hong, Soon Jun; Lim, Do-Sun

2016-01-01

Cardiac stem cells (CSCs) were known to secrete diverse paracrine factors leading to functional improvement and beneficial left ventricular remodeling via activation of the endogenous pro-survival signaling pathway. However, little is known about the paracrine factors secreted by CSCs and their roles in cardiomyocyte survival during hypoxic condition mimicking the post-myocardial infarction environment. We established Sca-1+/CD31− human telomerase reverse transcriptase-immortalized CSCs (Sca-1+/CD31− CSCshTERT), evaluated their stem cell properties, and paracrine potential in cardiomyocyte survival during hypoxia-induced injury. Sca-1+/CD31− CSCshTERT sustained proliferation ability even after long-term culture exceeding 100 population doublings, and represented multi-differentiation potential into cardiomyogenic, endothelial, adipogenic, and osteogenic lineages. Dominant factors secreted from Sca-1+/CD31− CSCshTERT were EGF, TGF-β1, IGF-1, IGF-2, MCP-1, HGF R, and IL-6. Among these, MCP-1 was the most predominant factor in Sca-1+/CD31− CSCshTERT conditioned medium (CM). Sca-1+/CD31− CSCshTERT CM increased survival and reduced apoptosis of HL-1 cardiomyocytes during hypoxic injury. MCP-1 silencing in Sca-1+/CD31− CSCshTERT CM resulted in a significant reduction in cardiomyocyte apoptosis. We demonstrated that Sca-1+/CD31− CSCshTERT exhibited long-term proliferation capacity and multi-differentiation potential. Sca-1+/CD31− CSCshTERT CM protected cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Thus, they are valuable sources for in vitro and in vivo studies in the cardiovascular field. PMID:27231894

Functional interaction between bicarbonate transporters and carbonic anhydrase modulates lactate uptake into mouse cardiomyocytes.

PubMed

Peetz, Jan; Barros, L Felipe; San Martín, Alejandro; Becker, Holger M

2015-07-01

Blood-derived lactate is a precious energy substrate for the heart muscle. Lactate is transported into cardiomyocytes via monocarboxylate transporters (MCTs) together with H(+), which couples lactate uptake to cellular pH regulation. In this study, we have investigated how the interplay between different acid/base transporters and carbonic anhydrases (CA), which catalyze the reversible hydration of CO2, modulates the uptake of lactate into isolated mouse cardiomyocytes. Lactate transport was estimated both as lactate-induced acidification and as changes in intracellular lactate levels measured with a newly developed Förster resonance energy transfer (FRET) nanosensor. Recordings of intracellular pH showed an increase in the rate of lactate-induced acidification when CA was inhibited by 6-ethoxy-2-benzothiazolesulfonamide (EZA), while direct measurements of lactate flux demonstrated a decrease in MCT transport activity, when CA was inhibited. The data indicate that catalytic activity of extracellular CA increases lactate uptake and counteracts intracellular lactate-induced acidification. We propose a hypothetical model, in which HCO3 (-), formed from cell-derived CO2 at the outer surface of the cardiomyocyte plasma membrane by membrane-anchored, extracellular CA, is transported into the cell via Na(+)/HCO3 (-) cotransport to counteract intracellular acidification, while the remaining H(+) stabilizes extracellular pH at the surface of the plasma membrane during MCT activity to enhance lactate influx into cardiomyocytes.

Labdane diterpenes protect against anoxia/reperfusion injury in cardiomyocytes: involvement of AKT activation

PubMed Central

Cuadrado, I; Fernández-Velasco, M; Boscá, L; de las Heras, B

2011-01-01

Several labdane diterpenes exert anti-inflammatory and cytoprotective actions; therefore, we have investigated whether these molecules protect cardiomyocytes in an anoxia/reperfusion (A/R) model, establishing the molecular mechanisms involved in the process. The cardioprotective activity of three diterpenes (T1, T2 and T3) was studied in the H9c2 cell line and in isolated rat cardiomyocyte subjected to A/R injury. In both cases, treatment with diterpenes T1 and T2 protected from A/R-induced apoptosis, as deduced by a decrease in the percentage of apoptotic and caspase-3 active positive cells, a decrease in the Bcl-2/Bax ratio and an increase in the expression of antiapoptotic proteins. Analysis of cell survival signaling pathways showed that diterpenes T1 and T2 added after A/R increased phospho-AKT and phospho-ERK 1/2 levels. These cardioprotective effects were lost when AKT activity was pharmacologically inhibited. Moreover, the labdane-induced cardioprotection involves activation of AMPK, suggesting a role for energy homeostasis in their mechanism of action. Labdane diterpenes (T1 and T2) also exerted cardioprotective effects against A/R-induced injury in isolated cardiomyocytes and the mechanisms involved activation of specific survival signals (PI3K/AKT pathways, ERK1/2 and AMPK) and inhibition of apoptosis. PMID:22071634

Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury

PubMed Central

Chen, Yi-Wen; Chou, Hsiu-Chuan; Lin, Szu-Ting; Chen, You-Hsuan; Chang, Yu-Jung; Chen, Linyi; Chan, Hong-Lin

2013-01-01

Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3), caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP), Ena/VASP-like protein (Evl), and isopentenyl-diphosphate delta-isomerase 1 (Idi-1) were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes. PMID:23573126

Differential Regenerative Capacity of Neonatal Mouse Hearts after Cryoinjury

PubMed Central

Darehzereshki, Ali; Rubin, Nicole; Gamba, Laurent; Kim, Jieun; Fraser, James; Huang, Ying; Billings, Joshua; Mohammadzadeh, Robabeh; Wood, John; Warburton, David; Kaartinen, Vesa; Lien, Ching-Ling

2015-01-01

Neonatal mouse hearts fully regenerate after ventricular resection similar to adult zebrafish. We established cryoinjury models to determine if different types and varying degrees of severity in cardiac injuries trigger different responses in neonatal mouse hearts. In contrast to ventricular resection, neonatal mouse hearts fail to regenerate and show severe impairment of cardiac function post transmural cryoinjury. However, neonatal hearts fully recover after non-transmural cryoinjury. Interestingly, cardiomyocyte proliferation does not significantly increase in neonatal mouse hearts after cryoinjuries. Epicardial activation and new coronary vessel formation occur after cryoinjury. The profibrotic marker PAI-1 is highly expressed after transmural but not non-transmural cryoinjuries, which may contribute to the differential scarring. Our results suggest that regenerative medicine strategies for heart injuries should vary depending on the nature of the injury. PMID:25555840

Moderate ethanol administration accentuates cardiomyocyte contractile dysfunction and mitochondrial injury in high fat diet-induced obesity.

PubMed

Yuan, Fang; Lei, Yonghong; Wang, Qiurong; Esberg, Lucy B; Huang, Zaixing; Scott, Glenda I; Li, Xue; Ren, Jun

2015-03-18

Light to moderate drinking confers cardioprotection although it remains unclear with regards to the role of moderate drinking on cardiac function in obesity. This study was designed to examine the impact of moderate ethanol intake on myocardial function in high fat diet intake-induced obesity and the mechanism(s) involved with a focus on mitochondrial integrity. C57BL/6 mice were fed low or high fat diet for 16 weeks prior to ethanol challenge (1g/kg/d for 3 days). Cardiac contractile function, intracellular Ca(2+) homeostasis, myocardial histology, and mitochondrial integrity [aconitase activity and the mitochondrial proteins SOD1, UCP-2 and PPARγ coactivator 1α (PGC-1α)] were assessed 24h after the final ethanol challenge. Fat diet intake compromised cardiomyocyte contractile and intracellular Ca(2+) properties (depressed peak shortening and maximal velocities of shortening/relengthening, prolonged duration of relengthening, dampened intracellular Ca(2+) rise and clearance without affecting duration of shortening). Although moderate ethanol challenge failed to alter cardiomyocyte mechanical property under low fat diet intake, it accentuated high fat diet intake-induced changes in cardiomyocyte contractile function and intracellular Ca(2+) handling. Moderate ethanol challenge failed to affect fat diet intake-induced cardiac hypertrophy as evidenced by H&E staining. High fat diet intake reduced myocardial aconitase activity, downregulated levels of mitochondrial protein UCP-2, PGC-1α, SOD1 and interrupted intracellular Ca(2+) regulatory proteins, the effect of which was augmented by moderate ethanol challenge. Neither high fat diet intake nor moderate ethanol challenge affected protein or mRNA levels as well as phosphorylation of Akt and GSK3β in mouse hearts. Taken together, our data revealed that moderate ethanol challenge accentuated high fat diet-induced cardiac contractile and intracellular Ca(2+) anomalies as well as mitochondrial injury. Copyright �

Cardiomyocyte-released factors stimulate oligodendrocyte precursor cells proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuroda, Mariko; Muramatsu, Rieko; Precursory Research for Embryonic Science and Technology

The heart produces multiple diffusible factors that are involved in a number of physiological processes, but the action of these factors on the central nervous system is not well understood. In this study, we found that one or more factors released by cardiomyocytes promote oligodendrocyte precursor cell (OPC) proliferation in vitro. Mouse OPCs co-cultured with mouse cardiomyocytes showed higher proliferative ability than OPCs cultured alone. In addition, cardiomyocyte-conditioned media was sufficient to promote OPC proliferation. The phosphorylation of phosphatidylinositol (PI) 3-kinase and extracellular signal-regulated kinase (ERK) in OPCs is necessary for the enhancement of OPC proliferation by cardiomyocyte-conditioned media. These datamore » indicate that heart-derived factors have the ability to directly regulate the function of central nervous system (CNS) cells.« less

Involvement of Rictor/mTORC2 in cardiomyocyte differentiation of mouse embryonic stem cells in vitro

PubMed Central

Zheng, Bei; Wang, Jiadan; Tang, Leilei; Tan, Chao; Zhao, Zhe; Xiao, Yi; Ge, Renshan; Zhu, Danyan

2017-01-01

Rictor is a key regulatory/structural subunit of the mammalian target of rapamycin complex 2 (mTORC2) and is required for phosphorylation of Akt at serine 473. It plays an important role in cell survival, actin cytoskeleton organization and other processes in embryogenesis. However, the role of Rictor/mTORC2 in the embryonic cardiac differentiation has been uncovered. In the present study, we examined a possible link between Rictor expression and cardiomyocyte differentiation of the mouse embryonic stem (mES) cells. Knockdown of Rictor by shRNA significantly reduced the phosphorylation of Akt at serine 473 followed by a decrease in cardiomyocyte differentiation detected by beating embryoid bodies. The protein levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein) and α-Actinin (cardiomyocyte biomarker) decreased in Rictor knockdown group during cardiogenesis. Furthermore, knockdown of Rictor specifically inhibited the ventricular-like cells differentiation of mES cells with reduced level of ventricular-specific protein, MLC-2v. Meanwhile, patch-clamp analysis revealed that shRNA-Rictor significantly increased the number of cardiomyocytes with abnormal electrophysiology. In addition, the expressions and distribution patterns of cell-cell junction proteins (Cx43/Desmoplakin/N-cadherin) were also affected in shRNA-Rictor cardiomyocytes. Taken together, the results demonstrated that Rictor/mTORC2 might play an important role in the cardiomyocyte differentiation of mES cells. Knockdown of Rictor resulted in inhibiting ventricular-like myocytes differentiation and induced arrhythmias symptom, which was accompanied by interfering the expression and distribution patterns of cell-cell junction proteins. Rictor/mTORC2 might become a new target for regulating cardiomyocyte differentiation and a useful reference for application of the induced pluripotent stem cells. PMID:28123351

Nerves Regulate Cardiomyocyte Proliferation and Heart Regeneration.

PubMed

Mahmoud, Ahmed I; O'Meara, Caitlin C; Gemberling, Matthew; Zhao, Long; Bryant, Donald M; Zheng, Ruimao; Gannon, Joseph B; Cai, Lei; Choi, Wen-Yee; Egnaczyk, Gregory F; Burns, Caroline E; Burns, C Geoffrey; MacRae, Calum A; Poss, Kenneth D; Lee, Richard T

2015-08-24

Some organisms, such as adult zebrafish and newborn mice, have the capacity to regenerate heart tissue following injury. Unraveling the mechanisms of heart regeneration is fundamental to understanding why regeneration fails in adult humans. Numerous studies have revealed that nerves are crucial for organ regeneration, thus we aimed to determine whether nerves guide heart regeneration. Here, we show using transgenic zebrafish that inhibition of cardiac innervation leads to reduction of myocyte proliferation following injury. Specifically, pharmacological inhibition of cholinergic nerve function reduces cardiomyocyte proliferation in the injured hearts of both zebrafish and neonatal mice. Direct mechanical denervation impairs heart regeneration in neonatal mice, which was rescued by the administration of neuregulin 1 (NRG1) and nerve growth factor (NGF) recombinant proteins. Transcriptional analysis of mechanically denervated hearts revealed a blunted inflammatory and immune response following injury. These findings demonstrate that nerve function is required for both zebrafish and mouse heart regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

Heat shock protein 70 inhibits cardiomyocyte necroptosis through repressing autophagy in myocardial ischemia/reperfusion injury.

PubMed

Liu, Xiaojuan; Zhang, Chao; Zhang, Chi; Li, Jingjing; Guo, Wanwan; Yan, Daliang; Yang, Chen; Zhao, Jianhua; Xia, Tian; Wang, Yuqing; Xu, Rong; Wu, Xiang; Shi, Jiahai

2016-06-01

Irreversible damage of cardiac function arisen from myocardial ischemia/reperfusion injury (MIRI) leads to an emerging challenge in the treatments of cardiac ischemic diseases. Molecular chaperone heat shock protein 70 (HSP70) attenuates heat-stimulated cell autophagy, apoptosis, and damage in the heart. Under specific conditions, autophagy may, directly or indirectly, induce cell death including necroptosis. Whether HSP70 inhibits cardiomyocyte necroptosis via suppressing autophagy during MIRI is unknown. In our study, HSP70 expression was opposite to necroptosis marker RIP1 and autophagy marker LC3A/B expression after myocardial ischemia/reperfusion (MIR) in vivo. Furthermore, in vitro primary rat cardiomyocytes mimicked MIRI by hypoxia/reoxygenation (H/R) treatment. Knockdown of HSP70 expression promoted cardiomyocyte autophagy and necroptosis following H/R treatment, while the increase tendency was downregulated by autophagy inhibitor 3-MA, showing that autophagy-induced necroptosis could be suppressed by HSP70. In summary, HSP70 downregulates cardiomyocyte necroptosis through suppressing autophagy during myocardial IR, revealing the novel protective mechanism of HSP70 and supplying a novel molecular target for the treatment of heart ischemic diseases.

Preconditioning by isoflurane elicits mitochondrial protective mechanisms independent of sarcolemmal KATP channel in mouse cardiomyocytes

PubMed Central

Muravyeva, Maria; Sedlic, Filip; Dolan, Nicholas; Bosnjak, Zeljko J; Stadnicka, Anna

2013-01-01

Cardiac mitochondria and the sarcolemmal (sarc)KATP channels contribute to cardioprotective signaling of anesthetic-induced preconditioning (APC). Changes in mitochondrial bioenergetics influence the sarcKATP channel function, but whether this channel has impacts on mitochondria is uncertain. We used the mouse model with deleted pore-forming Kir6.2 subunit of sarcKATP channel (Kir6.2 KO) to investigate whether the functional sarcKATP channels are necessary for isoflurane activation of mitochondrial protective mechanisms. Ventricular cardiomyocytes were isolated from C57Bl6 wild type (WT) and Kir6.2 KO mouse hearts. Flavoprotein autofluorescence, mitochondrial ROS production and mitochondrial membrane potential were monitored by laser-scanning confocal microscopy in intact cardiomyocytes. Cell survival was assessed using H2O2-induced stress. Isoflurane (0.5 mM) increased flavoprotein fluorescence to 180±14% and 190±15% and ROS production to 118±2% and 124±6% of baseline in WT and Kir6.2 KO myocytes, respectively. TMRE fluorescence decreased to 84±6% in WT and to 86±4% in Kir6.2 KO myocytes. This effect was abolished by 5HD. Pretreatment with isoflurane decreased the stress-induced cell death from 31±1% to 21±1% in WT and from 44±2% to 35±2% in Kir6.2 KO myocytes. In conclusion, Kir6.2 deletion increases sensitivity of intact cardiomyocytes t o oxidative stress, but does not alter the isoflurane-elicited protective mitochondrial mechanisms, suggesting independent roles for cardiac mitochondria and sarcKATP channels in APC by isoflurane. PMID:23318991

NF45 inhibits cardiomyocyte apoptosis following myocardial ischemia-reperfusion injury.

PubMed

Liu, Xiaojuan; Zhang, Chi; Qian, Long; Zhang, Chao; Wu, Kunpeng; Yang, Chen; Yan, Daliang; Wu, Xiang; Shi, Jiahai

2015-12-01

Cardiomyocyte apoptosis, which occurs during ischemia and reperfusion injury, can cause irreversible damage to cardiac function. There is accumulating evidence that nuclear factor 45 (NF45) and regulatory pathways are important in understanding reparative processes in the myocardium. NF45 is a multifunctional regulator of gene expression that participates in the regulation of DNA break repair. Recently, NF45 has been proved to be associated with tumor cell apoptosis in various human malignancies. However, the underlying mechanism of NF45 regulating myocardial ischemia-reperfusion (I/R) injury remains unclear. In this study, western blot showed that NF45 expression decreased after myocardial I/R in vivo. Double immunofluorescent staining revealed that NF45, located in the nucleus of cardiomyocyes, was correlated with cardiomyocyte apoptosis. Furthermore, NF45 expression decreased in H9c2 cells after hypoxia-reoxygenation (H/R) treatment in vitro, which was in line with the results in vivo. Overexpression of NF45 in H9c2 cells reduced cell apoptosis, as evidenced by increased Bcl-2 level, as well as decreased cleaved caspase-3, p53 and p21 expression. The expression of NF45 was reduced by LY294002 (a PI3K/Akt inhibitor), but not SB203580 (a p38 inhibitor), suggesting that NF45 prevented H/R-induced H9c2 cell apoptosis via PI3K/Akt pathway. Our data may supply a novel molecular target for acute myocardial infarction (AMI) therapy. Copyright © 2015 Elsevier GmbH. All rights reserved.

Altering CO2 during reperfusion of ischemic cardiomyocytes modifies mitochondrial oxidant injury.

PubMed

Lavani, Romeen; Chang, Wei-Tien; Anderson, Travis; Shao, Zuo-Hui; Wojcik, Kimberly R; Li, Chang-Qing; Pietrowski, Robert; Beiser, David G; Idris, Ahamed H; Hamann, Kimm J; Becker, Lance B; Vanden Hoek, Terry L

2007-07-01

Acute changes in tissue CO2 and pH during reperfusion of the ischemic heart may affect ischemia/reperfusion injury. We tested whether gradual vs. acute decreases in CO2 after cardiomyocyte ischemia affect reperfusion oxidants and injury. Comparative laboratory investigation. Institutional laboratory. Embryonic chick cardiomyocytes. Microscope fields of approximately 500 chick cardiomyocytes were monitored throughout 1 hr of simulated ischemia (PO2 of 3-5 torr, PCO2 of 144 torr, pH 6.8), followed by 3 hrs of reperfusion (PO2 of 149 torr, PCO2 of 36 torr, pH 7.4), and compared with cells reperfused with relative hypercarbia (PCO2 of 71 torr, pH 6.8) or hypocarbia (PCO2 of 7 torr, pH 7.9). The measured outcomes included cell viability (via propidium iodide) and oxidant generation (reactive oxygen species via 2',7'-dichlorofluorescin oxidation and nitric oxide [NO] via 4,5-diaminofluorescein diacetate oxidation). Compared with normocarbic reperfusion, hypercarbia significantly reduced cell death from 54.8% +/- 4.0% to 26.3% +/- 2.8% (p < .001), significantly decreased reperfusion reactive oxygen species (p < .05), and increased NO at a later phase of reperfusion (p < .01). The NO synthase inhibitor N-nitro-L-arginine methyl ester (200 microM) reversed this oxidant attenuation (p < .05), NO increase (p < .05), and the cardioprotection conferred by hypercarbic reperfusion (increasing death to 54.3% +/- 6.0% [p < .05]). Conversely, hypocarbic reperfusion increased cell death to 80.4% +/- 4.5% (p < .01). It also increased reactive oxygen species by almost two-fold (p = .052), without affecting the NO level thereafter. Increased reactive oxygen species was attenuated by the mitochondrial complex III inhibitor stigmatellin (20 nM) when given at reperfusion (p < .05). Cell death also decreased from 85.9% +/- 4.5% to 52.2% +/- 6.5% (p < .01). The nicotinamide adenine dinucleotide phosphate oxidase inhibitor apocynin (300 microM) had no effect on reperfusion reactive oxygen

A Proliferative Burst During Preadolescence Establishes the Final Cardiomyocyte Number

PubMed Central

Naqvi, Nawazish; Li, Ming; Calvert, John W.; Tejada, Thor; Lambert, Jonathan P.; Wu, Jianxin; Kesteven, Scott H.; Holman, Sara R.; Matsuda, Torahiro; Lovelock, Joshua D.; Howard, Wesley W.; Iismaa, Siiri E.; Chan, Andrea Y.; Crawford, Brian H.; Wagner, Mary B.; Martin, David I. K.; Lefer, David J.; Graham, Robert M.; Husain, Ahsan

2014-01-01

SUMMARY It is widely believed that perinatal cardiomyocyte terminal differentiation blocks cytokinesis, thereby causing binucleation and limiting regenerative repair after injury. This suggests that heart growth should occur entirely by cardiomyocyte hypertrophy during preadolescence when, in mice, cardiac mass increases many-fold over a few weeks. Here we show thata thyroid hormone surge activates the IGF-1/IGF1-R/Akt pathway on postnatal day-15andinitiates a brief but intense proliferative burst of predominantly binuclear cardiomyocytes. This proliferation increases cardiomyocyte numbers by ~40%, causing a major disparity between heart and cardiomyocyte growth. Also, the response to cardiac injury at postnatal day15 is intermediate between that observed at postnatal day-2 and -21, further suggesting persistence of cardiomyocyte proliferative capacity beyond the perinatal period. If replicated in humans, this may allow novel regenerative therapies for heart diseases. PMID:24813607

Integrins protect cardiomyocytes from ischemia/reperfusion injury

PubMed Central

Okada, Hideshi; Lai, N. Chin; Kawaraguchi, Yoshitaka; Liao, Peter; Copps, Jeffrey; Sugano, Yasuo; Okada-Maeda, Sunaho; Banerjee, Indroneal; Schilling, Jan M.; Gingras, Alexandre R.; Asfaw, Elizabeth K.; Suarez, Jorge; Kang, Seok-Min; Perkins, Guy A.; Au, Carol G.; Israeli-Rosenberg, Sharon; Manso, Ana Maria; Liu, Zheng; Milner, Derek J.; Kaufman, Stephen J.; Patel, Hemal H.; Roth, David M.; Hammond, H. Kirk; Taylor, Susan S.; Dillmann, Wolfgang H.; Goldhaber, Joshua I.; Ross, Robert S.

2013-01-01

Ischemic damage is recognized to cause cardiomyocyte (CM) death and myocardial dysfunction, but the role of cell-matrix interactions and integrins in this process has not been extensively studied. Expression of α7β1D integrin, the dominant integrin in normal adult CMs, increases during ischemia/reperfusion (I/R), while deficiency of β1 integrins increases ischemic damage. We hypothesized that the forced overexpression of integrins on the CM would offer protection from I/R injury. Tg mice with CM-specific overexpression of integrin α7β1D exposed to I/R had a substantial reduction in infarct size compared with that of α5β1D-overexpressing mice and WT littermate controls. Using isolated CMs, we found that α7β1D preserved mitochondrial membrane potential during hypoxia/reoxygenation (H/R) injury via inhibition of mitochondrial Ca2+ overload but did not alter H/R effects on oxidative stress. Therefore, we assessed Ca2+ handling proteins in the CM and found that β1D integrin colocalized with ryanodine receptor 2 (RyR2) in CM T-tubules, complexed with RyR2 in human and rat heart, and specifically bound to RyR2 amino acids 165–175. Integrins stabilized the RyR2 interdomain interaction, and this stabilization required integrin receptor binding to its ECM ligand. These data suggest that α7β1D integrin modifies Ca2+ regulatory pathways and offers a means to protect the myocardium from ischemic injury. PMID:24091324

Comparison of cardioprotective effects of labeled and unlabeled oleanoic acids with new BOPIM dye on primary neonatal rat cardiomyocytes following hypoxia/reoxygenation injury.

PubMed

Wang, Sa; He, Hai-bo; Xiao, Shu-zhang; Wang, Jun-zhi; Bai, Cai-hong; Wei, Na; Zou, Kun

2014-08-01

It is well known that fluorescent labeling has recently become a major research tool in molecular and cellular biology for demonstrating therapeutic mechanisms and metabolic pathways. However, few studies have reported the use of fluorescent labeling of natural products. We recently explored the boron 2-(2'-pyridyl) imidazole (BOPIM) derivative analogs, which are highly fluorescent, non-aggregated, and nontoxic. In the present study, the natural product oleanolic acid (OA) was functionalized and labeled with BOPIM, thus yielding a highly fluorescent probe, the comparison of cardioprotective effects of labeled and unlabeled OAs with BOPIM on primary neonatal rat cardiomyocytes with hypoxia/reoxygenation (H/R) injury were investigated. Pretreatment with OA and BOPIM-OA significantly prevented the H/R induced cell death in primary neonatal rat cardiomyocytes. However, BOPIM exhibited no improvements on the H/R injury cardiomyocytes, and which were similar to those of the H/R group. The results of comparison of cardioprotective effects between labeled and unlabeled OAs with BOPIM showed that introducing the BOPIM chromophore did not make a difference with H/R injury cardiomyocytes. BOPIM chromophore is a suitable probe for investigating the pharmacological mechanisms of natural products. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

5-Aminolevulinic acid with sodium ferrous citrate induces autophagy and protects cardiomyocytes from hypoxia-induced cellular injury through MAPK-Nrf-2-HO-1 signaling cascade

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Mingyi; Guangdong Cardiovascular Institute, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou; Department of Pediatrics, The Third Xiangya Hospital, Central South University, Changsha

Background: Hypoxia causes cardiac disease via oxidative stress and mitochondrial dysfunction. 5-Aminolevulinic acid in combination with sodium ferrous citrate (ALA/SFC) has been shown to up-regulate heme oxygenase-1 (HO-1) and decrease macrophage infiltration and renal cell apoptosis in renal ischemia injury mice. However, its underlying mechanism remains largely unknown. The aim of this study was to investigate whether ALA/SFC could protect cardiomyocytes from hypoxia-induced apoptosis by autophagy via HO-1 signaling. Materials & methods: Murine atrial cardiomyocyte HL-1 cells were pretreated with ALA/SFC and then exposed to hypoxia. Results: ALA/SFC pretreatment significantly attenuated hypoxia-induced cardiomyocyte apoptosis, reactive oxygen species production, and mitochondrial injury,more » while it increased cell viability and autophagy levels. HO-1 expression by ALA/SFC was associated with up-regulation and nuclear translocation of Nrf-2, whereas Nrf-2 siRNA dramatically reduced HO-1 expression. ERK1/2, p38, and SAPK/JNK pathways were activated by ALA/SFC and their specific inhibitors significantly reduced ALA/SFC-mediated HO-1 upregulation. Silencing of either Nrf-2 or HO-1and LY294002, inhibitor of autophagy, abolished the protective ability of ALA/AFC against hypoxia-induced injury and reduced ALA/SFC-induced autophagy. Conclusion: Taken together, our data suggest that ALA/SFC induces autophagy via activation of MAPK/Nrf-2/HO-1 signaling pathway to protect cardiomyocytes from hypoxia-induced apoptosis. - Highlights: • ALA/SFC attenuates hypoxia-induced cardiomyocyte apoptosis, reactive oxygen species production, and mitochondrial injury. • ALA/SFC increases the heme oxygenase-1 expression via Nrf-2 and ERK1/2, p38, and SAPK/JNK pathways. • ALA/SFC induces autophagy and inhibition of autophagy prevent ALA/SFC-mediated suppression of hypoxia-induced injury.« less

Identification of chemicals inducing cardiomyocyte proliferation in developmental stage-specific manner with pluripotent stem cells.

PubMed

Uosaki, Hideki; Magadum, Ajit; Seo, Kinya; Fukushima, Hiroyuki; Takeuchi, Ayako; Nakagawa, Yasuaki; Moyes, Kara White; Narazaki, Genta; Kuwahara, Koichiro; Laflamme, Michael; Matsuoka, Satoshi; Nakatsuji, Norio; Nakao, Kazuwa; Kwon, Chulan; Kass, David A; Engel, Felix B; Yamashita, Jun K

2013-12-01

The proliferation of cardiomyocytes is highly restricted after postnatal maturation, limiting heart regeneration. Elucidation of the regulatory machineries for the proliferation and growth arrest of cardiomyocytes is imperative. Chemical biology is efficient to dissect molecular mechanisms of various cellular events and often provides therapeutic potentials. We have been investigating cardiovascular differentiation with pluripotent stem cells. The combination of stem cell and chemical biology can provide novel approaches to investigate the molecular mechanisms and manipulation of cardiomyocyte proliferation. To identify chemicals that regulate cardiomyocyte proliferation, we performed a screening of a defined chemical library based on proliferation of mouse pluripotent stem cell-derived cardiomyocytes and identified 4 chemical compound groups: inhibitors of glycogen synthase kinase-3, p38 mitogen-activated protein kinase, and Ca(2+)/calmodulin-dependent protein kinase II, and activators of extracellular signal-regulated kinase. Several appropriate combinations of chemicals synergistically enhanced proliferation of cardiomyocytes derived from both mouse and human pluripotent stem cells, notably up to a 14-fold increase in mouse cardiomyocytes. We also examined the effects of identified chemicals on cardiomyocytes in various developmental stages and species. Whereas extracellular signal-regulated kinase activators and Ca(2+)/calmodulin-dependent protein kinase II inhibitors showed proliferative effects only on cardiomyocytes in early developmental stages, glycogen synthase kinase-3 and p38 mitogen-activated protein kinase inhibitors substantially and synergistically induced re-entry and progression of cell cycle in neonatal but also as well as adult cardiomyocytes. Our approach successfully uncovered novel molecular targets and mechanisms controlling cardiomyocyte proliferation in distinct developmental stages and offered pluripotent stem cell-derived cardiomyocytes

Identification of Chemicals Inducing Cardiomyocyte Proliferation in Developmental Stage-Specific Manner with Pluripotent Stem Cells

PubMed Central

Uosaki, Hideki; Magadum, Ajit; Seo, Kinya; Fukushima, Hiroyuki; Takeuchi, Ayako; Nakagawa, Yasuaki; Moyes, Kara White; Narazaki, Genta; Kuwahara, Koichiro; Laflamme, Michael; Matsuoka, Satoshi; Nakatsuji, Norio; Nakao, Kazuwa; Kwon, Chulan; Kass, David A.; Engel, Felix B.; Yamashita, Jun K.

2013-01-01

Background The proliferation of cardiomyocytes is highly restricted after postnatal maturation, limiting heart regeneration. Elucidation of the regulatory machineries for the proliferation and growth arrest of cardiomyocytes is imperative. Chemical biology is efficient to dissect molecular mechanisms of various cellular events and often provide therapeutic potentials. We have been investigating cardiovascular differentiation with pluripotent stem cells (PSCs). The combination of stem cell and chemical biology can provide novel approaches to investigate the molecular mechanisms and manipulation of cardiomyocyte proliferation. Methods and Results To identify chemicals that regulate cardiomyocyte proliferation, we performed a screening of a defined chemical library based on proliferation of mouse PSC-derived cardiomyocytes and identified 4 chemical compound groups - inhibitors of glycogen synthase kinase-3 (GSK3), p38 mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), and activators of extracellular signal-regulated kinase (ERK). Several appropriate combinations of chemicals synergistically enhanced proliferation of cardiomyocytes derived from both mouse and human PSCs, notably up to a 14-fold increase in mouse cardiomyocytes. We also examined the effects of identified chemicals on cardiomyocytes in various developmental stages and species. Whereas ERK activators and CaMKII inhibitors showed proliferative effects only on cardiomyocytes in early developmental stages, GSK3 and p38 MAPK inhibitors substantially and synergistically induced reentry and progression of cell cycle in not only neonatal but also adult cardiomyocytes. Conclusions Our approach successfully uncovered novel molecular targets and mechanisms controlling cardiomyocyte proliferation in distinct developmental stages and offered PSC-derived cardiomyocytes as a potent tool to explore chemical-based cardiac regenerative strategies. PMID:24141057

Dimethyl sulfoxide attenuates hydrogen peroxide-induced injury in cardiomyocytes via heme oxygenase-1.

PubMed

Man, Wang; Ming, Ding; Fang, Du; Chao, Liang; Jing, Cang

2014-06-01

The antioxidant property of dimethyl sulfoxide (DMSO) was formerly attributed to its direct effects. Our former study showed that DMSO is able to induce heme oxygenase-1 (HO-1) expression in endothelial cells, which is a potent antioxidant enzyme. In this study, we hypothesized that the antioxidant effects of DMSO in cardiomyocytes are mediated or partially mediated by increased HO-1 expression. Therefore, we investigated whether DMSO exerts protective effects against H2 O2 -induced oxidative damage in cardiomyocytes, and whether HO-1 is involved in DMSO-imparted protective effects, and we also explore the underlying mechanism of DMSO-induced HO-1 expression. Our study demonstrated that DMSO pretreatment showed a cytoprotective effect against H2 O2 -induced oxidative damage (impaired cell viability, increased apopototic cells rate and caspase-3 level, and increased release of LDH and CK) and this process is partially mediated by HO-1 upregulation. Furthermore, our data showed that the activation of p38 MAPK and Nrf2 translocation are involved in the HO-1 upregulation induced by DMSO. This study reports for the first time that the cytoprotective effect of DMSO in cardiomyocytes is partially mediated by HO-1, which may further explain the mechanisms by which DMSO exerts cardioprotection on H2 O2 injury. J. Cell. Biochem. 115: 1159-1165, 2014. © 2013 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

A Novel Atypical PKC-Iota Inhibitor, Echinochrome A, Enhances Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells.

PubMed

Kim, Hyoung Kyu; Cho, Sung Woo; Heo, Hye Jin; Jeong, Seung Hun; Kim, Min; Ko, Kyung Soo; Rhee, Byoung Doo; Mishchenko, Natalia P; Vasileva, Elena A; Fedoreyev, Sergey A; Stonik, Valentin A; Han, Jin

2018-06-02

Echinochrome A (EchA) is a marine bioproduct extracted from sea urchins having antioxidant, antimicrobial, anti-inflammatory, and chelating effects, and is the active component of the clinical drug histochrome. We investigated the potential use of Ech A for inducing cardiomyocyte differentiation from mouse embryonic stem cells (mESCs). We also assessed the effects of Ech A on mitochondrial mass, inner membrane potential (Δψm), reactive oxygen species generation, and levels of Ca 2+ . To identify the direct target of Ech A, we performed in vitro kinase activity and surface plasmon resonance binding assays. Ech A dose-dependently enhanced cardiomyocyte differentiation with higher beating rates. Ech A (50 μM) increased the mitochondrial mass and membrane potential but did not alter the mitochondrial superoxide and Ca 2+ levels. The in vitro kinase activity of the atypical protein kinase C-iota (PKCι) was significantly decreased by 50 μM of Ech A with an IC 50 for PKCι activity of 107 μM. Computational protein-ligand docking simulation results suggested the direct binding of Ech A to PKCι, and surface plasmon resonance confirmed the direct binding with a low K D of 6.3 nM. Therefore, Ech A is a potential drug for enhancing cardiomyocyte differentiation from mESCs through direct binding to PKCι and inhibition of its activity.

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling

PubMed Central

Yu, Zhi-Bin; Wei, Hongguang

2012-01-01

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213–C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97–H105, 2010). Here we characterized Ca2+-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca2+ was unchanged, but the peak Ca2+ transient was lowered and the durations of Ca2+ rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca2+ transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca2+, resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences. PMID:22538236

Cardiomyocyte injury assessed by a highly sensitive troponin assay and sudden cardiac death in the community: the Cardiovascular Health Study.

PubMed

Hussein, Ayman A; Gottdiener, John S; Bartz, Traci M; Sotoodehnia, Nona; deFilippi, Christopher; Dickfeld, Timm; Deo, Rajat; Siscovick, David; Stein, Phyllis K; Lloyd-Jones, Donald

2013-12-03

This study sought to determine the association between markers of cardiomyocyte injury in ambulatory subjects and sudden cardiac death (SCD). The pathophysiology of SCD is complex but is believed to be associated with an abnormal cardiac substrate in most cases. The association between biomarkers of cardiomyocyte injury in ambulatory subjects and SCD has not been investigated. Levels of cardiac troponin T, a biomarker of cardiomyocyte injury, were measured by a highly sensitive assay (hsTnT) in 4,431 ambulatory participants in the Cardiovascular Health Study, a longitudinal community-based prospective cohort study. Serial measures were obtained in 3,089 subjects. All deaths, including SCD, were adjudicated by a central events committee. Over a median follow-up of 13.1 years, 246 participants had SCD. Baseline levels of hsTnT were significantly associated with SCD (hazard ratio [HR] for +1 log(hsTnT): 2.04, 95% confidence interval [CI]: 1.78 to 2.34]. This association persisted in covariate-adjusted Cox analyses accounting for baseline risk factors (HR: 1.30, 95% CI: 1.05 to 1.62), as well as for incident heart failure and myocardial infarction (HR: 1.26, 95% CI: 1.01 to 1.57). The population was also categorized into 3 groups based on baseline hsTnT levels and SCD risk [fully adjusted HR: 1.89 vs. 1.55 vs. 1 (reference group) for hsTnT ≥12.10 vs. 5.01 to 12.09 vs. ≤ 5.00 pg/ml, respectively; p trend = 0.005]. On serial measurements, change in hsTnT levels was also associated with SCD risk (fully adjusted HR for +1 pg/ml per year increase from baseline: 1.03, 95% CI: 1.01 to 1.06). The findings suggest an association between cardiomyocyte injury in ambulatory subjects and SCD risk beyond that of traditional risk factors. Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Lipoxin A4-Induced Heme Oxygenase-1 Protects Cardiomyocytes against Hypoxia/Reoxygenation Injury via p38 MAPK Activation and Nrf2/ARE Complex

PubMed Central

Chen, Xiao-Qing; Wu, Sheng-Hua; Zhou, Yu; Tang, Yan-Rong

2013-01-01

Objective To investigate whether lipoxin A4 (LXA4) increases expression of heme oxygenase-1(HO-1) in cardiomyocytes, whether LXA4-induced HO-1 protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury, and what are the mechanisms involved in the LXA4-induced HO-1 induction. Methods Rat cardiomyocytes were exposed to H/R injury with or without preincubation with LXA4 or HO-1 inhibitor ZnPP-IX or various signal molecule inhibitors. Expressions of HO-1 protein and mRNA were analyzed by using Western blot and RT-PCR respectively. Activity of nuclear factor E2-related factor 2 (Nrf2) binding to the HO-1 E1 enhancer was assessed by chromatin immunoprecipitation. Nrf2 binding to the HO-1 antioxidant responsive element (ARE) were measured by using electrophoretic mobility shift assay. Results Pretreatment of the cells undergoing H/R lesion with LXA4 significantly reduced the lactate dehydrogenase and creatine kinase productions, increased the cell viability, and increased the expressions of HO-1 protein and mRNA and HO-1 promoter activity. HO-1 inhibition abolished the protective role of LXA4 on the cells undergoing H/R lesion. LXA4 increased p38 mitogen-activated protein kinase (p38 MAPK) activation, nuclear translocation of Nrf2, Nrf2 binding to the HO-1 ARE and E1 enhancer in cardiomyocytes with or without H/R exposure. Conclusion The protection role of LXA4 against H/R injury of cardiomyocytes is related to upregulation of HO-1, via activation of p38 MAPK pathway and nuclear translocation of Nrf2 and Nrf2 binding to the HO-1 ARE and E1 enhancer, but not via activation of phosphatidyinositol-3-kinase or extracellular signal-regulated kinase pathway. PMID:23826208

The HMGB1-TLR4 axis contributes to myocardial ischemia/reperfusion injury via regulation of cardiomyocyte apoptosis.

PubMed

Ding, Hua-Sheng; Yang, Jun; Chen, Ping; Yang, Jian; Bo, Sun-Qing; Ding, Jia-Wang; Yu, Qin-Qin

2013-09-15

Toll-like receptor 4 (TLR4) and its ligand high mobility group box 1 (HMGB1), are known for playing central roles in ischemia-reperfusion injury in myocardium. However, the detailed mechanisms of TLR4 and HMGB1 are not fully understood. The aim of this study was to investigate the effects and possible mechanisms of the HMGB1-TLR4 axis and cardiomyocyte apoptosis on myocardial ischemic damage. Artificial oxygen ventilated anesthetized C3H/HeN mice and C3H/HeJ mice were subjected to 30 min of left anterior descending coronary artery occlusion followed by 6h of reperfusion. The myocardial infarct size, HMGB1 levels, apoptosis index, Bax, Bcl-2 and TNF-α mRNA levels were assessed. The results showed that a lowered amount of cardiomyocyte apoptosis and infarct size in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency notably inhibited the expression of HMGB1 and TNF-a, both of which were up-regulated by ischemia/reperfusion. These findings suggest that the HMGB1-TLR4 axis plays a pathogenic role in triggering cardiomyocyte apoptosis during myocardial I/R injury and that the possible mechanism for this process is the result of released cytokines and inflammatory response involved in the HMGB1/TLR4-related pathway. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Akt1/protein kinase B enhances transcriptional reprogramming of fibroblasts to functional cardiomyocytes

PubMed Central

Zhou, Huanyu; Dickson, Matthew E.; Kim, Min Soo; Bassel-Duby, Rhonda; Olson, Eric N.

2015-01-01

Conversion of fibroblasts to functional cardiomyocytes represents a potential approach for restoring cardiac function after myocardial injury, but the technique thus far has been slow and inefficient. To improve the efficiency of reprogramming fibroblasts to cardiac-like myocytes (iCMs) by cardiac transcription factors [Gata4, Hand2, Mef2c, and Tbx5 (GHMT)], we screened 192 protein kinases and discovered that Akt/protein kinase B dramatically accelerates and amplifies this process in three different types of fibroblasts (mouse embryo, adult cardiac, and tail tip). Approximately 50% of reprogrammed mouse embryo fibroblasts displayed spontaneous beating after 3 wk of induction by Akt plus GHMT. Furthermore, addition of Akt1 to GHMT evoked a more mature cardiac phenotype for iCMs, as seen by enhanced polynucleation, cellular hypertrophy, gene expression, and metabolic reprogramming. Insulin-like growth factor 1 (IGF1) and phosphoinositol 3-kinase (PI3K) acted upstream of Akt whereas the mitochondrial target of rapamycin complex 1 (mTORC1) and forkhead box o3 (Foxo3a) acted downstream of Akt to influence fibroblast-to-cardiomyocyte reprogramming. These findings provide insights into the molecular basis of cardiac reprogramming and represent an important step toward further application of this technique. PMID:26354121

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes.

PubMed

Wiencierz, Anne Maria; Kernbach, Manuel; Ecklebe, Josephine; Monnerat, Gustavo; Tomiuk, Stefan; Raulf, Alexandra; Christalla, Peter; Malan, Daniela; Hesse, Michael; Bosio, Andreas; Fleischmann, Bernd K; Eckardt, Dominik

2015-01-01

Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers. In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis. Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications.

c-kit+ Cells Minimally Contribute Cardiomyocytes to the Heart

PubMed Central

van Berlo, Jop H.; Kanisicak, Onur; Maillet, Marjorie; Vagnozzi, Ronald J.; Karch, Jason; Lin, Suh-Chin J.; Middleton, Ryan C.; Marbán, Eduardo; Molkentin, Jeffery D.

2014-01-01

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine if endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with aging or after injury in adulthood. A cDNA encoding either Cre recombinase or a tamoxifen inducible MerCreMer chimeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of ≈0.03% or less, and if a preponderance towards cellular fusion is considered, the percentage falls below ≈0.008%. In contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although likely at a functionally insignificant level. PMID:24805242

Drp1 loss-of-function reduces cardiomyocyte oxygen dependence protecting the heart from ischemia-reperfusion injury.

PubMed

Zepeda, Ramiro; Kuzmicic, Jovan; Parra, Valentina; Troncoso, Rodrigo; Pennanen, Christian; Riquelme, Jaime A; Pedrozo, Zully; Chiong, Mario; Sánchez, Gina; Lavandero, Sergio

2014-06-01

Mitochondria are key organelles for ATP production in cardiomyocytes, which is regulated by processes of fission and fusion. We hypothesized that the mitochondria fusion protein dynamin-related protein 1 (Drp1) inhibition, attenuates ischemia-reperfusion (I/R) injury through modifications in mitochondrial metabolism. Rats were subjected to I/R through coronary artery ligation, and isolated cardiomyocytes were treated with an ischemia-mimicking solution. In vivo, cardiac function, myocardial infarction area, and mitochondrial morphology were determined, whereas in vitro, viability, mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption rate (OCR) were assessed. In both models, an adenovirus expressing Drp1 dominant-negative K38A (Drp1K38A) was used to induce Drp1 loss-of-function. Our results showed that I/R stimulated mitochondrial fission. Myocardial infarction size and cell death induced by I/R were significantly reduced, whereas cardiac function after I/R was improved in Drp1K38A-treated rats compared with controls. Drp1K38A-transduced cardiomyocytes showed lower OCR with no decrease in intracellular ATP levels, and on I/R, a larger decrease in OCR with a smaller reduction in intracellular ATP level was observed. However, proton leak-associated oxygen consumption was comparatively higher in Drp1K38A-treated cardiomyocytes, suggesting a protective mitochondrial uncoupling effect against I/R. Collectively, our results show that Drp1 inhibition triggers cardioprotection by reducing mitochondrial metabolism during I/R.

Differential Expression Levels of Integrin α6 Enable the Selective Identification and Isolation of Atrial and Ventricular Cardiomyocytes

PubMed Central

Wiencierz, Anne Maria; Kernbach, Manuel; Ecklebe, Josephine; Monnerat, Gustavo; Tomiuk, Stefan; Raulf, Alexandra; Christalla, Peter; Malan, Daniela; Hesse, Michael; Bosio, Andreas; Fleischmann, Bernd K.; Eckardt, Dominik

2015-01-01

Rationale Central questions such as cardiomyocyte subtype emergence during cardiogenesis or the availability of cardiomyocyte subtypes for cell replacement therapy require selective identification and purification of atrial and ventricular cardiomyocytes. However, current methodologies do not allow for a transgene-free selective isolation of atrial or ventricular cardiomyocytes due to the lack of subtype specific cell surface markers. Methods and Results In order to develop cell surface marker-based isolation procedures for cardiomyocyte subtypes, we performed an antibody-based screening on embryonic mouse hearts. Our data indicate that atrial and ventricular cardiomyocytes are characterized by differential expression of integrin α6 (ITGA6) throughout development and in the adult heart. We discovered that the expression level of this surface marker correlates with the intracellular subtype-specific expression of MLC-2a and MLC-2v on the single cell level and thereby enables the discrimination of cardiomyocyte subtypes by flow cytometry. Based on the differential expression of ITGA6 in atria and ventricles during cardiogenesis, we developed purification protocols for atrial and ventricular cardiomyocytes from mouse hearts. Atrial and ventricular identities of sorted cells were confirmed by expression profiling and patch clamp analysis. Conclusion Here, we introduce a non-genetic, antibody-based approach to specifically isolate highly pure and viable atrial and ventricular cardiomyocytes from mouse hearts of various developmental stages. This will facilitate in-depth characterization of the individual cellular subsets and support translational research applications. PMID:26618511

In vitro chronotropic effects of Erythrina senegalensis DC (Fabaceae) aqueous extract on mouse heart slice and pluripotent stem cell-derived cardiomyocytes.

PubMed

Nembo, Erastus Nembu; Atsamo, Albert Donatien; Nguelefack, Télesphore Benoît; Kamanyi, Albert; Hescheler, Jürgen; Nguemo, Filomain

2015-05-13

Erythrina senegalensis DC (Fabaceae) bark is commonly used in sub-Saharan traditional medicine for the treatment of many diseases including gastrointestinal disorders and cardiovascular diseases. In this study, we investigated the effect of the aqueous extract of the stem bark of Erythrina senegalensis on the contractile properties of mouse ventricular slices and human induced pluripotent stem (hiPS) cell-derived cardiomyocytes. We also investigated the cytotoxic effect of the extract on mouse embryonic stem (ES) cells differentiating into cardiomyocytes (CMs). We used well-established electrophysiological technologies to assess the effect of Erythrina senegalensis aqueous extract (ESAE) on the beating activity of mouse ventricular slices, mouse ES and hiPS cell-derived CMs. To study the cytotoxic effect of our extract, differentiating mouse ES cells were exposed to different concentrations of ESAE. EB morphology was assessed by microscopy at different stages of differentiation whereas cell viability was measured by flow cytometry, fluorometry and immunocytochemistry. The electrical activity of CMs and heart slices were respectively captured by the patch clamp technique and microelectrode array (MEA) method following ESAE acute exposure. Our findings revealed that ESAE exhibits a biphasic chronotropic activity on mouse ventricular slices with an initial low dose (0.001 and 0.01 µg/mL) decrease in beating activity followed by a corresponding significant increase in chronotropic activity at higher doses above 10 µg/mL. The muscarinic receptor blocker, atropine abolished the negative chronotropic activity of ESAE, while propranolol successfully blocked its positive chronotropic activity. ESAE showed a significant dose-dependent positive chronotropic activity on hiPS cell-derived CMs. Also, though not significantly, ESAE decreased cell viability and increased total caspase-3/7 activity of mouse ES cells in a concentration-dependent manner. Erythrina senegalensis

Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1.

PubMed

Song, Chun-Li; Liu, Bin; Diao, Hong-Ying; Shi, Yong-Feng; Zhang, Ji-Chang; Li, Yang-Xue; Liu, Ning; Yu, Yun-Peng; Wang, Guan; Wang, Jin-Peng; Li, Qian

2016-06-28

Insulin-like growth factor-1 (IGF-1) is an important regulator of cardiomyocyte homeostasis and cardiac structure, and the prosurvival and antiapoptotic effects of IGF-1 have been investigated. However, the effect of microRNA-320 (miR-320) in ischemia and reperfusion (I/R) by targeting IGF-1 is rarely discussed. We investigated the role of miR-320 in I/R injury. A total of 192 healthy female Wistar rats were divided into eight groups (n = 24). Rat heart I/R model was established. Hemodynamics, infarct size weight (ISW), heart function, and rat cardiomyocyte apoptosis were measured. Hypoxia-reoxygenation (H/R) in rat cardiomyocyte was used to simulate the I/R process. The mRNA levels of miR-320 and IGF-1, and proteins levels of IGF-1, IGF-1R, p-IGF-1R, p-ASK1, p-JNK, p-p38, Bcl-2, Bax and Caspase-3 were measured. In vivo inhibition of miR-320 expression significantly increased IGF-1 and IGF-1R mRNA levels, elevated the absolute values of SBP, DBP, MAP, ± dp/dtmax, LVEF and LVFS, decreased ISW, LVESD and LVEDd and the number of TUNEL positive cells, lowered the levels of p-ASK1, p-JNK, p-p38, Bax and Caspase-3 and increased expression of Bcl-2 compared to the I/R + NC group. Compared to H/R + NC group in vitro, miR-320 inhibition increased IGF-1 mRNA levels, inhibited cardiomyocyte apoptosis, down-regulated p-ASK, p-JNK, p-p38, Bax and Caspase-3 levels, and up-regulated Bcl-2 level. MiR-320 inhibition target elevated IGF-1 mRNA and protein levels, suppress early cardiomyocyte apoptosis of I/R, and inhibited ASK1-JNK/p38 pathway, which provides a new target for clinical study of I/R injury.

Down-regulation of microRNA-320 suppresses cardiomyocyte apoptosis and protects against myocardial ischemia and reperfusion injury by targeting IGF-1

PubMed Central

Song, Chun-Li; Liu, Bin; Diao, Hong-Ying; Shi, Yong-Feng; Zhang, Ji-Chang; Li, Yang-Xue; Liu, Ning; Yu, Yun-Peng; Wang, Guan; Wang, Jin-Peng; Li, Qian

2016-01-01

Insulin-like growth factor-1 (IGF-1) is an important regulator of cardiomyocyte homeostasis and cardiac structure, and the prosurvival and antiapoptotic effects of IGF-1 have been investigated. However, the effect of microRNA-320 (miR-320) in ischemia and reperfusion (I/R) by targeting IGF-1 is rarely discussed. We investigated the role of miR-320 in I/R injury. A total of 192 healthy female Wistar rats were divided into eight groups (n = 24). Rat heart I/R model was established. Hemodynamics, infarct size weight (ISW), heart function, and rat cardiomyocyte apoptosis were measured. Hypoxia-reoxygenation (H/R) in rat cardiomyocyte was used to simulate the I/R process. The mRNA levels of miR-320 and IGF-1, and proteins levels of IGF-1, IGF-1R, p-IGF-1R, p-ASK1, p-JNK, p-p38, Bcl-2, Bax and Caspase-3 were measured. In vivo inhibition of miR-320 expression significantly increased IGF-1 and IGF-1R mRNA levels, elevated the absolute values of SBP, DBP, MAP, ± dp/dtmax, LVEF and LVFS, decreased ISW, LVESD and LVEDd and the number of TUNEL positive cells, lowered the levels of p-ASK1, p-JNK, p-p38, Bax and Caspase-3 and increased expression of Bcl-2 compared to the I/R + NC group. Compared to H/R + NC group in vitro, miR-320 inhibition increased IGF-1 mRNA levels, inhibited cardiomyocyte apoptosis, down-regulated p-ASK, p-JNK, p-p38, Bax and Caspase-3 levels, and up-regulated Bcl-2 level. MiR-320 inhibition target elevated IGF-1 mRNA and protein levels, suppress early cardiomyocyte apoptosis of I/R, and inhibited ASK1-JNK/p38 pathway, which provides a new target for clinical study of I/R injury. PMID:27175593

Rac1 modulates cardiomyocyte adhesion during mouse embryonic development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abu-Issa, Radwan, E-mail: rabuissa@umich.edu

2015-01-24

Highlights: • Conditional knockout of Rac1 using Nkx2.5 Cre line is lethal at E13.5. • The myocardium of the mutant is thin and disorganized. • The phenotype is not due to cardiomyocyte low proliferation or apoptosis. • The phenotype is due to specific defect in cardiomyocyte adhesion. - Abstract: Rac1, a member of the Rho subfamily of small GTPases, is involved in morphogenesis and differentiation of many cell types. Here we define a role of Rac1 in cardiac development by specifically deleting Rac1 in the pre-cardiac mesoderm using the Nkx2.5-Cre transgenic driver line. Rac1-conditional knockout embryos initiate heart development normallymore » until embryonic day 11.5 (E11.5); their cardiac mesoderm is specified, and the heart tube is formed and looped. However, by E12.5-E13.5 the mutant hearts start failing and embryos develop edema and hemorrhage which is probably the cause for the lethality observed soon after. The hearts of Rac1-cKO embryos exhibit disorganized and thin myocardial walls and defects in outflow tract alignment. No significant differences of cardiomyocyte death or proliferation were found between developing control and mutant embryos. To uncover the role of Rac1 in the heart, E11.5 primary heart cells were cultured and analyzed in vitro. Rac1-deficient cardiomyocytes were less spread, round and loosely attached to the substrate and to each other implying that Rac1-mediated signaling is required for appropriate cell–cell and/or cellmatrix adhesion during cardiac development.« less

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes

PubMed Central

Tarnawski, Laura; Xian, Xiaojie; Monnerat, Gustavo; Macaulay, Iain C.; Malan, Daniela; Borgman, Andrew; Wu, Sean M.; Fleischmann, Bernd K.; Jovinge, Stefan

2015-01-01

In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes. PMID:26323090

Long-term supplementation with eicosapentaenoic acid salvages cardiomyocytes from hypoxia/reoxygenation-induced injury in rats fed with fish-oil-deprived diet.

PubMed

Nasa, Y; Hayashi, M; Sasaki, H; Hayashi, J; Takeo, S

1998-06-01

Dietary supplementation of fish oil containing eicosapentaenoic acid (C20:5 n-3, EPA) and docosahexaenoic acid (C22:6 n-3, DHA) has been shown to exert protective effects on ischemic/reperfused hearts. We determined whether deprivation of fish oil from the diet paradoxically enhances susceptibility of cardiomyocytes to hypoxia/reoxygenation-induced injury and whether supplementation with either EPA or DHA overcomes such alterations. Rats were fed with fish-oil-rich (FOR) diet, fish-oil-deprived (FOD) diet alone, FOD diet with EPA (1 g/kg/day), or FOD diet with DHA (1 g/kg/day) for 4 weeks. The FOD diet reduced n-3 polyunsaturated fatty acids (PUFAs) and increased n-6 PUFAs such as linoleic (C18:2) and arachidonic acids (C20:4) in myocardial phospholipids. EPA or DHA supplementation increased its incorporation into phospholipid pools. Cardiomyocytes isolated by treatment with collagenase were subjected to 150 min of hypoxia and subsequent reoxygenation for 15 min. In the FOD diet group, the number of surviving rod-shaped cells after hypoxia and reoxygenation was smaller than that of the FOR group. Supplementation with EPA did not affect the number of rod-shaped cells, but attenuated reoxygenation-induced reduction in the number of square-shaped cells. In contrast, DHA supplementation did not afford any protection. The results suggest that deprivation of fish oil from dietary intake enhances the susceptibility of cardiomyocytes to hypoxic injury, and EPA, but not DHA, is capable of salvaging cardiomyocytes from hypoxia/reoxygenation-induced damage.

HCN4-Overexpressing Mouse Embryonic Stem Cell-Derived Cardiomyocytes Generate a New Rapid Rhythm in Rats with Bradycardia.

PubMed

Saito, Yukihiro; Nakamura, Kazufumi; Yoshida, Masashi; Sugiyama, Hiroki; Takano, Makoto; Nagase, Satoshi; Morita, Hiroshi; Kusano, Kengo F; Ito, Hiroshi

2018-05-30

A biological pacemaker is expected to solve the persisting problems of an artificial cardiac pacemaker including short battery life, lead breaks, infection, and electromagnetic interference. We previously reported HCN4 overexpression enhances pacemaking ability of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) in vitro. However, the effect of these cells on bradycardia in vivo has remained unclear. Therefore, we transplanted HCN4-overexpressing mESC-CMs into bradycardia model animals and investigated whether they could function as a biological pacemaker. The rabbit Hcn4 gene was transfected into mouse embryonic stem cells and induced HCN4-overexpressing mESC-CMs. Non-cardiomyocytes were removed under serum/glucose-free and lactate-supplemented conditions. Cardiac balls containing 5 × 10 3 mESC-CMs were made by using the hanging drop method. One hundred cardiac balls were injected into the left ventricular free wall of complete atrioventricular block (CAVB) model rats. Heart beats were evaluated using an implantable telemetry system 7 to 30 days after cell transplantation. The result showed that ectopic ventricular beats that were faster than the intrinsic escape rhythm were often observed in CAVB model rats transplanted with HCN4-overexpressing mESC-CMs. On the other hand, the rats transplanted with non-overexpressing mESC-CMs showed sporadic single premature ventricular contraction but not sustained ectopic ventricular rhythms. These results indicated that HCN4-overexpressing mESC-CMs produce rapid ectopic ventricular rhythms as a biological pacemaker.

Higenamine protects ischemia/reperfusion induced cardiac injury and myocyte apoptosis through activation of β2-AR/PI3K/AKT signaling pathway

PubMed Central

Wu, Mei-ping; Zhang, Yi-shuai; Zhou, Qian-mei; Xiong, Jian; Dong, Yao-rong; Yan, Chen

2016-01-01

Cardiomyocyte apoptosis contributes to ischemic cardiac injury and the development of heart failure. Higenamine is a key component of the Chinese herb aconite root that has been prescribed for treating symptoms of heart failure for thousands of years in the oriental Asian countries. It has been shown that higenamine has anti-apoptotic effects in a few cell types including cardiomyocytes. However, the pharmacological target and molecular mechanism of higenamine in the heart are still not fully illustrated. Herein, we report that higenamine protected myocyte apoptosis and ischemia/reperfusion (I/R) injury through selective activation of beta2-adrenergic receptor (β2-AR). In particular, we show that higenamine significantly reduced I/R-induced myocardial infarction in mice. In both primary neonatal rat and adult mouse ventricular myocytes, we show higenamine inhibited cell apoptosis and also reduced biochemical markers of apoptosis such as cleaved caspase 3 and 9. More importantly, we show that the anti-apoptotic effects of higenamine in cardiomyocytes were completely abolished by β2-AR but not β1-AR antagonism. Furthermore, we confirmed that higenamine attenuated I/R-induced myocardial injury and reduced cleaved caspases in a β2-AR dependent manner in intact mouse hearts. Higenamine stimulated AKT phosphorylation and required PI3K activation for the anti-apoptotic effect in cardiomyocytes. These findings together suggest that anti-apoptotic and cardiac protective effects of higenamine are mediated by the β2-AR/PI3K/AKT cascade. PMID:26746354

Influence of contrast agent dose and ultrasound exposure on cardiomyocyte injury induced by myocardial contrast echocardiography in rats.

PubMed

Miller, Douglas L; Li, Peng; Dou, Chunyan; Gordon, David; Edwards, Chris A; Armstrong, William F

2005-10-01

To detect specific cardiomyocyte injury induced by myocardial contrast material-enhanced echocardiography (ie, myocardial contrast echocardiography) in rats and to ascertain the influences of contrast material dose and ultrasound exposure on this injury. All animal procedures were approved by the university committee for the use and care of animals. Myocardial contrast echocardiography with 1:4 electrocardiographic (ECG) triggering was performed at 1.5 MHz in 61 anesthetized rats. Evans blue (EB) dye was injected as the vital stain for cardiomyocyte injury. At the start of myocardial contrast echocardiography, which lasted 10 minutes, perflutren lipid microsphere-based contrast material was infused through the tail vein for 5 minutes. Premature heartbeats were counted from the ECG record. The numbers of EB-stained cells counted on sections of heart specimens obtained 24 hours after myocardial contrast echocardiography and then either fresh frozen or embedded in paraffin were determined by using fluorescence microscopy. Results were compared statistically by using t tests and Mann-Whitney rank sum tests. EB-stained cells were concentrated in the anterior region of the myocardium. In the paraffin-embedded specimens, EB-stained cells were often accompanied by but largely separate from areas of inflammatory cell infiltration. At end-systolic triggering with a 50 microL/kg dose of microsphere contrast material, the EB-stained cell count increased with increasing peak rarefactional pressure amplitude, with significantly increased cell counts at 1.6 MPa (P < .02) and 2.0 MPa (P < .005) relative to the cell counts at sham myocardial contrast echocardiography. Premature heartbeats had a similar exposure-response relationship; however, number of premature heartbeats and EB-stained cell count did not appear to be directly related (coefficient of determination r2 = 0.03). The EB-stained cell counts at end-diastolic triggering were not significantly different from those at end

Cardiac resident macrophages are involved in hypoxia-induced postnatal cardiomyocyte proliferation

PubMed Central

Liu, Bo; Zhang, Hua-Gang; Zhu, Yun; Jiang, Yun-Han; Luo, Gui-Ping; Tang, Fu-Qin; Jian, Zhao; Xiao, Ying-Bin

2017-01-01

Induction of cardiomyocyte proliferation, the most promising approach to reverse myocardial attrition, has been gaining importance as a therapy for cardiovascular disease. Hypoxia and macrophages were previously independently reported to promote cardiomyocyte proliferation in mice. However, whether hypoxia promotes cardiomyocyte proliferation in humans, and the association between hypoxia and macrophages in cardiomyocyte proliferation, have not to the best of our knowledge been previously investigated. The present study investigated the cardiomyocyte proliferation in 22 acyanotic and 29 cyanotic patients. Cardiomyocyte proliferation in a hypoxic mouse model (15% O2) was subsequently performed and the macrophage subsets were analyzed. A C-C chemokine receptor type 2 (CCR2) inhibitor was used to increase the number of resident macrophages in order to investigate the effect of macrophages on cardiomyocyte proliferation. The results demonstrated that cardiomyocyte proliferation in the cyanotic infant group was significantly increased compared with the acyanotic infant group and the hypoxia-treated C57BL/6J neonates confirmed the hypoxia-induced cardiomyocyte proliferation. However, hypoxia did not induce the proliferation of isolated cardiomyocytes. Notably, hypoxia treatment increased the number of cardiac resident macrophages in neonate hearts. Furthermore, increasing the number of resident macrophages significantly enhanced cardiomyocyte proliferation. In conclusion, postnatal hypoxia promoted cardiomyocyte proliferation in humans and animals, and cardiac resident macrophages may be involved in this process. Therefore, this novel mechanism may provide a promising strategy for cardiovascular disease treatment. PMID:28393210

Macrophage Colony-Stimulating Factor Improves Cardiac Function after Ischemic Injury by Inducing Vascular Endothelial Growth Factor Production and Survival of Cardiomyocytes

PubMed Central

Okazaki, Tatsuma; Ebihara, Satoru; Asada, Masanori; Yamanda, Shinsuke; Saijo, Yoshifumi; Shiraishi, Yasuyuki; Ebihara, Takae; Niu, Kaijun; Mei, He; Arai, Hiroyuki; Yambe, Tomoyuki

2007-01-01

Macrophage colony-stimulating factor (M-CSF), known as a hematopoietic growth factor, induces vascular endothelial growth factor (VEGF) production from skeletal muscles. However, the effects of M-CSF on cardiomyocytes have not been reported. Here, we show M-CSF increases VEGF production from cardiomyocytes, protects cardiomyocytes and myotubes from cell death, and improves cardiac function after ischemic injury. In mice, M-CSF increased VEGF production in hearts and in freshly isolated cardiomyocytes, which showed M-CSF receptor expression. In rat cell line H9c2 cardiomyocytes and myotubes, M-CSF induced VEGF production via the Akt signaling pathway, and M-CSF pretreatment protected these cells from H2O2-induced cell death. M-CSF activated Akt and extracellular signal-regulated kinase signaling pathways and up-regulated downstream anti-apoptotic Bcl-xL expression in these cells. Using goats as a large animal model of myocardial infarction, we found that M-CSF treatment after the onset of myocardial infarction by permanent coronary artery ligation promoted angiogenesis in ischemic hearts but did not reduce the infarct area. M-CSF pretreatment of the goat myocardial infarction model by coronary artery occlusion-reperfusion improved cardiac function, as assessed by hemodynamic parameters and echocardiography. These results suggest M-CSF might be a novel therapeutic agent for ischemic heart disease. PMID:17717142

Intramyocardial Injection of siRNAs Can Efficiently Establish Myocardial Tissue-Specific Renalase Knockdown Mouse Model.

PubMed

Huang, Kun; Liu, Ju; Zhang, Hui; Wang, Jiliang; Li, Huili

2016-01-01

Ischaemia/reperfusion (I/R) injury will cause additional death of cardiomyocytes in ischaemic heart disease. Recent studies revealed that renalase was involved in the I/R injury. So, the myocardial tissue-specific knockdown mouse models were needed for the investigations of renalase. To establish the mouse models, intramyocardial injection of siRNAs targeting renalase was performed in mice. The wild distribution and high transfection efficiency of the siRNAs were approved. And the renalase expression was efficiently suppressed in myocardial tissue. Compared with the high cost, time consumption, and genetic compensation risk of the Cre/loxP technology, RNA interference (RNAi) technology is much cheaper and less time-consuming. Among the RNAi technologies, injection of siRNAs is safer than virus. And considering the properties of the I/R injury mouse models, the efficiency and durability of injection with siRNAs are acceptable for the studies. Altogether, intramyocardial injection of siRNAs targeting renalase is an economical, safe, and efficient method to establish myocardial tissue-specific renalase knockdown mouse models.

Acute Pre-/Post-Treatment with 8th Day SOD-Like Supreme (a Free Radical Scavenging Health Product) Protects against Oxidant-Induced Injury in Cultured Cardiomyocytes and Hepatocytes In Vitro as Well as in Mouse Myocardium and Liver In Vivo.

PubMed

Leong, Pou Kuan; Chen, Jihang; Chan, Wing Man; Leung, Hoi Yan; Chan, Lincoln; Ko, Kam Ming

2017-04-10

8th Day superoxide dismutase (SOD)-Like Supreme (SOD-Like Supreme, a free radical scavenging health product) is an antioxidant-enriched fermentation preparation with free radical scavenging properties. In the present study, the cellular/tissue protective actions of SOD-Like Supreme against menadione toxicity in cultured H9c2 cardiomyocytes and in AML12 hepatocytes as well as oxidant-induced injury in the mouse myocardium and liver were investigated. SOD-Like Supreme was found to possess potent free radical scavenging activity in vitro as assessed by an oxygen radical absorbance capacity assay. Incubation with SOD-Like Supreme (0.5-3% (v/v)) was shown to protect against menadione-induced toxicity in H9c2 and AML12 cells, as evidenced by increases in cell viability. The ability of SOD-Like Supreme to protect against menadione cytotoxicity was associated with an elevation in the cellular reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in menadione-challenged cells. Consistent with the cell-based studies, pre-/post-treatment with SOD-Like Supreme (0.69 and 2.06 mL/kg, three intermittent doses per day for two consecutive days) was found to protect against isoproterenol-induced myocardial injury and carbon tetrachloride hepatotoxicity in mice. The cardio/hepatoprotection afforded by SOD-Like Supreme was also paralleled by increases in myocardial/hepatic mitochondrial GSH/GSSG ratios in the SOD-Like Supreme-treated/oxidant-challenged mice. In conclusion, incubation/treatment with SOD-Like Supreme was found to protect against oxidant-induced injury in vitro and in vivo, presumably by virtue of its free radical scavenging activity.

Reprogramming of mouse fibroblasts into cardiomyocyte-like cells in vitro.

PubMed

Qian, Li; Berry, Emily C; Fu, Ji-dong; Ieda, Masaki; Srivastava, Deepak

2013-06-01

Cardiac fibroblasts can be reprogrammed to cardiomyocyte-like cells by the introduction of three transcription factors: Gata4, Mef2c and Tbx5 (collectively referred to here as GMT). Resident cardiac fibroblasts can be converted in vivo into induced cardiomyocyte-like cells (iCMs) that closely resemble endogenous cardiomyocytes and electrically integrate with the host myocardium. In contrast, in vitro reprogramming yields many partially reprogrammed iCMs, with a few that reprogram fully into contracting myocytes (~3 out of 10,000 GMT-transduced cells). iCMs can be observed as early as 3 d after viral infection, and they continue to mature over 2 months before beating is observed. Despite the success of multiple groups, the inefficiency of in vitro reprogramming has made it challenging for others. However, given the advantages of in vitro iCMs for performing mechanistic studies and, if refined, for testing drugs or small molecules for personalized medicine and modeling cardiac disease in a dish, it is important to standardize the protocol to improve reproducibility and enhance the technology further. Here we describe a detailed step-by-step protocol for in vitro cardiac reprogramming using retroviruses encoding GMT.

Necroptosis may be a novel mechanism for cardiomyocyte death in acute myocarditis.

PubMed

Zhou, Fei; Jiang, Xuejun; Teng, Lin; Yang, Jun; Ding, Jiawang; He, Chao

2018-05-01

In this study, we investigated the roles of RIP1/RIP3 mediated cardiomyocyte necroptosis in CVB3-induced acute myocarditis. Serum concentrations of creatinine kinase (CK), CK-MB, and cardiac troponin I were detected using a Hitachi Automatic Biochemical Analyzer in a mouse model of acute VMC. Histological changes in cardiac tissue were observed by light microscope and expression levels of RIP1/RIP3 in the cardiac tissue were detected via Western blot and immunohistochemistry. The data showed that RIP1/RIP3 was highly expressed in cardiomyocytes in the acute VMC mouse model and that the necroptosis pathway specific blocker, Nec-1, dramatically reduced the myocardial damage by downregulating the expression of RIP1/RIP3. These findings provide evidence that necroptosis plays a significant role in cardiomyocyte death and it is a major pathway for cell death in acute VMC. Blocking the necroptosis pathway may serve as a new therapeutic option for the treatment of acute viral myocarditis.

HDAC Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy

PubMed Central

Xie, Min; Kong, Yongli; Tan, Wei; May, Herman; Battiprolu, Pavan K.; Pedrozo, Zully; Wang, Zhao; Morales, Cyndi; Luo, Xiang; Cho, Geoffrey; Jiang, Nan; Jessen, Michael E.; Warner, John J.; Lavandero, Sergio; Gillette, Thomas G.; Turer, Aslan T.; Hill, Joseph A.

2014-01-01

Background Reperfusion accounts for a substantial fraction of the myocardial injury occurring with ischemic heart disease. Yet, no standard therapies are available targeting reperfusion injury. Here, we tested the hypothesis that SAHA, a histone deacetylase (HDAC) inhibitor FDA-approved for cancer treatment, will blunt reperfusion injury. Methods and Results Twenty-one rabbits were randomized into 3 groups: a) vehicle control, b) SAHA pretreatment (one day prior and at surgery), and c) SAHA treatment at the time of reperfusion only. Each arm was subjected to ischemia/reperfusion surgery (I/R, 30min coronary ligation, 24h reperfusion). Additionally cultured neonatal and adult rat ventricular cardiomyocytes were subjected to simulated I/R (sI/R) to probe mechanism. SAHA reduced infarct (those reduction inhibitor, SAHA, infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during I/R occur, at least in part, through induction of autophagic flux. assayed in both rabbit myocardium and in mice harboring an RFP-GFP-LC3 transgene. In cultured myocytes subjected to sI/R, SAHA pretreatment reduced cell death by 40%. This eduction in cell death correlated with increased autophagic activity in SAHA-treated cells. RNAi-mediated knockdown of ATG7 and ATG5, essential autophagy proteins, abolished SAHA's cardioprotective effects. Conclusions The FDS-approved anti-cancer HDAC inhibitor, SAHA, reduces myocardial infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during I/R occur, at least in part, through induction of autophagic flux. PMID:24396039

Sulfiredoxin-1 protects against simulated ischaemia/reperfusion injury in cardiomyocyte by inhibiting PI3K/AKT-regulated mitochondrial apoptotic pathways.

PubMed

Zhang, Jiankai; He, Zhangyou; Guo, Jinhua; Li, Zhe; Wang, Xiaohong; Yang, Chun; Cui, Xiaojun

2016-01-01

Reactive oxygen species (ROS)-triggered cardiac cell injury is recognized as the major contributor for the pathogenesis progression of ischaemic cardiovascular diseases. Sulfiredoxin-1 (Srx-1) is an endogenous antioxidant and exerts the crucial neuroprotective effects in cerebral ischaemia. However, its function and the underlying mechanism in ischaemic heart diseases remain poorly defined. Here, a dramatical decrease in Srx-1 was validated in H9c2 cardiomyocytes upon simulated ischaemia-reperfusion (SI/R) injury. Moreover, Srx-1 protected H9c2 cells from SI/R-injured injury as the evidences that Srx-1 up-regulation attenuated the inhibitory effects on cell viability, lactate dehydrogenase (LDH) and cell apoptosis upon SI/R treatment. Knockdown of Srx-1 accelerated cell injury upon SI/R. Mechanism assay corroborated that SI/R treatment noticeably aggravated the loss of mitochondrial membrane potential (Δψm), which was remarkably abated in Ad-Srx-1 groups. Importantly, Srx-1 elevation substantially reduced cytochrome c release, the activity of caspase-9 and caspase-3, accompany with the subsequent decrease in the cleavage of poly (ADP ribose) polymerase (PARP). Concomitantly, overexpression of Srx-1 also decreased the expression of pro-apoptosis protein Bax and increased anti-apoptotic Bcl-2 expression. Further analysis substantiated that Srx-1 treatment remarkably induced the activation of PI3K/AKT signalling. Preconditioning with LY294002 dramatically decreased Srx-1-enhanced cell resistance to SI/R injury. Importantly, LY294002 mitigated the inhibitory effects of Srx-1 on Δψm loss, cytochrome c release, caspase-9/3 activity, and the expression of Bcl-2 family. Together, these results suggested that Srx-1 might protect cardiomyocyte injury upon SI/R by suppressing PI3K/AKT-mediated mitochondria dependent apoptosis, revealing a promising therapeutic agent against ischaemic cardiovascular diseases. © 2016 The Author(s).

Cardioprotective effect of breviscapine: inhibition of apoptosis in H9c2 cardiomyocytes via the PI3K/Akt/eNOS pathway following simulated ischemia/reperfusion injury.

PubMed

Wang, Jun; Ji, Shu-Yun; Liu, Si-Zhu; Jing, Rui; Lou, Wei-Juan

2015-09-01

Breviscapine (BE) is a standardized Chinese herbal medicine extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been widely used to treat cardiovascular and cerebrovascular diseases. However, there are no reports on the protective effects and underlying molecular mechanisms of BE action on myocardial ischemia/reperfusion (MI/R)-induced cardiomyocyte apoptosis. In the present study, we aimed to confirm the cardioprotective effect of BE from MI/R injury in vivo, and investigate the potential molecular mechanisms against simulated ischemia/reperfusion (SI/R)-induced cardiomyocyte apoptosis in vitro. The rat model of MI/R injury was induced by 30 min of transient vessel occlusion followed by 3 h of reperfusion. BE significantly reduced the myocardium infarct size and production of cardiac troponin (cTnl) in serum. In an in vitro experiment, H9c2 cardiomyocytes were incubated with vehicle or ischemic buffer during hypoxia; then, they were reoxygenated with or without BE. BE markedly improved the cell viability and decreased lactate dehydrogenase (LDH) release. We confirmed the anti-apoptotic effect of BE with the Hoechst 33258 staining assay, and this effect was associated with an increase in Bcl-2 and a decrease in active caspase-3 expression. Western blot analysis also showed that BE increased the phosphorylation of Akt and eNOS in H9c2 cells, and the protective effects of BE were partially inhibited by the phosphatidylinositol 3'-kinase (PI3K) specific inhibitor LY294002. Our results suggested that BE could provide significant cardioprotection against MI/R injury, and the potential mechanisms might involve suppression of cardiomyocyte apoptosis through activating the PI3K/Akt/eNOS signaling pathway.

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaja, Ivan; Bai, Xiaowen, E-mail: xibai@mcw.edu; Liu, Yanan

Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1more » (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

Viral myocarditis: potential defense mechanisms within the cardiomyocyte against virus infection.

PubMed

Yajima, Toshitaka

2011-05-01

Virus infection can inflict significant damage on cardiomyocytes through direct injury and secondary immune reactions, leading to myocarditis and dilated cardiomyopathy. While viral myocarditis or cardiomyopathy is a complication of systemic infection of cardiotropic viruses, most individuals infected with the viruses do not develop significant cardiac disease. However, some individuals proceed to develop severe virus-mediated heart disease. Recent studies have shown that viral infection of cardiomyocytes is required for the development of myocarditis and subsequent cardiomyopathy. This suggests that viral infection of cardiomyocytes can be an important step that determines the pathogenesis of viral myocarditis during systemic infection. Accordingly, this article focuses on potential defense mechanisms within the cardiomyocyte against virus infection. Understanding of the cardiomyocyte defense against invading viruses may give us novel insights into the pathophysiology of viral myocarditis, and enable us to develop innovative strategies of diagnosis and treatment for this challenging clinical entity.

MicroRNA-214 protects the mouse heart from ischemic injury by controlling Ca2+ overload and cell death

PubMed Central

Aurora, Arin B.; Mahmoud, Ahmed I.; Luo, Xiang; Johnson, Brett A.; van Rooij, Eva; Matsuzaki, Satoshi; Humphries, Kenneth M.; Hill, Joseph A.; Bassel-Duby, Rhonda; Sadek, Hesham A.; Olson, Eric N.

2012-01-01

Early reperfusion of ischemic cardiac tissue remains the most effective intervention for improving clinical outcome following myocardial infarction. However, abnormal increases in intracellular Ca2+ during myocardial reperfusion can cause cardiomyocyte death and consequent loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Therapeutic modulation of Ca2+ handling provides some cardioprotection against the paradoxical effects of restoring blood flow to the heart, highlighting the significance of Ca2+ overload to IR injury. Cardiac IR is also accompanied by dynamic changes in the expression of microRNAs (miRNAs); for example, miR-214 is upregulated during ischemic injury and heart failure, but its potential role in these processes is unknown. Here, we show that genetic deletion of miR-214 in mice causes loss of cardiac contractility, increased apoptosis, and excessive fibrosis in response to IR injury. The cardioprotective roles of miR-214 during IR injury were attributed to repression of the mRNA encoding sodium/calcium exchanger 1 (Ncx1), a key regulator of Ca2+ influx; and to repression of several downstream effectors of Ca2+ signaling that mediate cell death. These findings reveal a pivotal role for miR-214 as a regulator of cardiomyocyte Ca2+ homeostasis and survival during cardiac injury. PMID:22426211

Histone deacetylase inhibition blunts ischemia/reperfusion injury by inducing cardiomyocyte autophagy.

PubMed

Xie, Min; Kong, Yongli; Tan, Wei; May, Herman; Battiprolu, Pavan K; Pedrozo, Zully; Wang, Zhao V; Morales, Cyndi; Luo, Xiang; Cho, Geoffrey; Jiang, Nan; Jessen, Michael E; Warner, John J; Lavandero, Sergio; Gillette, Thomas G; Turer, Aslan T; Hill, Joseph A

2014-03-11

Reperfusion accounts for a substantial fraction of the myocardial injury occurring with ischemic heart disease. Yet, no standard therapies are available targeting reperfusion injury. Here, we tested the hypothesis that suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor approved for cancer treatment by the US Food and Drug Administration, will blunt reperfusion injury. Twenty-one rabbits were randomly assigned to 3 groups: (1) vehicle control, (2) SAHA pretreatment (1 day before and at surgery), and (3) SAHA treatment at the time of reperfusion only. Each arm was subjected to ischemia/reperfusion surgery (30 minutes coronary ligation, 24 hours reperfusion). In addition, cultured neonatal and adult rat ventricular cardiomyocytes were subjected to simulated ischemia/reperfusion to probe mechanism. SAHA reduced infarct size and partially rescued systolic function when administered either before surgery (pretreatment) or solely at the time of reperfusion. SAHA plasma concentrations were similar to those achieved in patients with cancer. In the infarct border zone, SAHA increased autophagic flux, assayed in both rabbit myocardium and in mice harboring an RFP-GFP-LC3 transgene. In cultured myocytes subjected to simulated ischemia/reperfusion, SAHA pretreatment reduced cell death by 40%. This reduction in cell death correlated with increased autophagic activity in SAHA-treated cells. RNAi-mediated knockdown of ATG7 and ATG5, essential autophagy proteins, abolished SAHA's cardioprotective effects. The US Food and Drug Administration-approved anticancer histone deacetylase inhibitor, SAHA, reduces myocardial infarct size in a large animal model, even when delivered in the clinically relevant context of reperfusion. The cardioprotective effects of SAHA during ischemia/reperfusion occur, at least in part, through the induction of autophagic flux.

Induction of cardiomyocyte-like cells in infarct hearts by gene transfer of Gata4, Mef2c, and Tbx5.

PubMed

Inagawa, Kohei; Miyamoto, Kazutaka; Yamakawa, Hiroyuki; Muraoka, Naoto; Sadahiro, Taketaro; Umei, Tomohiko; Wada, Rie; Katsumata, Yoshinori; Kaneda, Ruri; Nakade, Koji; Kurihara, Chitose; Obata, Yuichi; Miyake, Koichi; Fukuda, Keiichi; Ieda, Masaki

2012-10-12

After myocardial infarction (MI), massive cell death in the myocardium initiates fibrosis and scar formation, leading to heart failure. We recently found that a combination of 3 cardiac transcription factors, Gata4, Mef2c, and Tbx5 (GMT), reprograms fibroblasts directly into functional cardiomyocytes in vitro. To investigate whether viral gene transfer of GMT into infarcted hearts induces cardiomyocyte generation. Coronary artery ligation was used to generate MI in the mouse. In vitro transduction of GMT retrovirus converted cardiac fibroblasts from the infarct region into cardiomyocyte-like cells with cardiac-specific gene expression and sarcomeric structures. Injection of the green fluorescent protein (GFP) retrovirus into mouse hearts, immediately after MI, infected only proliferating noncardiomyocytes, mainly fibroblasts, in the infarct region. The GFP expression diminished after 2 weeks in immunocompetent mice but remained stable for 3 months in immunosuppressed mice, in which cardiac induction did not occur. In contrast, injection of GMT retrovirus into α-myosin heavy chain (αMHC)-GFP transgenic mouse hearts induced the expression of αMHC-GFP, a marker of cardiomyocytes, in 3% of virus-infected cells after 1 week. A pooled GMT injection into the immunosuppressed mouse hearts induced cardiac marker expression in retrovirus-infected cells within 2 weeks, although few cells showed striated muscle structures. To transduce GMT efficiently in vivo, we generated a polycistronic retrovirus expressing GMT separated by 2A "self-cleaving" peptides (3F2A). The 3F2A-induced cardiomyocyte-like cells in fibrotic tissue expressed sarcomeric α-actinin and cardiac troponin T and had clear cross striations. Quantitative RT-PCR also demonstrated that FACS-sorted 3F2A-transduced cells expressed cardiac-specific genes. GMT gene transfer induced cardiomyocyte-like cells in infarcted hearts.

Cardiomyocyte-expressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion injury

PubMed Central

Pu, Jun; Yuan, Ancai; Shan, Peiren; Gao, Erhe; Wang, Xiaoliang; Wang, Yajing; Lau, Wayne Bond; Koch, Walter; Ma, Xin-Liang; He, Ben

2013-01-01

Aims Emerging evidence indicates that nuclear receptors play a critical regulatory role in cardiovascular physiology/pathology. Recently, farnesoid-X-receptor (FXR), a member of the metabolic nuclear receptor superfamily, has been demonstrated to be expressed in vascular cells, with important roles in vascular physiology/pathology. However, the potential cardiac function of FXR remains unclear. We investigated the cardiac expression and biological function of FXR. Methods and results Farnesoid-X-receptor was detected in both isolated neonatal rat cardiac myocytes and fibroblasts. Natural and synthetic FXR agonists upregulated cardiac FXR expression, stimulated myocyte apoptosis, and reduced myocyte viability dose- and time-dependently. Mechanistic studies demonstrated that FXR agonists disrupted mitochondria, characterized by mitochondrial permeability transition pores activation, mitochondrial potential dissipation, cytochrome c release, and both caspase-9 and -3 activation. Such mitochondrial apoptotic responses were abolished by siRNA-mediated silencing of endogenous FXR or pharmacological inhibition of mitochondrial death signalling. Furthermore, low levels of FXR were detected in the adult mouse heart, with significant (∼2.0-fold) upregulation after myocardial ischaemia/reperfusion (MI/R). Pharmacological inhibition or genetic ablation of FXR significantly reduced myocardial apoptosis by 29.0–53.4%, decreased infarct size by 23.4–49.7%, and improved cardiac function in ischaemic/reperfused myocardium. Conclusion These results demonstrate that nuclear receptor FXR acts as a novel functional receptor in cardiac tissue, regulates apoptosis in cardiomyocytes, and contributes to MI/R injury. PMID:22307460

Dual effects of VEGF-B on activating cardiomyocytes and cardiac stem cells to protect the heart against short- and long-term ischemia-reperfusion injury.

PubMed

Li, Guo-Hua; Luo, Bin; Lv, Yan-Xia; Zheng, Fei; Wang, Lu; Wei, Meng-Xi; Li, Xian-Yu; Zhang, Lei; Wang, Jia-Ning; Chen, Shi-You; Tang, Jun-Ming; He, Xiaohua

2016-05-04

To investigate whether vascular endothelial growth factor B (VEGF-B) improves myocardial survival and cardiac stem cell (CSC) function in the ischemia-reperfusion (I/R) heart and promotes CSC mobilization and angiogenesis. One hour after myocardial ischemia and infarction, rats were treated with recombinant human VEGF-B protein following 24 h or 7 days of myocardial reperfusion. Twenty-four hours after myocardial I/R, VEGF-B increased pAkt and Bcl-2 levels, reduced p-p38MAPK, LC3-II/I, beclin-1, CK, CK-MB and cTnt levels, triggered cardiomyocyte protection against I/R-induced autophagy and apoptosis, and contributed to the decrease of infarction size and the improvement of heart function during I/R. Simultaneously, an in vitro hypoxia-reoxygenation (H/R)-induced H9c2 cardiomyocyte injury model was used to mimic I/R injury model in vivo; in this model, VEGF-B decreased LDH release, blocked H/R-induced apoptosis by inhibiting cell autophagy, and these special effects could be abolished by the autophagy inducer, rapamycin. Mechanistically, VEGF-B markedly activated the Akt signaling pathway while slightly inhibiting p38MAPK, leading to the blockade of cell autophagy and thus protecting cardiomyocyte from H/R-induced activation of the intrinsic apoptotic pathway. Seven days after I/R, VEGF-B induced the expression of SDF-1α and HGF, resulting in the massive mobilization and homing of c-Kit positive cells, triggering further angiogenesis and vasculogenesis in the infracted heart and contributing to the improvement of I/R heart function. VEGF-B could contribute to a favorable short- and long-term prognosis for I/R via the dual manipulation of cardiomyocytes and CSCs.

Decreased inward rectifier potassium current IK1 in dystrophin-deficient ventricular cardiomyocytes.

PubMed

Rubi, Lena; Koenig, Xaver; Kubista, Helmut; Todt, Hannes; Hilber, Karlheinz

2017-03-04

Kir2.x channels in ventricular cardiomyocytes (most prominently Kir2.1) account for the inward rectifier potassium current I K1 , which controls the resting membrane potential and the final phase of action potential repolarization. Recently it was hypothesized that the dystrophin-associated protein complex (DAPC) is important in the regulation of Kir2.x channels. To test this hypothesis, we investigated potential I K1 abnormalities in dystrophin-deficient ventricular cardiomyocytes derived from the hearts of Duchenne muscular dystrophy mouse models. We found that I K1 was substantially diminished in dystrophin-deficient cardiomyocytes when compared to wild type myocytes. This finding represents the first functional evidence for a significant role of the DAPC in the regulation of Kir2.x channels.

Inefficient reprogramming of fibroblasts into cardiomyocytes using Gata4, Mef2c, Tbx5

PubMed Central

Chen, J.X.; Krane, M.; Deutsch, M. A.; Wang, L.; Rav-Acha, M.; Gregoire, S.; Engels, M. C.; Rajarajan, K.; Karra, R.; Abel, E. D.; Wu, J. C.; Milan, D.; Wu, S. M.

2012-01-01

Rationale Direct reprogramming of fibroblasts into cardiomyocytes is a novel strategy for cardiac regeneration. However, the key determinants involved in this process are unknown. Objective To assess the efficiency of direct fibroblast reprogramming via viral overexpression of GATA4, Mef2c, and Tbx5 (GMT). Methods and Results We induced GMT overexpression in murine tail tip fibroblasts (TTFs) and cardiac fibroblasts (CFs) from multiple lines of transgenic mice carrying different cardiomyocyte lineage reporters. We found that the induction of GMT overexpression in TTFs and CFs is inefficient at inducing molecular and electrophysiological phenotypes of mature cardiomyocytes. In addition, transplantation of GMT infected CFs into injured mouse hearts resulted in decreased cell survival with minimal induction of cardiomyocyte genes. Conclusions Significant challenges remain in our ability to convert fibroblasts into cardiomyocyte-like cells and a greater understanding of cardiovascular epigenetics is needed to increase the translational potential of this strategy. PMID:22581928

Both cell fusion and transdifferentiation account for the transformation of human peripheral blood CD34-positive cells into cardiomyocytes in vivo.

PubMed

Zhang, Sui; Wang, Dachun; Estrov, Zeev; Raj, Sean; Willerson, James T; Yeh, Edward T H

2004-12-21

Adult human peripheral blood CD34-positive (CD34+) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients. We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34+ cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34+ cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA+ and troponin T+ or Nkx2.5+ cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA-, troponin T+ cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34+ cells contained human X chromosome only. Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.

Increased susceptibility to structural acute kidney injury in a mouse model of presymptomatic cardiomyopathy.

PubMed

Pleasant, LaTawnya; Ma, Qing; Devarajan, Mahima; Parameswaran, Priyanka; Drake, Keri; Siroky, Brian; Shay-Winkler, Kritton; Robbins, Jeffrey; Devarajan, Prasad

2017-09-01

The early events that signal renal dysfunction in presymptomatic heart failure are unclear. We tested the hypothesis that functional and mechanistic changes occur in the kidney that precede the development of symptomatic heart failure. We employed a transgenic mouse model with cardiomyocyte-specific overexpression of mutant α-B-crystallin that develops slowly progressive cardiomyopathy. Presymptomatic transgenic mice displayed an increase in serum creatinine (1.17 ± 0.34 vs. wild type 0.65 ± 0.16 mg/dl, P < 0.05) and in urinary neutrophil gelatinase-associated lipocalin (NGAL; 278.92 ± 176.24 vs. wild type 49.11 ± 22.79 ng/ml, P < 0.05) but no renal fibrosis. Presymptomatic transgenic mouse kidneys exhibited a twofold upregulation of the Ren1 gene, marked overexpression of renin protein in the tubules, and a worsened response to ischemia-reperfusion injury based on serum creatinine (2.77 ± 0.66 in transgenic mice vs. 2.01 ± 0.58 mg/dl in wild type, P < 0.05), urine NGAL (9,198.79 ± 3,799.52 in transgenic mice vs. 3,252.94 ± 2,420.36 ng/ml in wild type, P < 0.05), tubule dilation score (3.4 ± 0.5 in transgenic mice vs. 2.6 ± 0.5 in wild type, P < 0.05), tubule cast score (3.2 ± 0.4 in transgenic mice vs. 2.5 ± 0.5 in wild type, P < 0.05), and TdT-mediated dUTP nick-end labeling (TUNEL)-positive nuclei (10.1 ± 2.1 in the transgenic group vs. 5.7 ± 1.6 per 100 cells counted in wild type, P < 0.01). Our findings indicate functional renal impairment, urinary biomarker elevations, and induction of renin gene and protein expression in the kidney that occur in early presymptomatic heart failure, which increase the susceptibility to subsequent acute kidney injury. Copyright © 2017 the American Physiological Society.

Role of angiopoietin-2 in the cardioprotective effect of fibroblast growth factor 21 on ischemia/reperfusion-induced injury in H9c2 cardiomyocytes

PubMed Central

Hu, Shuoqiang; Cao, Shujun; Liu, Jinghua

2017-01-01

Fibroblast growth factor 21 (FGF21) exerts a protective effect in ischemia/reperfusion (I/R)-induced cardiac injury. However, the exact molecular mechanism underlying the FGF21 action remains unclear. The present study aimed to evaluate the role of angiopoietin-2 (Angpt2) in the cardioprotective effect of FGF21. For this purpose, the H9C2 cell line was subjected to simulated I/R or aerobic conditions with or without FGF21 administration. Certain groups were also transfected with Angpt2 small interfering RNA (siRNA). Cell viability, apoptosis rate and cell migration were examined, and the expression levels of Angpt2, glucose transporter 1 (GLUT1) and caspase-3 were measured by quantitative polymerase chain reaction (qPCR) and western blot analyses. The results demonstrated that FGF21 administration suppressed apoptosis and increased the cell migration ability following I/R-induced injury. qPCR and western blot data showed a decreased level of GLUT1 after I/R-induced injury, which was reversed by FGF21 administration. Furthermore, inhibition of Angpt2 expression using siRNA enhanced the cardioprotective effect of FGF21 by upregulation of GLUT1. In conclusion, FGF21 administration protected against I/R-induced injury in cardiomyocytes, and further inhibition of Angpt2 with FGF21 administration induced the expression of GLUT1, which may promote the energy metabolism in cardiomyocytes, consequently resulting in a more efficient cardioprotective effect. These results suggested that FGF21 administration and inhibition of Angpt2 could be a novel therapeutic approach for I/R-induced cardiac injury. PMID:28672998

Neovascularization of Ischemic Myocardium by Newly Isolated Tannins Prevents Cardiomyocyte Apoptosis and Improves Cardiac Function

PubMed Central

Gu, Xuemei; Cheng, Lei; Chueng, Winghong L; Yao, Xinsheng; Liu, Hongwei; Qi, Guoqing; Li, Ming

2006-01-01

During remodeling progress post myocardial infarction, the contribution of neoangiogenesis to the infarct-bed capillary is insufficient to support the greater demands of the hypertrophied but viable myocardium resulting in further ischemic injury to the viable cardiomyocytes at risk. Here we reported the bio-assay-guided identification and isolation of angiogenic tannins (angio-T) from Geum japonicum that induced rapid revascularization of infarcted myocardium and promoted survival potential of the viable cardiomyocytes at risk after myocardial infarction. Our results demonstrated that angio-T displayed potent dual effects on up-regulating expression of angiogenic factors, which would contribute to the early revascularization and protection of the cardiomyocytes against further ischemic injury, and inducing antiapoptotic protein expression, which inhibited apoptotic death of cardiomyocytes in the infarcted hearts and limited infarct size. Echocardiographic studies demonstrated that angio-T-induced therapeutic effects on acute infarcted myocardium were accompanied by significant functional improvement by 2 days after infarction. This improvement was sustained for 14 days. These therapeutic properties of angio-T to induce early reconstitution of a blood supply network, prevent apoptotic death of cardiomyocytes at risk, and improve heart function post infarction appear entirely novel and may provide a new dimension for therapeutic angiogenesis medicine for the treatment of ischemic heart diseases. PMID:17380192

Optimization of Direct Fibroblast Reprogramming to Cardiomyocytes Using Calcium Activity as a Functional Measure of Success

PubMed Central

Addis, Russell C.; Ifkovits, Jamie L.; Pinto, Filipa; Kellam, Lori D.; Esteso, Paul; Rentschler, Stacey; Christoforou, Nicolas; Epstein, Jonathan A.; Gearhart, John D.

2013-01-01

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persists for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently

Optimization of direct fibroblast reprogramming to cardiomyocytes using calcium activity as a functional measure of success.

PubMed

Addis, Russell C; Ifkovits, Jamie L; Pinto, Filipa; Kellam, Lori D; Esteso, Paul; Rentschler, Stacey; Christoforou, Nicolas; Epstein, Jonathan A; Gearhart, John D

2013-07-01

Direct conversion of fibroblasts to induced cardiomyocytes (iCMs) has great potential for regenerative medicine. Recent publications have reported significant progress, but the evaluation of reprogramming has relied upon non-functional measures such as flow cytometry for cardiomyocyte markers or GFP expression driven by a cardiomyocyte-specific promoter. The issue is one of practicality: the most stringent measures - electrophysiology to detect cell excitation and the presence of spontaneously contracting myocytes - are not readily quantifiable in the large numbers of cells screened in reprogramming experiments. However, excitation and contraction are linked by a third functional characteristic of cardiomyocytes: the rhythmic oscillation of intracellular calcium levels. We set out to optimize direct conversion of fibroblasts to iCMs with a quantifiable calcium reporter to rapidly assess functional transdifferentiation. We constructed a reporter system in which the calcium indicator GCaMP is driven by the cardiomyocyte-specific Troponin T promoter. Using calcium activity as our primary outcome measure, we compared several published combinations of transcription factors along with novel combinations in mouse embryonic fibroblasts. The most effective combination consisted of Hand2, Nkx2.5, Gata4, Mef2c, and Tbx5 (HNGMT). This combination is >50-fold more efficient than GMT alone and produces iCMs with cardiomyocyte marker expression, robust calcium oscillation, and spontaneous beating that persist for weeks following inactivation of reprogramming factors. HNGMT is also significantly more effective than previously published factor combinations for the transdifferentiation of adult mouse cardiac fibroblasts to iCMs. Quantification of calcium function is a convenient and effective means for the identification and evaluation of cardiomyocytes generated by direct reprogramming. Using this stringent outcome measure, we conclude that HNGMT produces iCMs more efficiently than

In vitro three-dimensional coculturing poly3-hydroxybutyrate-co-3-hydroxyhexanoate with mouse-induced pluripotent stem cells for myocardial patch application.

PubMed

Shijun, Xu; Junsheng, Mu; Jianqun, Zhang; Ping, Bo

2016-03-01

Identifying a suitable polymeric biomaterial for myocardial patch repair following myocardial infarction, cerebral infarction, and cartilage injury is essential. This study aimed to investigate the effect of the novel polymer material, poly3-hydroxybutyrate-co-3-hydroxyhexanoate, on the adhesion, proliferation, and differentiation of mouse-induced pluripotent stem cells in vitro. Mouse-induced pluripotent stem cells were isolated, expanded, and cultured on either two-dimensional or three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films (membranes were perforated to imitate three-dimensional space). Following attachment onto the films, mouse-induced pluripotent stem cell morphology was visualized using scanning electron microscopy. Cell vitality was detected using the Cell Counting Kit-8 assay and cell proliferation was observed using fluorescent 4',6-diamidino-2-phenylindole (DAPI) staining. Mouse-induced pluripotent stem cells were induced into cardiomyocytes by differentiation medium containing vitamin C. A control group in the absence of an inducer was included. Mouse-induced pluripotent stem cell survival and differentiation were observed using immunofluorescence and flow cytometry, respectively. Mouse-induced pluripotent stem cells growth, proliferation, and differentiation were observed on both two-dimensional and three-dimensional poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Vitamin C markedly improved the efficiency of mouse-induced pluripotent stem cells differentiation into cardiomyocytes on poly3-hydroxybutyrate-co-3-hydroxyhexanoate films. Three-dimensional culture was better at promoting mouse-induced pluripotent stem cell proliferation and differentiation compared with two-dimensional culture. © The Author(s) 2016.

Rac1 Is Required for Cardiomyocyte Apoptosis During Hyperglycemia

PubMed Central

Shen, E.; Li, Yanwen; Li, Ying; Shan, Limei; Zhu, Huaqing; Feng, Qingping; Arnold, J. Malcolm O.; Peng, Tianqing

2009-01-01

OBJECTIVE Hyperglycemia induces reactive oxygen species (ROS) and apoptosis in cardiomyocytes, which contributes to diabetic cardiomyopathy. The present study was to investigate the role of Rac1 in ROS production and cardiomyocyte apoptosis during hyperglycemia. RESEARCH DESIGN AND METHODS Mice with cardiomyocyte-specific Rac1 knockout (Rac1-ko) were generated. Hyperglycemia was induced in Rac1-ko mice and their wild-type littermates by injection of streptozotocin (STZ). In cultured adult rat cardiomyocytes, apoptosis was induced by high glucose. RESULTS The results showed a mouse model of STZ-induced diabetes, 7 days of hyperglycemia-upregulated Rac1 and NADPH oxidase activation, elevated ROS production, and induced apoptosis in the heart. These effects of hyperglycemia were significantly decreased in Rac1-ko mice or wild-type mice treated with apocynin. Interestingly, deficiency of Rac1 or apocynin treatment significantly reduced hyperglycemia-induced mitochondrial ROS production in the heart. Deficiency of Rac1 also attenuated myocardial dysfunction after 2 months of STZ injection. In cultured cardiomyocytes, high glucose upregulated Rac1 and NADPH oxidase activity and induced apoptotic cell death, which were blocked by overexpression of a dominant negative mutant of Rac1, knockdown of gp91phox or p47phox, or NADPH oxidase inhibitor. In type 2 diabetic db/db mice, administration of Rac1 inhibitor, NSC23766, significantly inhibited NADPH oxidase activity and apoptosis and slightly improved myocardial function. CONCLUSIONS Rac1 is pivotal in hyperglycemia-induced apoptosis in cardiomyocytes. The role of Rac1 is mediated through NADPH oxidase activation and associated with mitochondrial ROS generation. Our study suggests that Rac1 may serve as a potential therapeutic target for cardiac complications of diabetes. PMID:19592621

Inhibition of Cardiomyocytes Differentiation of Mouse Embryonic Stem Cells by CD38/cADPR/Ca2+ Signaling Pathway*

PubMed Central

Wei, Wen-Jie; Sun, Hai-Ying; Ting, Kai Yiu; Zhang, Li-He; Lee, Hon-Cheung; Li, Gui-Rong; Yue, Jianbo

2012-01-01

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca2+ mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca2+ in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca2+ signaling pathway antagonizes the CM differentiation of mouse ES cells. PMID:22908234

Interleukin-37 ameliorates myocardial ischaemia/reperfusion injury in mice

PubMed Central

Wu, B; Meng, K; Ji, Q; Cheng, M; Yu, K; Zhao, X; Tony, H; Liu, Y; Zhou, Y; Chang, C; Zhong, Y; Zhu, Z; Zhang, W; Mao, X; Zeng, Q

2014-01-01

Innate immune and inflammatory responses are involved in myocardial ischaemia/reperfusion (I/R) injury. Interleukin (IL)-37 is a newly identified member of the IL-1 family, and functions as a fundamental inhibitor of innate immunity and inflammation. However, its role in myocardial I/R injury remains unknown. I/R or sham operations were performed on male C57BL/6J mice. I/R mice received an injection of recombinant human IL-37 or vehicle, immediately before reperfusion. Compared with vehicle treatment, mice treated with IL-37 showed an obvious amelioration of the I/R injury, as demonstrated by reduced infarct size, decreased cardiac troponin T level and improved cardiac function. This protective effect was associated with the ability of IL-37 to suppress production of proinflammatory cytokines, chemokines and neutrophil infiltration, which together contributed to a decrease in cardiomyocyte apoptosis and reactive oxygen species (ROS) generation. In addition, we found that IL-37 inhibited the up-regulation of Toll-like receptor (TLR)-4 expression and nuclear factor kappa B (NF-kB) activation after I/R, while increasing the anti-inflammatory IL-10 level. Moreover, the administration of anti-IL-10R antibody abolished the protective effects of IL-37 in I/R injury. In-vitro experiments further demonstrated that IL-37 protected cardiomyocytes from apoptosis under I/R condition, and suppressed the migration ability of neutrophils towards the chemokine LIX. In conclusion, IL-37 plays a protective role against mouse myocardial I/R injury, offering a promising therapeutic medium for myocardial I/R injury. PMID:24527881

Two dimensional electrophysiological characterization of human pluripotent stem cell-derived cardiomyocyte system

PubMed Central

Zhu, Huanqi; Scharnhorst, Kelsey S.; Stieg, Adam Z.; Gimzewski, James K.; Minami, Itsunari; Nakatsuji, Norio; Nakano, Haruko; Nakano, Atsushi

2017-01-01

Stem cell-derived cardiomyocytes provide a promising tool for human developmental biology, regenerative therapies, disease modeling, and drug discovery. As human pluripotent stem cell-derived cardiomyocytes remain functionally fetal-type, close monitoring of electrophysiological maturation is critical for their further application to biology and translation. However, to date, electrophysiological analyses of stem cell-derived cardiomyocytes has largely been limited by biologically undefined factors including 3D nature of embryoid body, sera from animals, and the feeder cells isolated from mouse. Large variability in the aforementioned systems leads to uncontrollable and irreproducible results, making conclusive studies difficult. In this report, a chemically-defined differentiation regimen and a monolayer cell culture technique was combined with multielectrode arrays for accurate, real-time, and flexible measurement of electrophysiological parameters in translation-ready human cardiomyocytes. Consistent with their natural counterpart, amplitude and dV/dtmax of field potential progressively increased during the course of maturation. Monolayer culture allowed for the identification of pacemaking cells using the multielectrode array platform and thereby the estimation of conduction velocity, which gradually increased during the differentiation of cardiomyocytes. Thus, the electrophysiological maturation of the human pluripotent stem cell-derived cardiomyocytes in our system recapitulates in vivo development. This system provides a versatile biological tool to analyze human heart development, disease mechanisms, and the efficacy/toxicity of chemicals. PMID:28266620

Atomic force microscopy observation of lipopolysaccharide-induced cardiomyocyte cytoskeleton reorganization.

PubMed

Wang, Liqun; Chen, Tangting; Zhou, Xiang; Huang, Qiaobing; Jin, Chunhua

2013-08-01

We applied atomic force microscopy (AFM) to observe lipopolysaccharide (LPS)-induced intracellular cytoskeleton reorganization in primary cardiomyocytes from neonatal mouse. The nonionic detergent Triton X-100 was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized by AFM. Using three-dimensional technique of AFM, we were able to quantify the changes of cytoskeleton by the "density" and total "volume" of the cytoskeleton fibers. Compared to the control group, the density of cytoskeleton was remarkably decreased and the volume of cytoskeleton was significantly increased after LPS treatment, which suggests that LPS may induce the cytoskeleton reorganization and change the cardiomyocyte morphology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Knockdown of CkrL by shRNA deteriorates hypoxia/reoxygenation-induced H9C2 cardiomyocyte apoptosis and survival inhibition Via Bax and downregulation of P-Erk1/2.

PubMed

Zhang, Zhi-Sheng; Yang, Dong-Yan; Fu, Yan-Bo; Zhang, Lei; Zhao, Qian-Ping; Li, Gang

2015-03-01

Integrin β1 subunit and its downstream molecule integrin-linked kinase and focal adhesion kinase have been confirmed to be essential to cell survival and inhibition of apoptosis and hypoxia/reoxygenation (H/R)-induced injuries in cardiomyocytes. However, it is still unclear whether CrkL [v-crk avian sarcoma virus CT-10 oncogene homolog (Crk)-like], which acts also as a component of the integrin pathway, could also affect H/R-induced injuries in the cardiomyocytes. The rat-derived H9C2 cardiomyocytes were infected with a CrkL small hairpin RNA interference recombinant lentivirus, which knockdowns the endogenous CrkL expression in the cardiomyocytes. Apoptosis, cell proliferation and survival were examined in the H9C2 cardiomyocytes treated with either H/R or not. Results showed that knockdown of CrkL could significantly increase apoptosis and inhibition of the cell proliferation and survival and deteriorate the previously mentioned injuries induced by H/R. In contrast, overexpression of human CrkL could relieve the exacerbation of the previously mentioned injuries induced by CrkL knockdown in the H9C2 cardiomyocytes via regulation of Bax and extracellular signal-regulated kinase1/2 (p-ERK1/2). In conclusion, these results confirmed that knockdown of CrkL could deteriorate H/R-induced apoptosis and cell survival inhibition in rat-derived H9C2 cardiomyocytes via Bax and downregulation of p-ERK1/2. It implies that CrkL could mitigate H/R-induced injuries in the cardiomyocytes. Copyright © 2015 John Wiley & Sons, Ltd.

The pro-angiogenic cytokine pleiotrophin potentiates cardiomyocyte apoptosis through inhibition of endogenous AKT/PKB activity.

PubMed

Li, Jinliang; Wei, Hong; Chesley, Alan; Moon, Chanil; Krawczyk, Melissa; Volkova, Maria; Ziman, Bruce; Margulies, Kenneth B; Talan, Mark; Crow, Michael T; Boheler, Kenneth R

2007-11-30

Pleiotrophin is a development-regulated cytokine and growth factor that can promote angiogenesis, cell proliferation, or differentiation, and it has been reported to have neovasculogenic effects in damaged heart. Developmentally, it is prominently expressed in fetal and neonatal hearts, but it is minimally expressed in normal adult heart. Conversely, we show in a rat model of myocardial infarction and in human dilated cardiomyopathy that pleiotrophin is markedly up-regulated. To elucidate the effects of pleiotrophin on cardiac contractile cells, we employed primary cultures of rat neonatal and adult cardiomyocytes. We show that pleiotrophin is released from cardiomyocytes in vitro in response to hypoxia and that the addition of recombinant pleiotrophin promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. Functionally, it potentiates the apoptotic response of neonatal cardiomyocytes to hypoxic stress and to ultraviolet irradiation and of adult cardiomyocytes to hypoxia-reoxygenation. Moreover, UV-induced apoptosis in neonatal cardiomyocytes can be partially inhibited by small interfering RNA-mediated knockdown of endogenous pleiotrophin. Mechanistically, pleiotrophin antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride-mediated inhibition of GSK3beta reduce the potentiated programmed cell death elicited by pleiotrophin. These latter data indicate that pleiotrophin potentiates cardiomyocyte cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel function for pleiotrophin on heart cells following injury. It fosters cardiomyocyte programmed cell death in response to pro-apoptotic stress, which may be critical to myocardial injury repair.

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection.

PubMed

Alhamdi, Yasir; Neill, Daniel R; Abrams, Simon T; Malak, Hesham A; Yahya, Reham; Barrett-Jolley, Richard; Wang, Guozheng; Kadioglu, Aras; Toh, Cheng-Hock

2015-05-01

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection

PubMed Central

Alhamdi, Yasir; Neill, Daniel R.; Abrams, Simon T.; Malak, Hesham A.; Yahya, Reham; Barrett-Jolley, Richard; Wang, Guozheng; Kadioglu, Aras; Toh, Cheng-Hock

2015-01-01

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac

Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes.

PubMed

Shao, Zuo-Hui; Becker, Lance B; Vanden Hoek, Terry L; Schumacker, Paul T; Li, Chang-Qing; Zhao, Danhong; Wojcik, Kim; Anderson, Travis; Qin, Yimin; Dey, Lucy; Yuan, Chun-Su

2003-06-01

This study sought to test whether grape seed proanthocyanidin extract (GSPE) attenuates exogenous and endogenous oxidant stress induced in chick cardiomyocytes and whether this cytoprotection is mediated by PKC activation, mito K(ATP) channel opening, NO production, oxidant scavenging, or iron chelating effects. Cells were exposed to hydrogen peroxide (H(2)O(2)) (exogenous oxidant stress, 0.5mM) or antimycin A (endogenous oxidant stress, 100 micro M) for 2h following pretreatment with GSPE at various concentrations for 2h. Cells were also pretreated with GSPE or with inhibitors of PKC (chelerytherine), mito K(ATP) channel (5-hydroxydecanoate), nitric oxide synthase (nitro-L-arginine methyl ester) for 2h. Oxidant stress was measured by 2',7'-dichlorofluorescin diacetate and cell viability was assessed using propidium iodide. Free radical scavenging and iron chelating ability was tested in vitro. GSPE dose-dependently attenuated oxidant formation and significantly improved cell survival and contractile function. However, inhibitors of PKC, mito K(ATP) channel or NO synthase failed to abolish the protective action of GSPE during H(2)O(2) or antimycin A exposure. In vitro studies suggested that GSPE scavenges H(2)O(2), hydroxyl radical and superoxide, and may chelate iron. These results indicate that GSPE confers cardioprotection against exogenous H(2)O(2)- or antimycin A-induced oxidant injury. Its effect does not require PKC, mito K(ATP) channel, or NO synthase, presumably because it acts by reactive oxygen species scavenging and iron chelating directly.

Involvement of miR-1 in the protective effect of hydrogen sulfide against cardiomyocyte apoptosis induced by ischemia/reperfusion.

PubMed

Kang, Bo; Hong, Jiang; Xiao, Jian; Zhu, Xiaoyan; Ni, Xin; Zhang, Yufeng; He, Bin; Wang, Zhinong

2014-10-01

The protective effect of hydrogen sulfide (H2S) against myocardial ischemia/reperfusion (IR) injury via anti-apoptotic signaling is well established, but the underlying mechanism remains unclear. Recently, miRNAs have been identified as important mediators of myocardial injury by regulating apoptosis-related genes. It was found in our previous preliminary study that microRNA-1 (miR-1) expression underwent a significant change in IR group compared to H2S preconditioned group, indicating that miR-1 possessed myocyte-specific properties. In the present study, we intended to see whether miR-1 participated in H2S protection of cardiomyocytes against IR-induced apoptosis by regulating apoptosis-related genes. Cardiomyocytes of neonatal rats were subjected to hypoxia/reoxygenation (HR) injury with or without H2S preconditioning, while the myocardium of adult SD rats was subjected to IR with or without H2S preconditioning. It was found that HR injury increased apoptosis of cardiac myocytes, up-regulated the expression of miR-1, and down-regulated the expression of Bcl-2. H2S preconditioning attenuated cardiomyocyte apoptosis and LDH release, as well as enhanced cell viability following HR injury. MiR-1 was up-regulated by HR and down-regulated by H2S preconditioning. In contrast, Bcl-2 was down-regulated by HR and up-regulated by H2S preconditioning. In addition, Bcl-2 protein was down-regulated by the miR-1 mimic in a dose-dependent manner. H2S also attenuated IR-induced cardiomyocyte apoptosis in vivo. MiR-1 regulated H2S protection of cardiomyocytes against IR-induced apoptosis by stimulating Bcl-2. These results implicate miR-1 as an important regulator of H2S on the IR myocardium.

Two inhibitory systems and CKIs regulate cell cycle exit of mammalian cardiomyocytes after birth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tane, Shoji; Okayama, Hitomi; Ikenishi, Aiko

Mammalian cardiomyocytes actively proliferate during embryonic stages, following which they exit their cell cycle after birth, and the exit is maintained. Previously, we showed that two inhibitory systems (the G1-phase inhibitory system: repression of cyclin D1 expression; the M-phase inhibitory system: inhibition of CDK1 activation) maintain the cell cycle exit of mouse adult cardiomyocytes. We also showed that two CDK inhibitors (CKIs), p21{sup Cip1} and p27{sup Kip1}, regulate the cell cycle exit in a portion of postnatal cardiomyocytes. It remains unknown whether the two inhibitory systems are involved in the cell cycle exit of postnatal cardiomyocytes and whether p21{sup Cip1}more » and p27{sup Kip1} also inhibit entry to M-phase. Here, we showed that more than 40% of cardiomyocytes entered an additional cell cycle by induction of cyclin D1 expression at postnatal stages, but M-phase entry was inhibited in the majority of cardiomyocytes. Marked cell cycle progression and endoreplication were observed in cardiomyocytes of p21{sup Cip1} knockout mice at 4 weeks of age. In addition, tri- and tetranucleated cardiomyocytes increased significantly in p21{sup Cip1} knockout mice. These data showed that the G1-phase inhibitory system and two CKIs (p21{sup Cip1} and p27{sup Kip1}) inhibit entry to an additional cell cycle in postnatal cardiomyocytes, and that the M-phase inhibitory system and p21{sup Cip1} inhibit M-phase entry of cardiomyocytes which have entered the additional cell cycle. - Highlights: • Many postnatal cardiomyocytes entered an additional cell cycle by cyclin D1 induction. • The majority of cardiomyocytes could not enter M-phase after cyclin D1 induction. • Cell cycle progressed markedly in p21{sup Cip1} knockout mice after postnatal day 14. • Tri- and tetranucleated cardiomyocytes increased in p21{sup Cip1} knockout mice.« less

Regression of Copper-Deficient Heart Hypertrophy: Reduction in the Size of Hypertrophic Cardiomyocytes

USDA-ARS?s Scientific Manuscript database

Dietary copper deficiency causes cardiac hypertrophy and its transition to heart failure in a mouse model. Copper repletion results in a rapid regression of cardiac hypertrophy and prevention of heart failure. The present study was undertaken to understand dynamic changes of cardiomyocytes in the hy...

Cardioprotection Via Modulation of Calcium Homeostasis by Thiopental in Hypoxia-Reoxygenated Neonatal Rat Cardiomyocytes

PubMed Central

Kim, Hyun-Soo; Hwang, Ki-Chul

2010-01-01

Purpose Ca2+ homeostasis plays an important role in myocardial cell injury induced by hypoxia-reoxygenation, and prevention of intracellular Ca2+ overload is key to cardioprotection. Even though thiopental is a frequently used anesthetic agent, little is known about its cardioprotective effects, particulary in association with Ca2+ homeostasis. We investigated whether thiopental protects cardiomyocytes against hypoxia-reoxygenation injury by regulating Ca2+ homeostasis. Materials and Methods Neonatal rat cardiomyocytes were isolated. Cardiomyocytes were exposed to different concentrations of thiopental and immediately replaced in the hypoxic chamber to maintain hypoxia. After 1 hour of exposure, a culture dish was transferred to the CO2 incubator and cells were incubated at 37℃ for 5 hours. At the end of the experiments, the authors assessed cell protection using immunoblot analysis and caspase activity. The mRNA of genes involved in Ca2+ homeostasis, mitochondrial membrane potential, and cellular Ca2+ levels were examined. Results In thiopental-treated cardiomyocytes, there was a decrease in expression of the proapoptotic protein Bax, caspase-3 activation, and intracellular Ca2+ content. In addition, both enhancement of anti-apoptotic protein Bcl-2 and activation of Erk concerned with survival were shown. Furthermore, thiopental attenuated alterations of genes involving Ca2+ regulation and significantly modulated abnormal changes of NCX and SERCA2a genes in hypoxia-reoxygenated neonatal cardiomyocytes. Thiopental suppressed disruption of mitochondrial membrane potential (ΔΨm) induced by hypoxia-reoxygenation. Conclusion Thiopental is likely to modulate expression of genes that regulate Ca2+ homeostasis, which reduces apoptotic cell death and results in cardioprotection. PMID:20191008

Pitavastatin attenuates AGEs-induced mitophagy via inhibition of ROS generation in the mitochondria of cardiomyocytes.

PubMed

Zha, Zhimin; Wang, Junhong; Li, Shiling; Guo, Yan

2017-11-01

This study aimed to investigate whether pitavastatin protected against injury induced by advanced glycation end products products (AGEs) in neonatal rat cardiomyocytes, and to examine the underlying mechanisms. Cardiomyocytes of neonatal rats were incubated for 48 hours with AGEs (100mg/mL), receptor for advanced glycation end products (RAGE), antibody (1 mg/mL) and pitavastatin (600 ng/mL). The levels of p62 and beclin1 were determined by Western blotting. Mitochondrial membrane potential (DYm) and the generation of reactive oxygen species (ROS) were measured through the JC-1 and DCFH-DA. In the AGEs group, the expression of beclin1 was remarkably increased compared to the control group, while the expression of p62 was significantly decreased. AGEs also markedly decreasedDYm and significantly increased ROS compared with the control group. After treatment with RAGE antibody or pitavastatin, the level of beclin1 was markedly decreased compared with the AGEs group, but the level of p62 was remarkably increased. In the AGEs+ RAGE antibody group and AGEs+ pitavastatin group,DYm was significantly increased and ROS was remarkably decreased compared with the AGEs group. In conclusion, AGEs-RAGE may induce autophagy of cardiomyocytes by generation of ROS and pitavastatin could protect against AGEs-induced injury against cardiomyocytes.

Hawthorn (Crataegus monogyna Jacq.) extract exhibits atropine-sensitive activity in a cultured cardiomyocyte assay.

PubMed

Salehi, Satin; Long, Shannon R; Proteau, Philip J; Filtz, Theresa M

2009-01-01

Hawthorn (Crataegus spp.) plant extract is used as a herbal alternative medicine for the prevention and treatment of various cardiovascular diseases. Recently, it was shown that hawthorn extract preparations caused negative chronotropic effects in a cultured neonatal murine cardiomyocyte assay, independent of beta-adrenergic receptor blockade. The aim of this study was to further characterize the effect of hawthorn extract to decrease the contraction rate of cultured cardiomyocytes. To test the hypothesis that hawthorn is acting via muscarinic receptors, the effect of hawthorn extract on atrial versus ventricular cardiomyocytes in culture was evaluated. As would be expected for activation of muscarinic receptors, hawthorn extract had a greater effect in atrial cells. Atrial and/or ventricular cardiomyocytes were then treated with hawthorn extract in the presence of atropine or himbacine. Changes in the contraction rate of cultured cardiomyocytes revealed that both muscarinic antagonists significantly attenuated the negative chronotropic activity of hawthorn extract. Using quinuclidinyl benzilate, L-[benzylic-4,4'-(3)H] ([(3)H]-QNB) as a radioligand antagonist, the effect of a partially purified hawthorn extract fraction to inhibit muscarinic receptor binding was quantified. Hawthorn extract fraction 3 dose-dependently inhibited [(3)H]-QNB binding to mouse heart membranes. Taken together, these findings suggest that decreased contraction frequency by hawthorn extracts in neonatal murine cardiomyocytes may be mediated via muscarinic receptor activation.

CaM Kinase II mediates maladaptive post-infarct remodeling and pro-inflammatory chemoattractant signaling but not acute myocardial ischemia/reperfusion injury

PubMed Central

Weinreuter, Martin; Kreusser, Michael M; Beckendorf, Jan; Schreiter, Friederike C; Leuschner, Florian; Lehmann, Lorenz H; Hofmann, Kai P; Rostosky, Julia S; Diemert, Nathalie; Xu, Chang; Volz, Hans Christian; Jungmann, Andreas; Nickel, Alexander; Sticht, Carsten; Gretz, Norbert; Maack, Christoph; Schneider, Michael D; Gröne, Hermann-Josef; Müller, Oliver J; Katus, Hugo A; Backs, Johannes

2014-01-01

CaMKII was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. Here, we investigated the roles of different CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury by the use of new genetic CaMKII mouse models. Although CaMKIIδC was upregulated 1 day after I/R injury, cardiac damage 1 day after I/R was neither affected in CaMKIIδ-deficient mice, CaMKIIδ-deficient mice in which the splice variants CaMKIIδB and C were re-expressed, nor in cardiomyocyte-specific CaMKIIδ/γ double knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction, which was associated with reduced leukocyte infiltration and attenuated expression of members of the chemokine (C-C motif) ligand family, in particular CCL3 (macrophage inflammatory protein-1α, MIP-1α). Intriguingly, CaMKII was sufficient and required to induce CCL3 expression in isolated cardiomyocytes, indicating a cardiomyocyte autonomous effect. We propose that CaMKII-dependent chemoattractant signaling explains the effects on post-I/R remodeling. Taken together, we demonstrate that CaMKII is not critically involved in acute I/R-induced damage but in the process of post-infarct remodeling and inflammatory processes. PMID:25193973

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

PubMed Central

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-01

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening. PMID:26237021

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

PubMed

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-08

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

Neonatal Heart-Enriched miR-708 Promotes Proliferation and Stress Resistance of Cardiomyocytes in Rodents

PubMed Central

Deng, Shengqiong; Zhao, Qian; Zhen, Lixiao; Zhang, Chuyi; Liu, Cuicui; Wang, Guangxue; Zhang, Lin; Bao, Luer; Lu, Ying; Meng, Lingyu; Lü, Jinhui; Yu, Ping; Lin, Xin; Zhang, Yuzhen; Chen, Yi-Han; Fan, Huimin; Cho, William C.; Liu, Zhongmin; Yu, Zuoren

2017-01-01

Adult heart has limited potential for regeneration after pathological injury due to the limited cell proliferation of cardiomyocytes and the quiescent status of progenitor cells. As such, induction of cell-cycle reentry of cardiomyocytes is one of the key strategies for regeneration of damaged heart. In this study, a subset of miRNAs including miR-708 were identified to be much more abundant in the embryonic and neonatal cardiomyocytes than that in adult rodents. Overexpression of miR-708 promoted cellular proliferation of H9C2 cells or primary cardiomyocytes from neonatal rats or mice in vitro. Lipid nanoparticle delivery of miR-708 promoted myocardial regeneration and heart function recovery in vivo. In addition, miR-708 protected cardiomyocytes against stress-induced apoptosis under hypoxia or isoproterenol treatments. miR-708 inhibited the expression of MAPK14, which has been demonstrated arresting the cell cycle in cardiomyocytes. The cell proliferation-promoting function of miR-708 was dependent at least partly on the expression of MAPK14. These findings strengthen the potential of applying miRNAs to reconstitute lost cardiomyocytes in injured hearts, and may provide a novel miRNA candidate for promoting heart regeneration. PMID:28638481

Chemical chaperone 4-phenylbutyric acid protects H9c2 cardiomyocytes from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis.

PubMed

Jian, Lian; Lu, Yuan; Lu, Shan; Lu, Chengzhi

2016-05-01

Myocardial ischemia/reperfusion (I/R) is a potential contributor to high rates of mortality in several cardiovascular diseases. I/R initiates the unfolded protein response and endoplasmic reticulum (ER) stress, which may lead to apoptotic pathways and exaggerate I/R injury. 4‑phenylbutyric acid (4‑PBA), a low molecular weight, terminal aromatic substituted fatty acid, has been reported to function as an ER chaperone. The aim of the present study was to investigate whether 4‑PBA is able to reduce ER stress‑induced apoptosis and prevent cardiomyocyte damage during the process of I/R in vitro. Accordingly, the rat cardiomyocyte line, H9c2, was treated with hypoxia/reoxygenation as an I/R model in vitro. Myocardium apoptosis was determined with TUNEL staining. The expression of ER stress‑related proteins were examined by western blotting. The resulting data showed that I/R activates the ER stress proteins, glucose‑regulated protein 78, activating transcription factor 6 and protein kinase RNA‑like endoplasmic reticulum kinase, which were all reduced by pretreatment with 4‑PBA. In addition, pretreatment with 4‑PBA significantly inhibited the expression levels of pro‑apoptotic proteins, C/EBP homologous protein, B cell lymphoma (Bcl‑2)‑associated X protein and phosphorylated c‑Jun N‑terminal kinase, and enhanced the expression of the anti‑apoptotic protein Bcl‑2 (n=3; P<0.05). The data demonstrated that I/R initiates ER stress‑associated apoptotic pathways, and 4‑PBA pretreatment protected the cardiomyocytes from I/R‑induced cell death. To the best of our knowledge, the present study is the first to report on the cell repair mechanism of 4‑PBA against I/R damage in cardiomyocytes based on ER stress‑associated apoptotic pathways.

Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription

PubMed Central

Murray, Thomas V.A.; Smyrnias, Ioannis; Schnelle, Moritz; Mistry, Rajesh K.; Zhang, Min; Beretta, Matteo; Martin, Daniel; Anilkumar, Narayana; de Silva, Shana M.; Shah, Ajay M.; Brewer, Alison C.

2015-01-01

Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell

MicroRNA-137 Negatively Regulates H2O2-Induced Cardiomyocyte Apoptosis Through CDC42

PubMed Central

Wang, Junnan; Xu, Rihao; Wu, Junduo; Li, Zhibo

2015-01-01

Background Oxidative stress, inducing cardiomyocyte apoptosis or myocardial ischemia, is the major denominator of many cardiac diseases. In this study, we intended to explore the regulatory function of microRNA-137 (miR-137) in oxidative stress-induced cardiomyocyte apoptosis. Material/Methods Cardiomyocytes were extracted from newborn C57BL/6 mice and cultured in vitro. Apoptosis was induced by H2O2, and evaluated by TUNEL assay. The effect of cardiomyocyte apoptosis on gene expression of miR-137 was evaluated by qRT-PCR. Lentivirus was used to stably down-regulate miR-137, and the subsequent effects of miR-137 down-regulation on cardiomyocyte apoptosis, its targeted gene CDC42, and caspase pathway were evaluated by TUNEL assay, dual-luciferase reporter assay, and Western blot assay, respectively. Finally, CDC42 was down-regulated by siRNA and its effect on miR-137-mediated cardiomyocyte apoptosis protection was examined. Results H2O2 induced significant apoptosis and up-regulated miR-137 in cardiomyocytes, whereas lentivirus-mediated miR-137 down-regulation protected against apoptosis. CDC42 was the direct target gene of miR-137 and proteins of CDC42, caspase-3, and caspase-9 were all regulated by miR-137 down-regulation in cardiomyocyte apoptosis. SiRNA-mediated CDC42 down-regulation reversed the protection of miR-137 down-regulation against cardiomyocyte apoptosis. Conclusions Our work demonstrated miR-137 and CDC42 are critical regulators in cardiomyocyte apoptosis. It may help to identify the molecular targets to prevent myocardial injury in human patients. PMID:26566162

CIP, a cardiac Isl1-interacting protein, represses cardiomyocyte hypertrophy

PubMed Central

Huang, Zhan-Peng; Seok, Hee Young; Zhou, Bin; Chen, Jinghai; Chen, Jian-Fu; Tao, Yazhong; Pu, William T.; Wang, Da-Zhi

2012-01-01

Rationale Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well established, the molecular events that inhibit or repress cardiac hypertrophy are less known. Objective To identify and investigate novel regulators that modulate cardiac hypertrophy. Methods and Results Here, we report the identification, characterization and functional examination of CIP, a novel cardiac Isl1-interacting protein. CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast-two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a co-factor of CIP. CIP directly interacted with Isl1 and we mapped the domains of these two proteins which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the MEF2C enhancer. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Conclusions Our studies therefore identify CIP a novel regulator of cardiac hypertrophy. PMID:22343712

CIP, a cardiac Isl1-interacting protein, represses cardiomyocyte hypertrophy.

PubMed

Huang, Zhan-Peng; Young Seok, Hee; Zhou, Bin; Chen, Jinghai; Chen, Jian-Fu; Tao, Yazhong; Pu, William T; Wang, Da-Zhi

2012-03-16

Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well-established, the molecular events that inhibit or repress cardiac hypertrophy are less known. To identify and investigate novel regulators that modulate cardiac hypertrophy. Here, we report the identification, characterization, and functional examination of a novel cardiac Isl1-interacting protein (CIP). CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization, and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a cofactor of CIP. CIP directly interacted with Isl1, and we mapped the domains of these two proteins, which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the myocyte enhancer factor 2C. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Our studies therefore identify CIP as a novel regulator of cardiac hypertrophy.

Leukemia Inhibitory Factor Enhances Endogenous Cardiomyocyte Regeneration after Myocardial Infarction

PubMed Central

Kanda, Masato; Nagai, Toshio; Takahashi, Toshinao; Liu, Mei Lan; Kondou, Naomichi; Naito, Atsuhiko T.; Akazawa, Hiroshi; Sashida, Goro; Iwama, Atsushi; Komuro, Issei; Kobayashi, Yoshio

2016-01-01

Cardiac stem cells or precursor cells regenerate cardiomyocytes; however, the mechanism underlying this effect remains unclear. We generated CreLacZ mice in which more than 99.9% of the cardiomyocytes in the left ventricular field were positive for 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal) staining immediately after tamoxifen injection. Three months after myocardial infarction (MI), the MI mice had more X-gal-negative (newly generated) cells than the control mice (3.04 ± 0.38/mm2, MI; 0.47 ± 0.16/mm2, sham; p < 0.05). The cardiac side population (CSP) cell fraction contained label-retaining cells, which differentiated into X-gal-negative cardiomyocytes after MI. We injected a leukemia inhibitory factor (LIF)-expression construct at the time of MI and identified a significant functional improvement in the LIF-treated group. At 1 month after MI, in the MI border and scar area, the LIF-injected mice had 31.41 ± 5.83 X-gal-negative cardiomyocytes/mm2, whereas the control mice had 12.34 ± 2.56 X-gal-negative cardiomyocytes/mm2 (p < 0.05). Using 5-ethynyl-2'-deoxyurinide (EdU) administration after MI, the percentages of EdU-positive CSP cells in the LIF-treated and control mice were 29.4 ± 2.7% and 10.6 ± 3.7%, respectively, which suggests that LIF influenced CSP proliferation. Moreover, LIF activated the Janus kinase (JAK)signal transducer and activator of transcription (STAT), mitogen-activated protein kinase/extracellular signal-regulated (MEK)extracellular signal-regulated kinase (ERK), and phosphatidylinositol 3-kinase (PI3K)–AKT pathways in CSPs in vivo and in vitro. The enhanced green fluorescent protein (EGFP)-bone marrow-chimeric CreLacZ mouse results indicated that LIF did not stimulate cardiogenesis via circulating bone marrow-derived cells during the 4 weeks following MI. Thus, LIF stimulates, in part, stem cell-derived cardiomyocyte regeneration by activating cardiac stem or precursor cells. This approach may represent a novel therapeutic

[Desmin content and transversal stiffness of the left ventricle mouse cardiomyocytes and skeletal muscle fibers after a 30-day space flight on board "BION-M1" biosatellite].

PubMed

Ogneva, I V; Maximova, M V; Larina, I M

2014-01-01

The aim of this study was to determine the transversal stiffness of the cortical cytoskeleton and the cytoskeletal protein desmin content in the left ventricle cardiomyocytes, fibers of the mouse soleus and tibialis anterior muscle after a 30-day space flight on board the "BION-M1" biosatellite (Russia, 2013). The dissection was made after 13-16.5 h after landing. The transversal stiffness was measured in relaxed and calcium activated state by, atomic force microscopy. The desmin content was estimated by western blotting, and the expression level of desmin-coding gene was detected using real-time PCR. The results indicate that, the transversal stiffness of the left ventricle cardiomyocytes and fibers of the soleus muscle in relaxed and activated states did not differ from the control. The transversal stiffness of the tibialis muscle fibers in relaxed and activated state was increased in the mice group after space flight. At the same time, in all types of studied tissues the desmin content and the expression level of desmin-coding gene did not differ from the control level.

Nicorandil, a Nitric Oxide Donor and ATP-Sensitive Potassium Channel Opener, Protects Against Dystrophin-Deficient Cardiomyopathy

PubMed Central

Afzal, Muhammad Z.; Reiter, Melanie; Gastonguay, Courtney; McGivern, Jered V.; Guan, Xuan; Ge, Zhi-Dong; Mack, David L.; Childers, Martin K.; Ebert, Allison D.; Strande, Jennifer L.

2016-01-01

Background Dystrophin-deficient cardiomyopathy is a growing clinical problem without targeted treatments. We investigated whether nicorandil promotes cardioprotection in human dystrophin-deficient induced pluripotent stem cell (iPSC)-derived cardiomyocytes and the muscular dystrophy mdx mouse heart. Methods and Results Dystrophin-deficient iPSC-derived cardiomyocytes had decreased levels of endothelial nitric oxide synthase and neuronal nitric oxide synthase. The dystrophin-deficient cardiomyocytes had increased cell injury and death after 2 hours of stress and recovery. This was associated with increased levels of reactive oxygen species and dissipation of the mitochondrial membrane potential. Nicorandil pretreatment was able to abolish these stress-induced changes through a mechanism that involved the nitric oxide–cyclic guanosine monophosphate pathway and mitochondrial adenosine triphosphate-sensitive potassium channels. The increased reactive oxygen species levels in the dystrophin-deficient cardiomyocytes were associated with diminished expression of select antioxidant genes and increased activity of xanthine oxidase. Furthermore, nicorandil was found to improve the restoration of cardiac function after ischemia and reperfusion in the isolated mdx mouse heart. Conclusion Nicorandil protects against stress-induced cell death in dystrophin-deficient cardiomyocytes and preserves cardiac function in the mdx mouse heart subjected to ischemia and reperfusion injury. This suggests a potential therapeutic role for nicorandil in dystrophin-deficient cardiomyopathy. PMID:26940570

The influence of rapamycin on the early cardioprotective effect of hypoxic preconditioning on cardiomyocytes

PubMed Central

Wang, Jiang; Maimaitili, YiLiyaer; Yu, Jin; Guo, Hai; Ma, Hai-Ping; Chen, Chun-ling

2016-01-01

Introduction The purpose of this study was to examine the effects of rapamycin on the cardioprotective effect of hypoxic preconditioning (HPC) and on the mammalian target of rapamycin (mTOR)-mediated hypoxia-inducible factor 1 (HIF-1) signaling pathway. Material and methods Primary cardiomyocytes were isolated from rat pups and underwent rapamycin and/or HPC, followed by hypoxia/re-oxygenation (H/R) injury. Cell viability and cell injury were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and qRT-PCR was used to measure HIF-1α and mTOR mRNA expression. A Langendorff heart perfusion model was conducted to observe the effect of rapamycin. Results Rapamycin treatment nearly abolished the cardioprotective effect of HPC in cardiomyocytes, reduced cell viability (p = 0.007) and increased cell damage (p = 0.032). HIF-1α and mTOR mRNA expression increased in cardiomyocytes undergoing I/R injury within 2 h after HPC. After rapamycin treatment, mTOR mRNA expression and HPC-induced HIF-1α mRNA expression were both reduced (p < 0.001). A Langendorff heart perfusion model in rat hearts showed that rapamycin greatly attenuated the cardioprotective effect of HPC in terms of heart rate, LVDP, and dp/dtmax (all, p < 0.029). Conclusions Rapamycin, through inhibition of mTOR, reduces the elevated HIF-1α expression at an early stage of HPC, and attenuates the early cardioprotective effect of HPC. PMID:28721162

[Differentiation of mesenchymal stem cells into cardiomyocytes induced by cardiomyocytes].

PubMed

Wang, Ting-Zhong; Ma, Ai-Qun; Xu, Zheng-Yun; Jiang, Wen-Hui; Du, Yuan

2005-06-01

To investigate the role of adult cardiomyocytes in the differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. Rat MSCs were isolated by a Percoll's gradient solution and cultured in low-glucose Dulbecco' s modified Eagle' s medium (DMEM). After 2 passages, cell-surface antigen CD34, CD71 and CD90 for rat MSCs were determined by flow cytometry, and these MSCs were transfected with pEGFP-N3 by Lipofectamine2000. Then those MSCs labeled with GFP, were cultured in contacted, nocontacted and conditioned with adult rat myocardiocytes. Immunofluorescence staining against alpha-actin, desmin, and troponin-T were performed after 1 week. Immunofluorescence staining was positive against alpha-actin, desmin, and troponin-T on MSCs in contacted culture group. In contrast, no alpha-actin, desmin, and troponin-T expression on MSCs were observed in the noncontacted culture group and the conditioned culture group. Direct cell-to-cell contact between MSCs and adult cardiomyocytes may induce differentiation of MSCs into cardiomyocytes.

Cell fusion contributes to the rescue of apoptotic cardiomyocytes by bone marrow cells

PubMed Central

Yang, Wei-Jian; Li, Shu-Hong; Weisel, Richard D; Liu, Shi-Ming; Li, Ren-Ke

2012-01-01

Cardiomyocyte apoptosis is an important contributor to the progressive cardiac dysfunction that culminates in congestive heart failure. Bone marrow cells (BMCs) restore cardiac function following ischaemia, and transplanted BMCs have been reported to fuse with cells of diverse tissues. We previously demonstrated that the myogenic conversion of bone marrow stromal cells increased nearly twofold when the cells were co-cultured with apoptotic (TNF-α treated) cardiomyocytes. We therefore hypothesized that cell fusion may be a major mechanism by which BMCs rescue cardiomyocytes from apoptosis. We induced cellular apoptosis in neonatal rat cardiomyocytes by treatment with hydrogen peroxide (H2O2). The TUNEL assay demonstrated an increase in apoptosis from 4.5 ± 1.3% in non-treated cells to 19.0 ± 4.4% (P < 0.05) in treated cells. We subsequently co-cultured the apoptotic cardiomyocytes with BMCs and assessed cell fusion using flow cytometry. Fusion was rare in the non-treated control cardiomyocytes (0.3%), whereas H2O2 treatment led to significantly higher fusion rates than the control group (P < 0.05), with the highest rate of 7.9 ± 0.3% occurring at 25 μM H2O2. We found an inverse correlation between cell fusion and completion of cardiomyocyte apoptosis (R2 = 0.9863). An in vivo mouse model provided evidence of cell fusion in the infarcted myocardium following the injection of BMCs. The percentage of cells undergoing fusion was significantly higher in mice injected with BMCs following infarction (8.8 ± 1.3%) compared to mice that did not undergo infarction (4.6 ± 0.6%, P < 0.05). Enhancing cell fusion may be one method to preserve cardiomyocytes following myocardial infarction, and this new approach may provide a novel target for cardiac regenerative therapies. PMID:22805279

Early biomarkers of doxorubicin-induced heart injury in a mouse model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Desai, Varsha G., E-mail: varsha.desai@fda.hhs.gov; Kwekel, Joshua C.; Vijay, Vikrant

Cardiac troponins, which are used as myocardial injury markers, are released in plasma only after tissue damage has occurred. Therefore, there is a need for identification of biomarkers of earlier events in cardiac injury to limit the extent of damage. To accomplish this, expression profiling of 1179 unique microRNAs (miRNAs) was performed in a chronic cardiotoxicity mouse model developed in our laboratory. Male B6C3F{sub 1} mice were injected intravenously with 3 mg/kg doxorubicin (DOX; an anti-cancer drug), or saline once a week for 2, 3, 4, 6, and 8 weeks, resulting in cumulative DOX doses of 6, 9, 12, 18,more » and 24 mg/kg, respectively. Mice were euthanized a week after the last dose. Cardiac injury was evidenced in mice exposed to 18 mg/kg and higher cumulative DOX dose whereas examination of hearts by light microscopy revealed cardiac lesions at 24 mg/kg DOX. Also, 24 miRNAs were differentially expressed in mouse hearts, with the expression of 1, 1, 2, 8, and 21 miRNAs altered at 6, 9, 12, 18, and 24 mg/kg DOX, respectively. A pro-apoptotic miR-34a was the only miRNA that was up-regulated at all cumulative DOX doses and showed a significant dose-related response. Up-regulation of miR-34a at 6 mg/kg DOX may suggest apoptosis as an early molecular change in the hearts of DOX-treated mice. At 12 mg/kg DOX, up-regulation of miR-34a was associated with down-regulation of hypertrophy-related miR-150; changes observed before cardiac injury. These findings may lead to the development of biomarkers of earlier events in DOX-induced cardiotoxicity that occur before the release of cardiac troponins. - Highlights: • Upregulation of miR-34a before doxorubicin-induced cardiac tissue injury • Apoptosis might be an early event in mouse heart during doxorubicin treatment. • Expression of miR-150 declined before doxorubicin-induced cardiac tissue injury.« less

Ibutilide protects against cardiomyocytes injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways.

PubMed

Wang, Yu; Wang, Yi-Li; Huang, Xia; Yang, Yang; Zhao, Ya-Jun; Wei, Cheng-Xi; Zhao, Ming

2017-02-01

Atrial fibrillation (AF) is a complex disease with multiple inter-relating causes culminating in rapid atrial activation and atrial structural remodeling. The contribution of endoplasmic reticulum and mitochondria stress to AF has been highlighted. As the class III antiarrhythmic agent, ibutilide are widely used to AF. This study was designed to explore whether ibutilide could treat AF by inhibiting endoplasmic reticulum stress pathways and mitochondria stress. The neonatal rat cardiomyocytes were isolated and exposed to H 2 O 2 , ibutilide was add to the culture medium 12 h. Then the cell viability, oxidative stress levels and apoptotic rate were analyzed. In addition, endoplasmic reticulum stress related protein (GRP78, GRP94, CHOP), mitochondria-dependent protein (Bax, Bcl-2) and caspase-3/9/12 were identified by real-time PCR and western blot analysis. In our results, remarkable decreased cell viability and oxidative stress levels were detected in cardiomyocytes after treating with H 2 O 2 . The apoptotic rate and the expression of proteins involved in mitochondrial stress and endoplasmic reticulum stress pathways increased. While ibutilide significantly inhibited these changes. These data suggested that ibutilide serves a protective role against H 2 O 2 -induced apoptosis of neonatal rat cardiomyocytes, and the mechanism is related to suppression of mitochondrial stress and endoplasmic reticulum stress.

Protective effects of circulating microvesicles derived from myocardial ischemic rats on apoptosis of cardiomyocytes in myocardial ischemia/reperfusion injury.

PubMed

Wang, Yao; Wei, Su; Wang, Yi-Lu; Liu, Miao; Shang, Man; Zhang, Qi; Wu, Yan-Na; Liu, Ming-Lin; Song, Jun-Qiu; Liu, Yan-Xia

2017-08-15

To investigate the effects of circulating microvesicles derived from myocardial ischemia (I-MVs) on apoptosis in myocardial ischemia/reperfusion (I/R) injury in rats. I-MVs from rats undergoing myocardial left anterior descending (LAD) coronary artery ligation were isolated by ultracentrifugation from circulating blood and characterized by flow cytometry. I-MVs were administered intravenously (4.8 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min of ischemia and 120-min of reperfusion of LAD in rats. Treatment with I-MVssignificantly reduced the size of myocardial infarction, the activities of serum CK-MB and LDH, and the number of apoptotic cardiomyocytes. The activities of caspase 3, caspase 9 and caspase 12 in myocardium were also decreased significantly with I-MVs treatment. Moreover, the expression of Bax was decreased but Bcl-2 was increased. The expression of glucose regulated protein 78 (GRP78), sarco/endoplasmic reticulum Ca 2+ -ATPase 2 (SERCA2) and phosphorylated phospholamban (p-PLB) were increased after being treated with I-MVs. I-MVs could protect hearts from I/R injury in rats through SERCA2 and p-PLB of calcium regulatory proteins to alleviate intrinsic myocardial apoptosis including mitochondrial and endoplasmic reticulum pathways.

Reperfusion injury intensifies the adaptive human T cell alloresponse in a human-mouse chimeric artery model.

PubMed

Yi, Tai; Fogal, Birgit; Hao, Zhengrong; Tobiasova, Zuzana; Wang, Chen; Rao, Deepak A; Al-Lamki, Rafia S; Kirkiles-Smith, Nancy C; Kulkarni, Sanjay; Bradley, John R; Bothwell, Alfred L M; Sessa, William C; Tellides, George; Pober, Jordan S

2012-02-01

Perioperative nonimmune injuries to an allograft can decrease graft survival. We have developed a model for studying this process using human materials. Human artery segments were transplanted as infrarenal aortic interposition grafts into an immunodeficient mouse host, allowed to "heal in" for 30 days, and then retransplanted into a second mouse host. To induce a reperfusion injury, the healed-in artery segments were incubated for 3 hours under hypoxic conditions ex vivo before retransplantation. To induce immunologic rejection, the animals receiving the retransplanted artery segment were adoptively transferred with human peripheral blood mononuclear cells or purified T cells from a donor allogeneic to the artery 1 week before surgery. To compare rejection of injured versus healthy tissues, these manipulations were combined. Results were analyzed ex vivo by histology, morphometry, immunohistochemistry, and mRNA quantitation or in vivo by ultrasound. Our results showed that reperfusion injury, which otherwise heals with minimal sequelae, intensifies the degree of allogeneic T cell-mediated injury to human artery segments. We developed a new human-mouse chimeric model demonstrating interactions of reperfusion injury and alloimmunity using human cells and tissues that may be adapted to study other forms of nonimmune injury and other types of adaptive immune responses.

Ghrelin protected neonatal rat cardiomyocyte against hypoxia/reoxygenation injury by inhibiting apoptosis through Akt-mTOR signal.

PubMed

Wang, Lifeng; Lu, Yingjie; Liu, Xian; Wang, Xiaoyun

2017-04-01

Reducing reperfusion period myocardial cell damage is efficient to reduce myocardial ischemia-reperfusion injury. Ghrelin can increase myocardial contractility, improve heart failure caused by myocardial infarction. This study aimed to investigate the protective effect of Ghrelin on myocardial hypoxia/reoxygenation (H/R) injury of neonatal rat cardiomyocytes (NRCMs) and to explore the mechanisms. We isolated the NRCMs, established myocardial H/R model, blocked growth hormone secretagogue receptor (GHSR) by siRNA technique, examined cell activity by MTT and LDH assay, detected apoptosis by Hoechst 33258 staining and flow cytometry and determined the expression levels of apoptosis related proteins and signaling pathway proteins by western blot. We found that Ghrelin can significantly improve cell activity and decrease apoptosis after H/R, however this effect was abolished by GHSR-siRNA. In addition, we found that Ghrelin can significantly increase the expression of Bcl-2 but inhibit the level of Bax and caspase-3. Further mechanism study found that the phosphorylation level of signaling pathway protein Akt and mTOR in Ghrelin treated group were significantly higher than that in other groups. In conclusion, Ghrelin can reduce the H/R damage on NRCMs and inhibit the apoptosis by activating Akt-mTOR signaling pathway.

BIN1 is Reduced and Cav1.2 Trafficking is Impaired in Human Failing Cardiomyocytes

PubMed Central

Hong, Ting-Ting; Smyth, James W.; Chu, Kevin Y.; Vogan, Jacob M.; Fong, Tina S.; Jensen, Brian C.; Fang, Kun; Halushka, Marc K.; Russell, Stuart D.; Colecraft, Henry; Hoopes, Charles W.; Ocorr, Karen; Chi, Neil C.; Shaw, Robin M.

2011-01-01

Background Heart failure is a growing epidemic and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T-tubules. BIN1 is a membrane scaffolding protein that causes Cav1.2 to traffic to T-tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. Objective To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. Methods Intact myocardium and freshly isolated cardiomyocytes from non-failing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after shRNA mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino mediated knockdown of BIN1. Results BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced 42% by imaging and biochemical T-tubule fraction of Cav1.2 is reduced 68%. Total calcium current is reduced 41% in a cell line expressing non-trafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. Conclusions The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility. PMID:22138472

Naturally Engineered Maturation of Cardiomyocytes

PubMed Central

Scuderi, Gaetano J.; Butcher, Jonathan

2017-01-01

Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This

Postnatal Ablation of Foxm1 from Cardiomyocytes Causes Late Onset Cardiac Hypertrophy and Fibrosis without Exacerbating Pressure Overload-Induced Cardiac Remodeling

PubMed Central

Bolte, Craig; Zhang, Yufang; York, Allen; Kalin, Tanya V.; Schultz, Jo El J.; Molkentin, Jeffery D.; Kalinichenko, Vladimir V.

2012-01-01

Heart disease remains a leading cause of morbidity and mortality in the industrialized world. Hypertrophic cardiomyopathy is the most common genetic cardiovascular disorder and the most common cause of sudden cardiac death. Foxm1 transcription factor (also known as HFH-11B, Trident, Win or MPP2) plays an important role in the pathogenesis of various cancers and is a critical mediator of post-injury repair in multiple organs. Foxm1 has been previously shown to be essential for heart development and proliferation of embryonic cardiomyocytes. However, the role of Foxm1 in postnatal heart development and in cardiac injury has not been evaluated. To delete Foxm1 in postnatal cardiomyocytes, αMHC-Cre/Foxm1fl/fl mice were generated. Surprisingly, αMHC-Cre/Foxm1fl/fl mice exhibited normal cardiomyocyte proliferation at postnatal day seven and had no defects in cardiac structure or function but developed cardiac hypertrophy and fibrosis late in life. The development of cardiomyocyte hypertrophy and cardiac fibrosis in aged Foxm1-deficient mice was associated with reduced expression of Hey2, an important regulator of cardiac homeostasis, and increased expression of genes critical for cardiac remodeling, including MMP9, αSMA, fibronectin and vimentin. We also found that following aortic constriction Foxm1 mRNA and protein were induced in cardiomyocytes. However, Foxm1 deletion did not exacerbate cardiac hypertrophy or fibrosis following chronic pressure overload. Our results demonstrate that Foxm1 regulates genes critical for age-induced cardiomyocyte hypertrophy and cardiac fibrosis. PMID:23144938

Uniaxial strain of cultured mouse and rat cardiomyocyte strands slows conduction more when its axis is parallel to impulse propagation than when it is perpendicular.

PubMed

Buccarello, A; Azzarito, M; Michoud, F; Lacour, S P; Kucera, J P

2018-05-01

Cardiac tissue deformation can modify tissue resistance, membrane capacitance and ion currents and hence cause arrhythmogenic slow conduction. Our aim was to investigate whether uniaxial strain causes different changes in conduction velocity (θ) when the principal strain axis is parallel vs perpendicular to impulse propagation. Cardiomyocyte strands were cultured on stretchable custom microelectrode arrays, and θ was determined during steady-state pacing. Uniaxial strain (5%) with principal axis parallel (orthodromic) or perpendicular (paradromic) to propagation was applied for 1 minute and controlled by imaging a grid of markers. The results were analysed in terms of cable theory. Both types of strain induced immediate changes of θ upon application and release. In material coordinates, orthodromic strain decreased θ significantly more (P < .001) than paradromic strain (2.2 ± 0.5% vs 1.0 ± 0.2% in n = 8 mouse cardiomyocyte cultures, 2.3 ± 0.4% vs 0.9 ± 0.5% in n = 4 rat cardiomyocyte cultures, respectively). The larger effect of orthodromic strain can be explained by the increase in axial myoplasmic resistance, which is not altered by paradromic strain. Thus, changes in tissue resistance substantially contributed to the changes of θ during strain, in addition to other influences (eg stretch-activated channels). Besides these immediate effects, the application of strain also consistently initiated a slow progressive decrease in θ and a slow recovery of θ upon release. Changes in cardiac conduction velocity caused by acute stretch do not only depend on the magnitude of strain but also on its orientation relative to impulse propagation. This dependence is due to different effects on tissue resistance. © 2017 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Cardiomyocyte cell cycle control and growth estimation in vivo--an analysis based on cardiomyocyte nuclei.

PubMed

Walsh, Stuart; Pontén, Annica; Fleischmann, Bernd K; Jovinge, Stefan

2010-06-01

Adult mammalian cardiomyocytes are traditionally viewed as being permanently withdrawn from the cell cycle. Whereas some groups have reported none, others have reported extensive mitosis in adult myocardium under steady-state conditions. Recently, a highly specific assay of 14C dating in humans has suggested a continuous generation of cardiomyocytes in the adult, albeit at a very low rate. Mice represent the most commonly used animal model for these studies, but their short lifespan makes them unsuitable for 14C studies. Herein, we investigate the cellular growth pattern for murine cardiomyocyte growth under steady-state conditions, addressed with new analytical and technical strategies, and we furthermore relate this to gene expression patterns. The observed levels of DNA synthesis in early life were associated with cardiomyocyte proliferation. Mitosis was prolonged into early life, longer than the most conservative previous estimates. DNA synthesis in neonatal life was attributable to bi-nucleation, therefore suggesting that cardiomyocytes withdraw from the cell cycle shortly after birth. No cell cycle activity was observed in adult cardiomyocytes and significant polyploidy was observed in cardiomyocyte nuclei. Gene analyses identified 32 genes whose expression was predicted to be particular to day 3-4 neonatal myocytes, compared with embryonic or adult cells. These cell cycle-associated genes are crucial to the understanding of the mechanisms of bi-nucleation and physiological cellular growth in the neonatal period.

Nanoparticle-Mediated Delivery of Mitochondrial Division Inhibitor 1 to the Myocardium Protects the Heart From Ischemia-Reperfusion Injury Through Inhibition of Mitochondria Outer Membrane Permeabilization: A New Therapeutic Modality for Acute Myocardial Infarction.

PubMed

Ishikita, Ayako; Matoba, Tetsuya; Ikeda, Gentaro; Koga, Jun-Ichiro; Mao, Yajing; Nakano, Kaku; Takeuchi, Osamu; Sadoshima, Junichi; Egashira, Kensuke

2016-07-22

Mitochondria-mediated cell death plays a critical role in myocardial ischemia-reperfusion (IR) injury. We hypothesized that nanoparticle-mediated drug delivery of mitochondrial division inhibitor 1 (Mdivi1) protects hearts from IR injury through inhibition of mitochondria outer membrane permeabilization (MOMP), which causes mitochondrial-mediated cell death. We formulated poly (lactic-co-glycolic acid) nanoparticles containing Mdivi1 (Mdivi1-NP). We recently demonstrated that these nanoparticles could be successfully delivered to the cytosol and mitochondria of cardiomyocytes under H2O2-induced oxidative stress that mimicked IR injury. Pretreatment with Mdivi1-NP ameliorated H2O2-induced cell death in rat neonatal cardiomyocytes more potently than Mdivi1 alone, as indicated by a lower estimated half-maximal effective concentration and greater maximal effect on cell survival. Mdivi1-NP treatment of Langendorff-perfused mouse hearts through the coronary arteries at the time of reperfusion reduced infarct size after IR injury more effectively than Mdivi1 alone. Mdivi1-NP treatment also inhibited Drp1-mediated Bax translocation to the mitochondria and subsequent cytochrome c leakage into the cytosol, namely, MOMP, in mouse IR hearts. MOMP inhibition was also observed in cyclophilin D knockout (CypD-KO) mice, which lack the mitochondrial permeability transition pore (MPTP) opening. Intravenous Mdivi1-NP treatment in vivo at the time of reperfusion reduced IR injury in wild-type and CypD-KO mice, but not Bax-KO mice. Mdivi1-NP treatment reduced IR injury through inhibition of MOMP, even in the absence of a CypD/MPTP opening. Thus, nanoparticle-mediated drug delivery of Mdivi1 may be a novel treatment strategy for IR injury. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: A sex-specific gene profiling analysis.

PubMed

Wang, Hao; Sun, Xuming; Chou, Jeff; Lin, Marina; Ferrario, Carlos M; Zapata-Sudo, Gisele; Groban, Leanne

2017-08-01

Activation of G protein-coupled estrogen receptor (GPER) by its agonist, G1, protects the heart from stressors such as pressure-overload, ischemia, a high-salt diet, estrogen loss, and aging, in various male and female animal models. Due to nonspecific effects of G1, the exact functions of cardiac GPER cannot be concluded from studies using systemic G1 administration. Moreover, global knockdown of GPER affects glucose homeostasis, blood pressure, and many other cardiovascular-related systems, thereby confounding interpretation of its direct cardiac actions. We generated a cardiomyocyte-specific GPER knockout (KO) mouse model to specifically investigate the functions of GPER in cardiomyocytes. Compared to wild type mice, cardiomyocyte-specific GPER KO mice exhibited adverse alterations in cardiac structure and impaired systolic and diastolic function, as measured by echocardiography. Gene deletion effects on left ventricular dimensions were more profound in male KO mice compared to female KO mice. Analysis of DNA microarray data from isolated cardiomyocytes of wild type and KO mice revealed sex-based differences in gene expression profiles affecting multiple transcriptional networks. Gene Set Enrichment Analysis (GSEA) revealed that mitochondrial genes are enriched in GPER KO females, whereas inflammatory response genes are enriched in GPER KO males, compared to their wild type counterparts of the same sex. The cardiomyocyte-specific GPER KO mouse model provides us with a powerful tool to study the functions of GPER in cardiomyocytes. The gene expression profiles of the GPER KO mice provide foundational information for further study of the mechanisms underlying sex-specific cardioprotection by GPER. Copyright © 2016 Elsevier B.V. All rights reserved.

RNase1 prevents the damaging interplay between extracellular RNA and tumour necrosis factor-α in cardiac ischaemia/reperfusion injury.

PubMed

Cabrera-Fuentes, H A; Ruiz-Meana, M; Simsekyilmaz, S; Kostin, S; Inserte, J; Saffarzadeh, M; Galuska, S P; Vijayan, V; Barba, I; Barreto, G; Fischer, S; Lochnit, G; Ilinskaya, O N; Baumgart-Vogt, E; Böning, A; Lecour, S; Hausenloy, D J; Liehn, E A; Garcia-Dorado, D; Schlüter, K-D; Preissner, K T

2014-12-01

Despite optimal therapy, the morbidity and mortality of patients presenting with an acute myocardial infarction (MI) remain significant, and the initial mechanistic trigger of myocardial "ischaemia/reperfusion (I/R) injury" remains greatly unexplained. Here we show that factors released from the damaged cardiac tissue itself, in particular extracellular RNA (eRNA) and tumour-necrosis-factor α (TNF-α), may dictate I/R injury. In an experimental in vivo mouse model of myocardial I/R as well as in the isolated I/R Langendorff-perfused rat heart, cardiomyocyte death was induced by eRNA and TNF-α. Moreover, TNF-α promoted further eRNA release especially under hypoxia, feeding a vicious cell damaging cycle during I/R with the massive production of oxygen radicals, mitochondrial obstruction, decrease in antioxidant enzymes and decline of cardiomyocyte functions. The administration of RNase1 significantly decreased myocardial infarction in both experimental models. This regimen allowed the reduction in cytokine release, normalisation of antioxidant enzymes as well as preservation of cardiac tissue. Thus, RNase1 administration provides a novel therapeutic regimen to interfere with the adverse eRNA-TNF-α interplay and significantly reduces or prevents the pathological outcome of ischaemic heart disease.

Spallanzani's mouse: a model of restoration and regeneration.

PubMed

Heber-Katz, E; Leferovich, J M; Bedelbaeva, K; Gourevitch, D

2004-01-01

The ability to regenerate is thought to be a lost phenotype in mammals, though there are certainly sporadic examples of mammalian regeneration. Our laboratory has identified a strain of mouse, the MRL mouse, which has a unique capacity to heal complex tissue in an epimorphic fashion, i.e., to restore a damaged limb or organ to its normal structure and function. Initial studies using through-and-through ear punches showed rapid full closure of the ear holes with cartilage growth, new hair follicles, and normal tissue architecture reminiscent of regeneration seen in amphibians as opposed to the scarring usually seen in mammals. Since the ear hole closure phenotype is a quantitative trait, this has been used to show-through extensive breeding and backcrossing--that the trait is heritable. Such analysis reveals that there is a complex genetic basis for this trait with multiple loci. One of the major phenotypes of the MRL mouse is a potent remodeling response with the absence or a reduced level of scarring. MRL healing is associated with the upregulation of the metalloproteinases MMP-2 and MMP-9 and the downregulation of their inhibitors TIMP-2 and TIMP-3, both present in inflammatory cells such as neutrophils and macrophages. This model has more recently been extended to the heart. In this case, a cryoinjury to the right ventricle leads to near complete scarless healing in the MRL mouse whereas scarring is seen in the control mouse. In the MRL heart, bromodeoxyuridine uptake by cardiomyocytes filling the wound site can be seen 60 days after injury. This does not occur in the control mouse. Function in the MRL heart, as measured by echocardiography, returns to normal.

The effects of L-arginine on cerebral hemodynamics after controlled cortical impact injury in the mouse.

PubMed

Liu, Hao; Goodman, J Clay; Robertson, Claudia S

2002-03-01

Traumatic brain injury (TBI) induces vascular changes that may influence neurological outcome by causing the brain to be more susceptible to secondary ischemic insults. In rat models of TBI, L-arginine administration has been shown to restore cerebral blood flow and improve neurological outcome. The purpose of this study was to determine if hypoperfusion occurs in a mouse model of TBI and if L-arginine administration has the same beneficial effects after injury in the mouse. C57BL6 mice were anesthetized with isoflurane, intubated and mechanically ventilated, and underwent a 3-m/sec, 1.5-mm deformation cortical impact injury. Five minutes after injury, L-arginine, 300 mg/kg, or saline were administered. Arterial blood pressure, intracranial pressure, and laser Doppler flow at the impact site were monitored for 3 h after the injury. The cerebral hemodynamic effects of the TBI induced by cortical impact injury were similar to that previously observed in rats. Intracranial hypertension, with ICP peaking at 46+/-2 mm Hg, and systemic hypotension both contributed to a reduction in CPP. In addition, LDF decreased significantly at the impact site. L-Arginine administration restored LDF to near baseline levels without increasing ICP. These studies demonstrate that cerebral hemodynamics can be measured in mouse models of TBI. The changes in cerebral hemodynamics are relatively simlar to those see in the rat model of cortical impact injury and suggest an important role for nitric oxide metabolism in the maintenance of cerebral blood flow following TBI.

Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway.

PubMed

Su, Hongyan; Li, Jingyuan; Chen, Tongshuai; Li, Na; Xiao, Jie; Wang, Shujian; Guo, Xiaobin; Yang, Yi; Bu, Peili

2016-11-01

Melatonin is well known for its cardioprotective effects; however, whether melatonin exerts therapeutic effects on cardiomyocyte hypertrophy remains to be investigated, as do the mechanisms underlying these effects, if they exist. Cyclophilin A (CyPA) and its corresponding receptor, CD147, which exists in a variety of cells, play crucial roles in modulating reactive oxygen species (ROS) production. In this study, we explored the role of the CyPA/CD147 signaling pathway in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and the protective effects exerted by melatonin against Ang II-induced injury in cultured H9C2 cells. Cyclosporine A, a specific CyPA/CD147 signaling pathway inhibitor, was used to manipulate CyPA/CD147 activity. H9C2 cells were then subjected to Ang II or CyPA treatment in either the absence or presence of melatonin. Our results indicate that Ang II induces cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway and promotes ROS production, which can be blocked by melatonin pretreatment in a concentration-dependent manner, in cultured H9C2 cells and that CyPA/CD147 signaling pathway inhibition protects against Ang II-induced cardiomyocyte hypertrophy. The protective effects of melatonin against Ang II-induced cardiomyocyte hypertrophy depend at least partially on CyPA/CD147 inhibition.

Local IGF-1 isoform protects cardiomyocytes from hypertrophic and oxidative stresses via SirT1 activity.

PubMed

Vinciguerra, Manlio; Santini, Maria Paola; Claycomb, William C; Ladurner, Andreas G; Rosenthal, Nadia

2009-12-10

Oxidative and hypertrophic stresses contribute to the pathogenesis of heart failure. Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a complex post-transcriptional regulation, generating distinct isoforms. Locally acting IGF-1 isoform (mIGF-1) helps the heart to recover from toxic injury and from infarct. In the murine heart, moderate overexpression of the NAD(+)-dependent deacetylase SirT1 was reported to mitigate oxidative stress. SirT1 is known to promote lifespan extension and to protect from metabolic challenges. Circulating IGF-1 and SirT1 play antagonizing biological roles and share molecular targets in the heart, in turn affecting cardiomyocyte physiology. However, how different IGF-1 isoforms may impact SirT1 and affect cardiomyocyte function is unknown. Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress. Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy. Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion. Thus, mIGF-1 protects cardiomyocytes from oxidative and hypertrophic stresses via SirT1 activity, and may represent a promising cardiac therapeutic.

Local IGF-1 isoform protects cardiomyocytes from hypertrophic and oxidative stresses via SirT1 activity

PubMed Central

Vinciguerra, Manlio; Santini, Maria Paola; Claycomb, William C.; Ladurner, Andreas G.; Rosenthal, Nadia

2010-01-01

Oxidative and hypertrophic stresses contribute to the pathogenesis of heart failure. Insulin-like growth factor-1 (IGF-1) is a peptide hormone with a complex post-transcriptional regulation, generating distinct isoforms. Locally acting IGF-1 isoform (mIGF-1) helps the heart to recover from toxic injury and from infarct. In the murine heart, moderate overexpression of the NAD+-dependent deacetylase SirT1 was reported to mitigate oxidative stress. SirT1 is known to promote lifespan extension and to protect from metabolic challenges. Circulating IGF-1 and SirT1 play antagonizing biological roles and share molecular targets in the heart, in turn affecting cardiomyocyte physiology. However, how different IGF-1 isoforms may impact SirT1 and affect cardiomyocyte function is unknown. Here we show that locally acting mIGF-1 increases SirT1 expression/activity, whereas circulating IGF-1 isoform does not affect it, in cultured HL-1 and neonatal cardiomyocytes. mIGF-1-induced SirT1 activity exerts protection against angiotensin II (Ang II)-triggered hypertrophy and against paraquat (PQ) and Ang II-induced oxidative stress. Conversely, circulating IGF-1 triggered itself oxidative stress and cardiomyocyte hypertrophy. Interestingly, potent cardio-protective genes (adiponectin, UCP-1 and MT-2) were increased specifically in mIGF-1-overexpressing cardiomyocytes, in a SirT1-dependent fashion. Thus, mIGF-1 protects cardiomyocytes from oxidative and hypertrophic stresses via SirT1 activity, and may represent a promising cardiac therapeutic. PMID:20228935

The protective effect of Hif3a RNA interference and HIF-prolyl hydroxylase inhibition on cardiomyocytes under anoxia-reoxygenation.

PubMed

Drevytska, T; Gonchar, E; Okhai, I; Lynnyk, O; Mankovska, I; Klionsky, D; Dosenko, V

2018-06-01

The aim of this study was to investigate the molecular mechanisms underlying the protective effects of hypoxia-inducible factor (HIF) signaling pathway activation in cardiomyocytes under anoxia-reoxygenation (A/R) injury. In this study, rat neonatal cardiomyocytes were pretreated with anti-Hif3A/Hif-3α siRNA or HIF-prolyl hydroxylase inhibitor prior to A/R injury. Our results showed that both HIF3A silencing and HIF-prolyl hydroxylase inhibition effectively increased the cell viability during A/R, led to changes in mRNA expression of HIF1-target genes, and reduced the loss of mitochondrial membrane potential (Δψ m ). Furthermore, application of anti-Hif3a siRNA led to an increase in mRNA expression of Epo, Igf1, Slc2a1/Glut-1, and Slc2a4/Glut-4. Similar results were observed with HIF-prolyl hydroxylase inhibition, which additionally upregulated the mRNA expression of Epor, Tert, and Pdk1. Hif3a RNA-interference and application of HIF-prolyl hydroxylase inhibitor during A/R modelling led to an increase of Δψ m on 11.5 and 11.9 mV respectively, compared to the control groups. Thus, Hif3a RNA interference and HIF-prolyl hydroxylase inhibition protect cardiomyocytes against A/R injury via the HIF signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

The proliferative and chronotropic effects of Brillantaisia nitens Lindau (Acanthaceae) extracts on pluripotent stem cells and their cardiomyocytes derivatives.

PubMed

Nembo, Erastus Nembu; Dimo, Theophile; Bopda, Orelien Sylvain Mtopi; Hescheler, Jürgen; Nguemo, Filomain

2014-10-28

Brillantaisia nitens Lindau (Acanthaceae) leaves are commonly used in traditional medicine in Africa for the treatment of many disorders including heart diseases and malaria. In this study, we therefore evaluated the effect of the methylene chloride/methanol leaf extract of Brillantaisia nitens on the proliferation of mouse pluripotent stem cells and their cardiomyocyte derivatives. In this study, we combined two emerging technologies, pluripotent stem cell-derived cardiomyocytes and modern electrophysiology systems (impedance-based real-time) to assess the cytotoxicity of Brillantaisia nitens extract (BNE). Undifferentiated pluripotent cells and cardiomyocytes were exposed to different concentrations of BNE. Cell viability and contraction were monitored by impedance using the xCELLigence system for short- and long-term treatment whereas the excitability of single cardiomyocytes was captured by patch clamp technique after BNE acute exposure. Brillantaisia nitens extract inhibited the proliferation and increased cytotoxicity of embryonic stem cells in a concentration-dependent manner. With the increase in concentration of BNE, beating rate and the contractile amplitude of cardiomyocytes changed significantly. Spontaneous rhythmic activity of cardiomyocytes was completely suppressed after 48 and 24h exposures to relatively low (4.16 mg/ml) and high (8.32 mg/ml) concentrations of BNE, respectively. Moreover, acute application of 4.16 mg/ml of BNE led to a significant alteration of action potential (AP) parameters such as beating frequency, amplitude and AP duration at 90% of repolarization. Brillantaisia nitens extract inhibits the proliferative capacity of pluripotent stem cells and reduces electrical activity of cardiomyocytes, confirming its depressant action on the heart. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Evidence for Cardiomyocyte Renewal in Humans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bergmann, O; Bhardwaj, R D; Bernard, S

2008-10-14

It has been difficult to establish whether we are limited to the heart muscle cells we are born with or if cardiomyocytes are generated also later in life. We have taken advantage of the integration of {sup 14}C, generated by nuclear bomb tests during the Cold War, into DNA to establish the age of cardiomyocytes in humans. We report that cardiomyocytes renew, with a gradual decrease from 1% turning over annually at the age of 20 to 0.3% at the age of 75. Less than 50% of cardiomyocytes are exchanged during a normal lifespan. The capacity to generate cardiomyocytes inmore » the adult human heart suggests that it may be rational to work towards the development of therapeutic strategies aiming to stimulate this process in cardiac pathologies.« less

Renal papillary tip extract stimulates BNP production and excretion from cardiomyocytes

PubMed Central

Hashizume, Ryotaro; Suzuki, Noboru; Ito, Rie; Yamanaka, Keiichi; Saito, Hiromitsu; Kiyonari, Hiroshi; Tawara, Isao; Kageyama, Yuki; Ogihara, Yoshito; Ali, Yusuf; Yamada, Norikazu; Katayama, Naoyuki; Ito, Masaaki

2018-01-01

Background Brain natriuretic peptide (BNP) is an important biomarker for patients with cardiovascular diseases, including heart failure, hypertension and cardiac hypertrophy. It is also known that BNP levels are relatively higher in patients with chronic kidney disease and no heart disease; however, the mechanism remains unclear. Methods and results We developed a BNP reporter mouse and occasionally found that this promoter was activated specifically in the papillary tip of the kidneys, and its activation was not accompanied by BNP mRNA expression. No evidence was found to support the existence of BNP isoforms or other nucleotide expression apart from BNP and tdTomato. The pBNP-tdTomato-positive cells were interstitial cells and were not proliferative. Unexpectedly, both the expression and secretion of BNP increased in primary cultured neonatal cardiomyocytes after their treatment with an extract of the renal papillary tip. Intraperitoneal injection of the extract of the papillary tips reduced blood pressure from 210 mmHg to 165 mmHg, the decrease being accompanied by an increase in serum BNP and urinary cGMP production in stroke-prone spontaneously hypertensive (SHR-SP) rats. Furthermore the induction of BNP by the papillary extract from rats with heart failure due to myocardial infarction was increased in cardiomyocytes. Conclusions These results suggested that the papillary tip express a substance that can stimulate BNP production and secretion from cardiomyocytes. PMID:29734386

Effects and mechanisms of apolipoprotein A-V on the regulation of lipid accumulation in cardiomyocytes.

PubMed

Luo, Jun; Xu, Li; Li, Jiang; Zhao, Shuiping

2018-03-12

Apolipoprotein (apo) A-V is a key regulator of triglyceride (TG) metabolism. We investigated effects of apoA-V on lipid metabolism in cardiomyocytes in this study. We first examined whether apoA-V can be taken up by cardiomyocytes and whether low density lipoprotein receptor family members participate in this process. Next, triglyceride (TG) content and lipid droplet changes were detected at different concentrations of apoA-V in normal and lipid-accumulation cells in normal and obese animals. Finally, we tested the levels of fatty acids (FAs) taken up into cardiomyocytes and lipid secretion through [ 14 C]-oleic acid. Our results show that heart tissue has apoA-V protein, and apoA-V is taken up by cardiomyocytes. When HL-1 cells were transfected with low density lipoprotein receptor (LDLR)-related protein 1(LRP1) siRNA, apoA-V intake decreased by 53% (P<0.05), while a 37% lipid accumulation in HL-1 cells remain unchanged. ApoA-V localized to the cytoplasm and was associated with lipid droplets in HL-1 cells. A 1200 and 1800 ng/mL apoA-V intervention decreased TG content by 28% and 45% in HL-1 cells, respectively and decreased TG content by 39% in mouse heart tissue (P<0.05). However, apoA-V had no effects on TG content in either normal HL-1 cells or mice. The levels of FAs taken up into cardiomyocytes decreased by 43% (P < 0.05), and the levels of TG and cholesterol ester secretion increased by 1.2-fold and 1.6-fold, respectively (P < 0.05). ApoA-V is a novel regulator of lipid metabolism in cardiomyocytes.

Notch3/Akt signaling contributes to OSM-induced protection against cardiac ischemia/reperfusion injury.

PubMed

Zhang, Mingming; Wang, Chen; Hu, Jianqiang; Lin, Jie; Zhao, Zhijing; Shen, Min; Gao, Haokao; Li, Na; Liu, Min; Zheng, Pengfei; Qiu, Cuiting; Gao, Erhe; Wang, Haichang; Sun, Dongdong

2015-09-01

Oncostatin M (OSM) exhibits many unique biological activities by activating the Oβ receptor. However, its role in myocardial ischemia/reperfusion injury (I/R injury) in mice remains unknown. We investigated whether Notch3/Akt signaling is involved in the regulation of OSM-induced protection against cardiac I/R injury. The effects of OSM were assessed in mice that underwent myocardial I/R injury by OSM treatment or by genetic deficiency of the OSM receptor Oβ. We investigated its effects on cardiomyocyte apoptosis and mitochondrial biogenesis and whether Notch3/Akt signaling was involved in the regulation of OSM-induced protection against cardiac I/R injury. The mice underwent 30 min of ischemia followed by 3 h of reperfusion and were randomized to be treated with Notch3 siRNA (siNotch3) or lentivirus carrying Notch3 cDNA (Notch3) 72 h before coronary artery ligation. Myocardial infarct size, cardiac function, cardiomyocyte apoptosis and mitochondria morphology in mice that underwent cardiac I/R injury were compared between groups. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through promotion of Notch3 production, thus activating the PI3K/Akt pathway. OSM enhanced mitochondrial biogenesis and mitochondrial function in mice subjected to cardiac I/R injury. In contrast, OSM receptor Oβ knock out exacerbated cardiac I/R injury, decreased Notch3 production, enhanced cardiomyocyte apoptosis, and impaired mitochondrial biogenesis in cardiac I/R injured mice. The mechanism of OSM on cardiac I/R injury is partly mediated by the Notch3/Akt pathway. These results suggest a novel role of Notch3/Akt signaling that contributes to OSM-induced protection against cardiac I/R injury.

Renal Impairment with Sublethal Tubular Cell Injury in a Chronic Liver Disease Mouse Model

PubMed Central

Ishida, Tokiko; Kotani, Hirokazu; Miyao, Masashi; Kawai, Chihiro; Jemail, Leila; Abiru, Hitoshi; Tamaki, Keiji

2016-01-01

The pathogenesis of renal impairment in chronic liver diseases (CLDs) has been primarily studied in the advanced stages of hepatic injury. Meanwhile, the pathology of renal impairment in the early phase of CLDs is poorly understood, and animal models to elucidate its mechanisms are needed. Thus, we investigated whether an existing mouse model of CLD induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) shows renal impairment in the early phase. Renal injury markers, renal histology (including immunohistochemistry for tubular injury markers and transmission electron microscopy), autophagy, and oxidative stress were studied longitudinally in DDC- and standard diet–fed BALB/c mice. Slight but significant renal dysfunction was evident in DDC-fed mice from the early phase. Meanwhile, histological examinations of the kidneys with routine light microscopy did not show definitive morphological findings, and electron microscopic analyses were required to detect limited injuries such as loss of brush border microvilli and mitochondrial deformities. Limited injuries have been recently designated as sublethal tubular cell injury. As humans with renal impairment, either with or without CLD, often show almost normal tubules, sublethal injury has been of particular interest. In this study, the injuries were associated with mitochondrial aberrations and oxidative stress, a possible mechanism for sublethal injury. Intriguingly, two defense mechanisms were associated with this injury that prevent it from progressing to apparent cell death: autophagy and single-cell extrusion with regeneration. Furthermore, the renal impairment of this model progressed to chronic kidney disease with interstitial fibrosis after long-term DDC feeding. These findings indicated that DDC induces renal impairment with sublethal tubular cell injury from the early phase, leading to chronic kidney disease. Importantly, this CLD mouse model could be useful for studying the pathophysiological mechanisms

Taurine ameliorated homocysteine-induced H9C2 cardiomyocyte apoptosis by modulating endoplasmic reticulum stress.

PubMed

Zhang, Zhimin; Zhao, Lianyou; Zhou, Yanfen; Lu, Xuanhao; Wang, Zhengqiang; Wang, Jipeng; Li, Wei

2017-05-01

Homocysteine (Hcy)-triggered endoplasmic reticulum (ER) stress-mediated endothelial cell apoptosis has been suggested as a cause of Hcy-dependent vascular injury. However, whether ER stress is the molecular mechanism linking Hcy and cardiomyocytes death is unclear. Taurine has been reported to exert cardioprotective effects via various mechanisms. However, whether taurine protects against Hcy-induced cardiomyocyte death by attenuating ER stress is unknown. This study aimed to evaluate the opposite effects of taurine on Hcy-induced cardiomyocyte apoptosis and their underlying mechanisms. Our results demonstrated that low-dose or short-term Hcy treatment increased the expression of glucose-regulated protein 78 (GRP78) and activated protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6), which in turn prevented apoptotic cell death. High-dose Hcy or prolonged Hcy treatment duration significantly up-regulated levels of C/EBP homologous protein (CHOP), cleaved caspase-12, p-c-Jun N-terminal kinase (JNK), and then triggered apoptotic events. High-dose Hcy also resulted in a decrease in mitochondrial membrane potential (Δψm) and an increase in cytoplasmic cytochrome C and the expression of cleaved caspase-9. Pretreatment of cardiomyocytes with sodium 4-phenylbutyric acid (an ER stress inhibitor) significantly inhibited Hcy-induced apoptosis. Furthermore, blocking the PERK pathway partly alleviated Hcy-induced ER stress-modulated cardiomyocyte apoptosis, and down-regulated the levels of Bax and cleaved caspase-3. Experimental taurine pretreatment inhibited the expression of ER stress-related proteins, and protected against apoptotic events triggered by Hcy-induced ER stress. Taken together, our results suggest that Hcy triggered ER stress in cardiomyocytes, which was the crucial molecular mechanism mediating Hcy-induced cardiomyocyte apoptosis, and the adverse effect of Hcy could be prevented by taurine.

Increased Postnatal Cardiac Hyperplasia Precedes Cardiomyocyte Hypertrophy in a Model of Hypertrophic Cardiomyopathy

PubMed Central

Farrell, Emily T.; Grimes, Adrian C.; de Lange, Willem J.; Armstrong, Annie E.; Ralphe, J. Carter

2017-01-01

Rationale: Hypertrophic cardiomyopathy (HCM) occurs in ~0.5% of the population and is a leading cause of sudden cardiac death (SCD) in young adults. Cardiomyocyte hypertrophy has been the accepted mechanism for cardiac enlargement in HCM, but the early signaling responsible for initiating hypertrophy is poorly understood. Mutations in cardiac myosin binding protein C (MYBPC3) are among the most common HCM-causing mutations. Ablation of Mybpc3 in an HCM mouse model (cMyBP-C−/−) rapidly leads to cardiomegaly by postnatal day (PND) 9, though hearts are indistinguishable from wild-type (WT) at birth. This model provides a unique opportunity to explore early processes involved in the dramatic postnatal transition to hypertrophy. Methods and Results: We performed microarray analysis, echocardiography, qPCR, immunohistochemistry (IHC), and isolated cardiomyocyte measurements to characterize the perinatal cMyBP-C−/− phenotype before and after overt hypertrophy. cMyBP-C−/− hearts showed elevated cell cycling at PND1 that transitioned to hypertrophy by PND9. An expanded time course revealed that increased cardiomyocyte cycling was associated with elevated heart weight to body weight ratios prior to cellular hypertrophy, suggesting that cell cycling resulted in cardiomyocyte proliferation. Animals heterozygous for the cMyBP-C deletion trended in the direction of the homozygous null, yet did not show increased heart size by PND9. Conclusions: Results indicate that altered regulation of the cell cycling pathway and elevated proliferation precedes hypertrophy in the cMyBP-C−/− HCM model, and suggests that increased cardiomyocyte number contributes to increased heart size in cMyBP-C−/− mice. This pre-hypertrophic period may reflect a unique time during which the commitment to HCM is determined and disease severity is influenced. PMID:28659827

BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes.

PubMed

Hong, Ting-Ting; Smyth, James W; Chu, Kevin Y; Vogan, Jacob M; Fong, Tina S; Jensen, Brian C; Fang, Kun; Halushka, Marc K; Russell, Stuart D; Colecraft, Henry; Hoopes, Charles W; Ocorr, Karen; Chi, Neil C; Shaw, Robin M

2012-05-01

Heart failure is a growing epidemic, and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T tubules. Bridging integrator 1 (BIN1) is a membrane scaffolding protein that causes Cav1.2 to traffic to T tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. Intact myocardium and freshly isolated cardiomyocytes from nonfailing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch-clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking-competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after small hairpin RNA-mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino-mediated knockdown of BIN1. BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced to 42% by imaging, and a biochemical T-tubule fraction of Cav1.2 is reduced to 68%. The total calcium current is reduced to 41% in a cell line expressing a nontrafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility. Copyright © 2012 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Histone hyperacetylation and exon skipping: a calcium-mediated dynamic regulation in cardiomyocytes

PubMed Central

Sharma, Alok; Nguyen, Hieu; Cai, Lu; Lou, Hua

2015-01-01

In contrast to cell type-specific pre-mRNA alternative splicing, mechanisms controlling activity-dependent alternative splicing is under-studied and not well understood. In a recent study, we conducted a comprehensive analysis of calcium-mediated mechanism that regulates alternative exon skipping in mouse cardiomyocytes. Our results reveal a strong link between histone hyperacetylation and skipping of cassette exons, and provide support to the kinetic coupling model of the epigenetic regulation of alternative splicing at the chromatin level. PMID:26325491

PAR-2 mediates increased inflammatory cell adhesion and neointima formation following vascular injury in the mouse.

PubMed

Tennant, Gail M; Wadsworth, Roger M; Kennedy, Simon

2008-05-01

Activation of PAR-2 in the vasculature affects vascular tone and adhesion of leukocytes to the endothelium. Since adhesion of leukocytes is increased following vascular injury and is important in determining the extent of neointima formation, we hypothesised that mice lacking PAR-2 may have reduced neointima formation following vascular injury. PAR-2 activating peptides and trypsin induced endothelium-dependent relaxation of mouse carotid artery which was absent in the knockout mouse. Lack of a PAR-2 receptor did not affect lymphocyte adhesion under basal conditions, but reduced the contractile response produced by lymphocytes. Twenty-eight days after denuding injury, vessel contraction to lymphocytes was reduced in both strains while lymphocyte adhesion was significantly greater in PAR-2(+/+) mice compared to the PAR-2 knockout mice. Neointimal area was markedly reduced in the PAR-2 knockout mouse. Our data show that PAR-2 modulates inflammatory cell adhesion when stimulated and in mice lacking the PAR-2 receptor, adhesion to injured vessels is reduced with a consequent reduction in neointima formation.

Herba Cistanches stimulates cellular glutathione redox cycling by reactive oxygen species generated from mitochondrial respiration in H9c2 cardiomyocytes.

PubMed

Wong, Hoi Shan; Ko, Kam Ming

2013-01-01

Earlier findings demonstrated that pretreatment of Herba Cistanches [the dried whole plant of Cistanche deserticola Y.C. Ma (Orobanchaceae)], a "Yang-invigorating" Chinese tonic herb, stimulated the ATP-generation capacity (ATP-GC) in mitochondria isolated from rat heart ex vivo. The enhancement of mitochondrial ATP-GC by Herba Cistanches was associated with induction of glutathione antioxidant status and protection against ischemia/reperfusion (I/R) injury in rat hearts. This study investigated the relationship between enhancements in mitochondrial ATP-GC and glutathione antioxidant status in H9c2 cardiomyocytes using a semipurified fraction of Herba Cistanches (HCF1). HCF1 (10-300 ng/mL) was tested for its effects on mitochondrial ATP generation, glutathione antioxidant status and protection against oxidant injury in H9c2 cardiomyocytes and rat hearts. HCF1 at 30 ng/mL increased mitochondrial ATP-GC and ADP-stimulated state 3 respiration (by 50 and 100%, respectively) in H9c2 cardiomyocytes. The stimulation of mitochondrial respiration was associated with the induction of mitochondrial uncoupling (27%) and enhancement of cellular glutathione redox cycling as well as protection against hypoxia/reoxygenation (hypox/reoxy)-induced apoptosis (by 60%). While HCF1 treatment increased reactive oxygen species generation from mitochondrial respiration in H9c2 cardiomyocytes, pretreatment with antioxidants (DMTU) abrogated the HCF1-induced cellular responses and the associated cytoprotective effect. HCF1 pretreatment (1.14 and 3.41 mg/kg × 14) also protected against myocardial I/R injury in rats (by 13 and 32%), presumably mediated by the induction of glutathione antioxidant response. The long-term intake of HCF1 may offer a prospect for the prevention of ischemic heart disease.

Inhibition of microRNA-497 ameliorates anoxia/reoxygenation injury in cardiomyocytes by suppressing cell apoptosis and enhancing autophagy

PubMed Central

Li, Xixian; Zeng, Zhi; Li, Qingman; Xu, Qiulin; Xie, Jiahe; Hao, Huixin; Luo, Guangjin; Liao, Wangjun; Bin, Jianping; Huang, Xiaobo; Liao, Yulin

2015-01-01

MiR-497 is predicted to target anti-apoptosis gene Bcl2 and autophagy gene microtubule-associated protein 1 light chain 3 B (LC3B), but the functional consequence of miR-497 in response to anoxia/reoxygenation (AR) or ischemia/reperfusion (IR) remains unknown. This study was designed to investigate the influences of miR-497 on myocardial AR or IR injury. We noted that miR-497 was enriched in cardiac tissues, while its expression was dynamically changed in murine hearts subjected to myocardial infarction and in neonatal rat cardiomyocytes (NRCs) subjected to AR. Forced expression of miR-497 (miR-497 mimic) induced apoptosis in NRCs as determined by Hoechst staining and TUNEL assay. In response to AR, silencing of miR-497 using a miR-497 sponge significantly reduced cell apoptosis and enhanced autophagic flux. Furthermore, the infarct size induced by IR in adenovirus (Ad)-miR-497 sponge infected mice was significantly smaller than in mice receiving Ad-vector or vehicle treatment, while Ad-miR-497 increased infarct size. The expression of Bcl-2 and LC3B-II in NRCs or in murine heart was significantly decreased by miR-497 mimic and enhanced by miR-497 sponge. These findings demonstrate that inhibition of miR-497 holds promise for limiting myocardial IR injury. PMID:26299920

Overexpression of BAG3 Attenuates Hypoxia-Induced Cardiomyocyte Apoptosis by Inducing Autophagy.

PubMed

Zhang, Jiankai; He, Zhangyou; Xiao, Wenjian; Na, Qingqing; Wu, Tianxiu; Su, Kaixin; Cui, Xiaojun

2016-01-01

Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes. BAG3 expression level was measured in H9c2 cells treated with hypoxia for 48 h. Cell proliferation and apoptosis were tested using MTT assay and Annexin V FITC-PI staining assay, respectively. The mRNA or protein expression level of BAG3, LC3-I, LC3-II, Atg5, NF-x03BA;B p65 and phosphorylated NF-x03BA;B p65 were assessed by qRT-PCR and western blot assay, respectively. Resluts: Overexpression of BAG3 inhibited cell apoptosis and promoted proliferation in hypoxia-injured H9c2 cells. Furthermore, autophagy and NF-x03BA;B were activated by BAG3 overexpression, and the NF-x03BA;B inhibitor PDTC could inhibit the activation of autophagy induced by BAG3 overexpression. In addition, the autophagy inhibitor 3-MA partly impeded the inhibitory effect of BAG3 on hypoxia-induced cardiomyocyte apoptosis. these results suggested that overexpression of BAG3 promoted cell proliferation and inhibited apoptosis by activating autophagy though the NF-x03BA;B signaling pathway in hypoxia-injured cardiomyocytes. © 2016 The Author(s) Published by S. Karger AG, Basel.

[Morphological signs of survival cultured adult rat cardiomyocytes].

PubMed

Chang, Hui; Zhang, Lin; Yu, Zhi-Bin

2011-02-01

To clarify the key morphological signs for the survival of adult rat cardiomyocytes in primary culture. The adult rat hearts were retrogradely superfused by Langendorff apparatus. Cardiomyocytes were digested by collagenase I and cultured in three groups: (1) Serum free medium + BA (Bongkrekic acid, apoptotic inhibitor), (2) 5% serum medium, and (3) 5% serum medium + BA. The morphological alterations were observed and the percentage of rod-shaped cardiomyocytes, the apoptotic rate of cells, the rate of pseudopodium formation and the nuclear distances of cardiomyocytes were detected during culture. (1) The percentage of rod-shaped cardiomyocytes decreased gradually in the first 3 days of cell culture. The percentage of rod-shaped cardiomyocytes cultured without fetal bovine serum (FBS) decreased more rapidly than those cultured with FBS. No differences were noticed between with and without the addition of apoptotic inhibitor BA. The apoptotic rate of cardiomyocytes increased in the first 3 days of cell culture, and the apoptotic rate of cells cultured without FBS increased more than that cultured with FBS. Also BA had no effect on apoptotic rate. (2) Cardiomyocytes cultured with FBS spread from the intercalated disk and extended pseudopodium on the second or third day of cell culture. Cardiomyocytes with thin membranous pseudopodium developed would survive and spread laterally at the 6th day of culture. Cells with the elongated morphology gradually spread extensively and took on a spheroidal shape. Myofibrils gradually lost their parallel. Cells cultured without FBS had no pseudopodium formation. The intercalated disk of cells gradually changed blunt. There was no effect on the rate of pseudopodium formation when added with apoptotic inhibitor BA. (3) Cytoskeletal remodeling occurred in survived cardiomyocytes. After 6 days of culture, cardiomyocytes exhibited characteristic of redifferentiation. (4) The distance between nuclei decreased in a single cardiomyocyte

In vivo characterization of early-stage radiation skin injury in a mouse model by two-photon microscopy

PubMed Central

Jang, Won Hyuk; Shim, Sehwan; Wang, Taejun; Yoon, Yeoreum; Jang, Won-Suk; Myung, Jae Kyung; Park, Sunhoo; Kim, Ki Hean

2016-01-01

Ionizing radiation (IR) injury is tissue damage caused by high energy electromagnetic waves such as X-ray and gamma ray. Diagnosis and treatment of IR injury are difficult due to its characteristics of clinically latent post-irradiation periods and the following successive and unpredictable inflammatory bursts. Skin is one of the many sensitive organs to IR and bears local injury upon exposure. Early-stage diagnosis of IR skin injury is essential in order to maximize treatment efficiency and to prevent the aggravation of IR injury. In this study, early-stage changes of the IR injured skin at the cellular level were characterized in an in vivo mouse model by two-photon microscopy (TPM). Various IR doses were applied to the mouse hind limbs and the injured skin regions were imaged daily for 6 days after IR irradiation. Changes in the morphology and distribution of the epidermal cells and damage of the sebaceous glands were observed before clinical symptoms. These results showed that TPM is sensitive to early-stage changes of IR skin injury and may be useful for its diagnosis. PMID:26755422

Newborn Hypoxia/Anoxia Inhibits Cardiomyocyte Proliferation and Decreases Cardiomyocyte Endowment in the Developing Heart: Role of Endothelin-1

PubMed Central

Paradis, Alexandra N.; Gay, Maresha S.; Wilson, Christopher G.; Zhang, Lubo

2015-01-01

In the developing heart, cardiomyocytes undergo terminal differentiation during a critical window around birth. Hypoxia is a major stress to preterm infants, yet its effect on the development and maturation of the heart remains unknown. We tested the hypothesis in a rat model that newborn anoxia accelerates cardiomyocyte terminal differentiation and results in reduced cardiomyocyte endowment in the developing heart via an endothelin-1-dependent mechanism. Newborn rats were exposed to anoxia twice daily from postnatal day 1 to 3, and hearts were isolated and studied at postnatal day 4 (P4), 7 (P7), and 14 (P14). Anoxia significantly increased HIF-1α protein expression and pre-proET-1 mRNA abundance in P4 neonatal hearts. Cardiomyocyte proliferation was significantly decreased by anoxia in P4 and P7, resulting in a significant reduction of cardiomyocyte number per heart weight in the P14 neonates. Furthermore, the expression of cyclin D2 was significantly decreased due to anoxia, while p27 expression was increased. Anoxia has no significant effect on cardiomyocyte binucleation or myocyte size. Consistently, prenatal hypoxia significantly decreased cardiomyocyte proliferation but had no effect on binucleation in the fetal heart. Newborn administration of PD156707, an ETA-receptor antagonist, significantly increased cardiomyocyte proliferation at P4 and cell size at P7, resulting in an increase in the heart to body weight ratio in P7 neonates. In addition, PD156707 abrogated the anoxia-mediated effects. The results suggest that hypoxia and anoxia via activation of endothelin-1 at the critical window of heart development inhibits cardiomyocyte proliferation and decreases myocyte endowment in the developing heart, which may negatively impact cardiac function later in life. PMID:25692855

Substance P Receptor Signaling Mediates Doxorubicin-Induced Cardiomyocyte Apoptosis and Triple-Negative Breast Cancer Chemoresistance

PubMed Central

Robinson, Prema; Kasembeli, Moses; Bharadwaj, Uddalak; Engineer, Nikita; Eckols, Kris T.; Tweardy, David J.

2016-01-01

Doxorubicin (DOX), an anthracycline, is broadly considered the most active single agent available for treating breast cancer but has been known to induce cardiotoxicity. Although DOX is highly effective in treating triple-negative breast cancer (TNBC), DOX can have poor outcomes owing to induction of chemoresistance. There is an urgent need to develop new therapies for TNBC aimed at improving DOX outcome and DOX-induced cardiotoxicity. Substance P (SP), a neuropeptide involved in pain transmission is known to stimulate production of reactive oxygen species (ROS). Elevated cardiac ROS is linked with heart injury and failure. We investigated the role of SP in chemotherapy-associated death of cardiomyocytes and chemoresistance. We showed that pretreating a cardiomyocyte cell line (H9C2) and a TNBC cell line (MDA-MB 231) with aprepitant, a SP receptor antagonist that is routinely used to treat chemotherapy-associated associated nausea, decreased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in cardiomyocytes and increased DOX-induced reduction of cell viability, apoptotic cell death, and ROS production in TNBC cells compared with cells treated with DOX alone. Our findings demonstrate the ability of aprepitant to decrease DOX-induced killing of cardiomyocytes and to increase cancer cell sensitivity to DOX, which has tremendous clinical significance. PMID:26981525

Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis.

PubMed

Huang, Zhan-Peng; Kataoka, Masaharu; Chen, Jinghai; Wu, Gengze; Ding, Jian; Nie, Mao; Lin, Zhiqiang; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Zhou, Bin; Wakimoto, Hiroko; Zeng, Chunyu; Kyselovic, Jan; Deng, Zhong-Liang; Seidman, Christine E; Seidman, J G; Pu, William T; Wang, Da-Zhi

2015-11-02

Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression.

Cardiomyocyte-enriched protein CIP protects against pathophysiological stresses and regulates cardiac homeostasis

PubMed Central

Huang, Zhan-Peng; Kataoka, Masaharu; Chen, Jinghai; Wu, Gengze; Ding, Jian; Nie, Mao; Lin, Zhiqiang; Liu, Jianming; Hu, Xiaoyun; Ma, Lixin; Zhou, Bin; Wakimoto, Hiroko; Zeng, Chunyu; Kyselovic, Jan; Deng, Zhong-Liang; Seidman, Christine E.; Seidman, J.G.; Pu, William T.; Wang, Da-Zhi

2015-01-01

Cardiomyopathy is a common human disorder that is characterized by contractile dysfunction and cardiac remodeling. Genetic mutations and altered expression of genes encoding many signaling molecules and contractile proteins are associated with cardiomyopathy; however, how cardiomyocytes sense pathophysiological stresses in order to then modulate cardiac remodeling remains poorly understood. Here, we have described a regulator in the heart that harmonizes the progression of cardiac hypertrophy and dilation. We determined that expression of the myocyte-enriched protein cardiac ISL1-interacting protein (CIP, also known as MLIP) is reduced in patients with dilated cardiomyopathy. As CIP is highly conserved between human and mouse, we evaluated the effects of CIP deficiency on cardiac remodeling in mice. Deletion of the CIP-encoding gene accelerated progress from hypertrophy to heart failure in several cardiomyopathy models. Conversely, transgenic and AAV-mediated CIP overexpression prevented pathologic remodeling and preserved cardiac function. CIP deficiency combined with lamin A/C deletion resulted in severe dilated cardiomyopathy and cardiac dysfunction in the absence of stress. Transcriptome analyses of CIP-deficient hearts revealed that the p53- and FOXO1-mediated gene networks related to homeostasis are disturbed upon pressure overload stress. Moreover, FOXO1 overexpression suppressed stress-induced cardiomyocyte hypertrophy in CIP-deficient cardiomyocytes. Our studies identify CIP as a key regulator of cardiomyopathy that has potential as a therapeutic target to attenuate heart failure progression. PMID:26436652

Heterogeneity of adult masseter muscle satellite cells with cardiomyocyte differentiation potential.

PubMed

Huang, Wei; Liang, Jialiang; Feng, Yuliang; Jia, Zhanfeng; Jiang, Lin; Cai, Wenfeng; Paul, Christian; Gu, Jianguo G; Stambrook, Peter J; Millard, Ronald W; Zhu, Xiao-Lan; Zhu, Ping; Wang, Yigang

2018-05-26

Although resident cardiac stem cells have been reported, regeneration of functional cardiomyocytes (CMs) remains a challenge. The present study identifies an alternative progenitor source for CM regeneration without the need for genetic manipulation or invasive heart biopsy procedures. Unlike limb skeletal muscles, masseter muscles (MM) in the mouse head are developed from Nkx2-5 mesodermal progenitors. Adult masseter muscle satellite cells (MMSCs) display heterogeneity in developmental origin and cell phenotypes. The heterogeneous MMSCs that can be characterized by cell sorting based on stem cell antigen-1 (Sca1) show different lineage potential. While cardiogenic potential is preserved in Sca1 + MMSCs as shown by expression of cardiac progenitor genes (including Nkx2-5), skeletal myogenic capacity is maintained in Sca1 - MMSCs with Pax7 expression. Sca1 + MMSC-derived beating cells express cardiac genes and exhibit CM-like morphology. Electrophysiological properties of MMSC-derived CMs are demonstrated by calcium transients and action potentials. These findings show that MMSCs could serve as a novel cell source for cardiomyocyte replacement. Copyright © 2018. Published by Elsevier Inc.

Effect of CPAP in a Mouse Model of Hyperoxic Neonatal Lung Injury

PubMed Central

Reyburn, Brent; Fiore, Juliann M. Di; Raffay, Thomas; Martin, Richard J.; Y.S., Prakash; Jafri, Anjum; MacFarlane, Peter M.

2015-01-01

Background Continuous positive airway pressure [CPAP] and supplemental oxygen have become the mainstay of neonatal respiratory support in preterm infants. Although oxygen therapy is associated with respiratory morbidities including bronchopulmonary dysplasia [BPD], the long-term effects of CPAP on lung function are largely unknown. We used a hyperoxia-induced mouse model of BPD to explore the effects of daily CPAP during the first week of life on later respiratory system mechanics. Objective To test the hypothesis that daily CPAP in a newborn mouse model of BPD improves longer term respiratory mechanics. Methods Mouse pups from C57BL/6 pregnant dams were exposed to room air [RA] or hyperoxia [50% O2, 24hrs/day] for the first postnatal week with or without exposure to daily CPAP [6cmH2O, 3hrs/day]. Respiratory system resistance [Rrs] and compliance [Crs] were measured following a subsequent 2 week period of room RA recovery. Additional measurements included radial alveolar counts and macrophage counts. Results Mice exposed to hyperoxia had significantly elevated Rrs, decreased Crs, reduced alveolarization, and increased macrophage counts at three weeks compared to RA treated mice. Daily CPAP treatment significantly improved Rrs, Crs and alveolarization, and decreased lung macrophage infiltration in hyperoxia-exposed pups. Conclusions We have demonstrated that daily CPAP had a longer term benefit on baseline respiratory system mechanics in a neonatal mouse model of BPD. We speculate that this beneficial effect of CPAP was the consequence of a decrease in the inflammatory response and resultant alveolar injury associated with hyperoxic newborn lung injury. PMID:26394387

Hemodynamic and morphologic responses in mouse brain during acute head injury imaged by multispectral structured illumination

NASA Astrophysics Data System (ADS)

Volkov, Boris; Mathews, Marlon S.; Abookasis, David

2015-03-01

Multispectral imaging has received significant attention over the last decade as it integrates spectroscopy, imaging, tomography analysis concurrently to acquire both spatial and spectral information from biological tissue. In the present study, a multispectral setup based on projection of structured illumination at several near-infrared wavelengths and at different spatial frequencies is applied to quantitatively assess brain function before, during, and after the onset of traumatic brain injury in an intact mouse brain (n=5). For the production of head injury, we used the weight drop method where weight of a cylindrical metallic rod falling along a metal tube strikes the mouse's head. Structured light was projected onto the scalp surface and diffuse reflected light was recorded by a CCD camera positioned perpendicular to the mouse head. Following data analysis, we were able to concurrently show a series of hemodynamic and morphologic changes over time including higher deoxyhemoglobin, reduction in oxygen saturation, cell swelling, etc., in comparison with baseline measurements. Overall, results demonstrates the capability of multispectral imaging based structured illumination to detect and map of brain tissue optical and physiological properties following brain injury in a simple noninvasive and noncontact manner.

A mouse model of ocular blast injury that induces closed globe anterior and posterior pole damage

PubMed Central

Hines-Beard, Jessica; Marchetta, Jeffrey; Gordon, Sarah; Chaum, Edward; Geisert, Eldon E.; Rex, Tonia S.

2012-01-01

We developed and characterized a mouse model of primary ocular blast injury. The device consists of: a pressurized air tank attached to a regulated paintball gun with a machined barrel; a chamber that protects the mouse from direct injury and recoil, while exposing the eye; and a secure platform that enables fine, controlled movement of the chamber in relation to the barrel. Expected pressures were calculated and the optimal pressure transducer, based on the predicted pressures, was positioned to measure output pressures at the location where the mouse eye would be placed. Mice were exposed to one of three blast pressures (23.6, 26.4, or 30.4psi). Gross pathology, intraocular pressure, optical coherence tomography, and visual acuity were assessed 0, 3, 7, 14, and 28 days after exposure. Contralateral eyes and non-blast exposed mice were used as controls. We detected increased damage with increased pressures and a shift in the damage profile over time. Gross pathology included corneal edema, corneal abrasions, and optic nerve avulsion. Retinal damage was detected by optical coherence tomography and a deficit in visual acuity was detected by optokinetics. Our findings are comparable to those identified in Veterans of the recent wars with closed eye injuries as a result of blast exposure. In summary, this is a relatively simple system that creates injuries with features similar to those seen in patients with ocular blast trauma. This is an important new model for testing the short-term and long-term spectrum of closed globe blast injuries and potential therapeutic interventions. PMID:22504073

MicroRNA-15b deteriorates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by downregulating Bcl-2 and MAPK3.

PubMed

Liu, Yaling; Yang, Liqun; Yin, Jiemin; Su, Diansan; Pan, Zhiying; Li, Peiying; Wang, Xiaodong

2018-01-01

To investigate the role of miRNA-15b in cardiomyocyte apoptosis after ischemia reperfusion injury in acute myocardial infarction (AMI), we conducted the AMI rat model by using left anterior descending ligation and performed hypoxia/reoxygenation experiments in H9c2 cells. MiRNA-15b was measured by quantitative reverse transcription PCR (qRT-PCR). Cardiomyocyte apoptosis was determined by terminal deoxynucleotide transferase dUTP nick end labeling staining. Synthesized miRNA-15b mimic and inhibitor were transfected into H9c2 cells by Lipofectamine regent. RNA expression of B cell lymphoma/leukemia-2 (Bcl-2) and mitogen-activated protein kinase 3 (MAPK3) was examined by qRT-PCR and their protein expression was determined by western blot. Ischemia reperfusion increased miRNA-15b expression in the ischemic rat heart and resulted more severe cardiomyocytes apoptosis. In H9c2 cells, hypoxia/reoxygenation induced increased miRNA-15b expression and augmented cardiomyocyte apoptosis observed at 24 hours after 24-hour hypoxia. Compared with the vehicle group, miRNA-15b mimic further raised miRNA-15b level and increased cardiomyocyte apoptosis, whereas miRNA-15b inhibitor suppressed miRNA-15b expression and protected cardiomyocytes from apoptosis. Although the mRNA expression of the target genes Bcl-2 and MAPK3 was not changed significantly, the protein expression of these two genes were markedly reduced after miRNA-15b mimic treatment and significantly increased after transfected with miRNA-15b inhibitors. In conclusion, miRNA-15b deteriorates cardiomyocyte apoptosis by post-transcriptionally downregulating the expression of Bcl-2 and MAPK3. © American Federation for Medical Research (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

The cardiomyocyte molecular clock, regulation of Scn5a, and arrhythmia susceptibility

PubMed Central

Lefta, Mellani; Zhang, Xiping; Bartos, Daniel; Feng, Han-Zhong; Zhao, Yihua; Patwardhan, Abhijit; Jin, Jian-Ping; Esser, Karyn A.; Delisle, Brian P.

2013-01-01

The molecular clock mechanism underlies circadian rhythms and is defined by a transcription-translation feedback loop. Bmal1 encodes a core molecular clock transcription factor. Germline Bmal1 knockout mice show a loss of circadian variation in heart rate and blood pressure, and they develop dilated cardiomyopathy. We tested the role of the molecular clock in adult cardiomyocytes by generating mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1). ECG telemetry showed that cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1−/−) in adult mice slowed heart rate, prolonged RR and QRS intervals, and increased episodes of arrhythmia. Moreover, isolated iCSΔBmal1−/− hearts were more susceptible to arrhythmia during electromechanical stimulation. Examination of candidate cardiac ion channel genes showed that Scn5a, which encodes the principle cardiac voltage-gated Na+ channel (NaV1.5), was circadianly expressed in control mouse and rat hearts but not in iCSΔBmal1−/− hearts. In vitro studies confirmed circadian expression of a human Scn5a promoter-luciferase reporter construct and determined that overexpression of clock factors transactivated the Scn5a promoter. Loss of Scn5a circadian expression in iCSΔBmal1−/− hearts was associated with decreased levels of NaV1.5 and Na+ current in ventricular myocytes. We conclude that disruption of the molecular clock in the adult heart slows heart rate, increases arrhythmias, and decreases the functional expression of Scn5a. These findings suggest a potential link between environmental factors that alter the cardiomyocyte molecular clock and factors that influence arrhythmia susceptibility in humans. PMID:23364267

Cardiomyocyte dysfunction during the chronic phase of Chagas disease.

PubMed

Roman-Campos, Danilo; Sales-Júnior, Policarpo; Duarte, Hugo Leonardo; Gomes, Eneas Ricardo; Guatimosim, Silvia; Ropert, Catherine; Gazzinelli, Ricardo Tostes; Cruz, Jader Santos

2013-04-01

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure.

Cardiomyocyte dysfunction during the chronic phase of Chagas disease

PubMed Central

Roman-Campos, Danilo; Sales-Júnior, Policarpo; Duarte, Hugo Leonardo; Gomes, Eneas Ricardo; Guatimosim, Silvia; Ropert, Catherine; Gazzinelli, Ricardo Tostes; Cruz, Jader Santos

2013-01-01

Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of heart failure. We investigated modifications in the cellular electrophysiological and calcium-handling characteristics of an infected mouse heart during the chronic phase of the disease. The patch-clamp technique was used to record action potentials (APs) and L-type Ca2+ and transient outward K+ currents. [Ca2+]i changes were determined using confocal microscopy. Infected ventricular cells showed prolonged APs, reduced transient outward K+ and L-type Ca2+ currents and reduced Ca2+ release from the sarcoplasmic reticulum. Thus, the chronic phase of Chagas disease is characterised by cardiomyocyte dysfunction, which could lead to heart failure. PMID:23579807

Intravenous Administration of Lycopene, a Tomato Extract, Protects against Myocardial Ischemia-Reperfusion Injury

PubMed Central

Tong, Chao; Peng, Chuan; Wang, Lianlian; Zhang, Li; Yang, Xiaotao; Xu, Ping; Li, Jinjin; Delplancke, Thibaut; Zhang, Hua; Qi, Hongbo

2016-01-01

Background: Oral uptake of lycopene has been shown to be beneficial for preventing myocardial ischemia-reperfusion (I/R) injury. However, the strong first-pass metabolism of lycopene influences its bioavailability and impedes its clinic application. In this study, we determined an intravenous (IV) administration dose of lycopene protects against myocardial infarction (MI) in a mouse model, and investigated the effects of acute lycopene administration on reactive oxygen species (ROS) production and related signaling pathways during myocardial I/R. Methods: In this study, we established both in vitro hypoxia/reoxygenation (H/R) cell model and in vivo regional myocardial I/R mouse model by ligating left anterior artery descending. TTC dual staining was used to assess I/R induced MI in the absence and presence of acute lycopene administration via tail vein injection. Results: Lycopene treatment (1 μM) before reoxygenation significantly reduced cardiomyocyte death induced by H/R. Intravenous administration of lycopene to achieve 1 μM concentration in circulating blood significantly suppressed MI, ROS production, and JNK phosphorylation in the cardiac tissue of mice during in vivo regional I/R. Conclusion: Elevating circulating lycopene to 1 μM via IV injection protects against myocardial I/R injury through inhibition of ROS accumulation and consequent inflammation in mice. PMID:26950150

Intravenous Administration of Lycopene, a Tomato Extract, Protects against Myocardial Ischemia-Reperfusion Injury.

PubMed

Tong, Chao; Peng, Chuan; Wang, Lianlian; Zhang, Li; Yang, Xiaotao; Xu, Ping; Li, Jinjin; Delplancke, Thibaut; Zhang, Hua; Qi, Hongbo

2016-03-03

Oral uptake of lycopene has been shown to be beneficial for preventing myocardial ischemia-reperfusion (I/R) injury. However, the strong first-pass metabolism of lycopene influences its bioavailability and impedes its clinic application. In this study, we determined an intravenous (IV) administration dose of lycopene protects against myocardial infarction (MI) in a mouse model, and investigated the effects of acute lycopene administration on reactive oxygen species (ROS) production and related signaling pathways during myocardial I/R. In this study, we established both in vitro hypoxia/reoxygenation (H/R) cell model and in vivo regional myocardial I/R mouse model by ligating left anterior artery descending. TTC dual staining was used to assess I/R induced MI in the absence and presence of acute lycopene administration via tail vein injection. Lycopene treatment (1 μM) before reoxygenation significantly reduced cardiomyocyte death induced by H/R. Intravenous administration of lycopene to achieve 1 μM concentration in circulating blood significantly suppressed MI, ROS production, and JNK phosphorylation in the cardiac tissue of mice during in vivo regional I/R. Elevating circulating lycopene to 1 μM via IV injection protects against myocardial I/R injury through inhibition of ROS accumulation and consequent inflammation in mice.

Multicolor Fluorescence Imaging of Traumatic Brain Injury in a Cryolesion Mouse Model

PubMed Central

2012-01-01

Traumatic brain injury is characterized by initial tissue damage, which then can lead to secondary processes such as cell death and blood-brain-barrier disruption. Clinical and preclinical studies of traumatic brain injury typically employ anatomical imaging techniques and there is a need for new molecular imaging methods that provide complementary biochemical information. Here, we assess the ability of a targeted, near-infrared fluorescent probe, named PSS-794, to detect cell death in a brain cryolesion mouse model that replicates certain features of traumatic brain injury. In short, the model involves brief contact of a cold rod to the head of a living, anesthetized mouse. Using noninvasive whole-body fluorescence imaging, PSS-794 permitted visualization of the cryolesion in the living animal. Ex vivo imaging and histological analysis confirmed PSS-794 localization to site of brain cell death. The nontargeted, deep-red Tracer-653 was validated as a tracer dye for monitoring blood-brain-barrier disruption, and a binary mixture of PSS-794 and Tracer-653 was employed for multicolor imaging of cell death and blood-brain-barrier permeability in a single animal. The imaging data indicates that at 3 days after brain cryoinjury the amount of cell death had decreased significantly, but the integrity of the blood-brain-barrier was still impaired; at 7 days, the blood-brain-barrier was still three times more permeable than before cryoinjury. PMID:22860222

Splicing factor NSSR1 reduces neuronal injury after mouse transient global cerebral ischemia.

PubMed

Qi, Yao; Li, Ya; Cui, Shi-Chao; Zhao, Jing-Jing; Liu, Xiao-Yan; Ji, Chun-Xia; Sun, Feng-Yan; Xu, Ping; Chen, Xian-Hua

2015-05-01

This study focuses on the function of NSSR1, a splicing factor, in neuronal injury in the ischemic mouse brain using the transient global cerebral ischemic mouse model and the cultured cells treated with oxygen-glucose deprivation (OGD). The results showed that the cerebral ischemia triggers the expression of NSSR1 in hippocampal astrocytes, predominantly the dephosphorylated NSSR1 proteins, and the Exon3 inclusive NCAM-L1 variant and the Exon4 inclusive CREB variant. While in the hippocampus of astrocyte-specific NSSR1 conditional knockdown (cKD) mice, where cerebral ischemia no longer triggers NSSR1 expression in astrocytes, the expression of Exon3 inclusive NCAM-L1 variant and Exon4 inclusive CREB variant were no longer triggered as well. In addition, the injury of hippocampal neurons was more severe in astrocyte-specific NSSR1 cKD mice compared with in wild-type mice after brain ischemia. Of note, the culture media harvested from the astrocytes with overexpression of NSSR1 or the Exon3 inclusive NCAM-L1 variant, or Exon4 inclusive CREB variant were all able to reduce the neuronal injury induced by OGD. The results provide the evidence demonstrating that: (1) Splicing factor NSSR1 is a new factor involved in reducing ischemic injury. (2) Ischemia induces NSSR1 expression in astrocytes, not in neurons. (3) NSSR1-mediated pathway in astrocytes is required for reducing ischemic neuronal injury. (4) NCAM-L1 and CREB are probably mediators in NSSR1-mediated pathway. In conclusion, our results suggest for the first time that NSSR1 may provide a novel mechanism for reducing neuronal injury after ischemia, probably through regulation on alternative splicing of NCAM-L1 and CREB in astrocytes. © 2014 Wiley Periodicals, Inc.

Liver Transplantation in the Mouse: Insights Into Liver Immunobiology, Tissue Injury and Allograft Tolerance

PubMed Central

Yokota, Shinichiro; Yoshida, Osamu; Ono, Yoshihiro; Geller, David A.; Thomson, Angus W.

2016-01-01

The surgically-demanding mouse orthotopic liver transplant model was first described in 1991. It has proved a powerful research tool for investigation of liver biology, tissue injury, the regulation of alloimmunity and tolerance induction and the pathogenesis of specific liver diseases. Liver transplantation in mice has unique advantages over transplantation of the liver in larger species, such as the rat or pig, since the mouse genome is well-characterized and there is much greater availability of both genetically-modified animals and research reagents. Liver transplant experiments using various transgenic or gene knockout mice has provided valuable mechanistic insights into the immuno- and pathobiology of the liver and the regulation of graft rejection and tolerance over the past 25 years. The molecular pathways identified in regulation of tissue injury and promotion of liver transplant tolerance provide new potential targets for therapeutic intervention to control adverse inflammatory responses/ immune-mediated events in the hepatic environment and systemically. Conclusion: Orthotopic liver transplantation in the mouse is a valuable model for gaining improved insights into liver biology, immunopathology and allograft tolerance that may result in therapeutic innovation in liver and other diseases. PMID:26709949

Analysis of cardiomyocyte movement in the developing murine heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Hisayuki; Yuasa, Shinsuke, E-mail: yuasa@a8.keio.jp; Tabata, Hidenori

The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cellmore » cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle. - Highlights: • We directly visualized cardiomyocyte movement inside the developing murine heart. • Cell cycle related genes were upregulated in the proliferating cardiomyocytes. • Time-lapse imaging revealed that proliferating murine cardiomyocytes stayed in place. • Murine ventricular cardiomyocytes proliferate on site during development.« less

Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

NASA Astrophysics Data System (ADS)

Arshi, Armin; Nakashima, Yasuhiro; Nakano, Haruko; Eaimkhong, Sarayoot; Evseenko, Denis; Reed, Jason; Stieg, Adam Z.; Gimzewski, James K.; Nakano, Atsushi

2013-04-01

While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell-cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

PubMed

Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

2016-02-01

Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Long noncoding RNA FTX regulates cardiomyocyte apoptosis by targeting miR-29b-1-5p and Bcl2l2.

PubMed

Long, Bo; Li, Na; Xu, Xi-Xia; Li, Xiao-Xin; Xu, Xin-Jie; Guo, Dan; Zhang, Dong; Wu, Zhi-Hong; Zhang, Shu-Yang

2018-01-01

Cardiomyocyte apoptosis correlates with the pathogenesis of heart disease. Long noncoding RNA (LncRNA) emerges as a class of noncoding RNAs that regulate gene expression and participate in various cellular processes. However, the role of lncRNAs in cardiomyocyte apoptosis remains to be elucidated. In our study, we found that lncRNA FTX is significantly down-regulated upon ischemia/reperfusion injury and hydrogen peroxide treatment. Enhanced expression of FTX inhibits cardiomyocyte apoptosis induced by hydrogen peroxide. miR-29b-1-5p was found to interact with FTX and regulate the expression of Bcl2l2. Inhibition of miR-29b-1-5p attenuated cardiomyocyte apoptosis upon hydrogen peroxide treatment. We then found that FTX functions as endogenous sponge for miR-29b-1-5p and regulates the activity of miR-29b-1-5p. The results demonstrate that FTX regulates cardiomyocyte apoptosis through modulating the expression of Bcl2l2 which is mediated by miR-29b-1-5p. Our findings reveal a novel regulatory model which is composed of FTX, miR-29b-1-5p and Bcl2l2. Manipulating of their levels may become a new approach to tackling cardiomyocyte apoptosis related heart diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Assessment of the cellular and electrophysiological response of cardiomyocytes to radiation

NASA Astrophysics Data System (ADS)

Helm, Alexander; Ritter, Sylvia; Durante, Marco; Friess, Johannes; Thielemann, Christiane; Mr; Frank, Simon

Cardiac disease is considered as a late effect resulting from an exposure during long-term space missions. Yet, the underlying mechanisms and the impact of radiation quality and dose are not well understood. To address this topic, we used cardiomyocytes derived from mouse embryonic stem cells (mESC) as a model system. This model has already been successfully used for cardiotoxicity screening of new drugs. Both, the cellular and electrophysiological response to X-ray irradiation were examined. Cellular endpoints such as the induction of micronuclei, apoptosis, number of binucleated cells and expression of connexin43 (Cx 43) were analyzed by standard techniques. For electrophysiological studies a microelectrode array (MEA) was used allowing non-invasive recordings of electrical signals such as signal amplitude and shape, beat rate and conduction velocity. Data analysis was performed using the MATLAB based software DrCell. As a first approach, cardiomyocytes were generated by differentiation of mESC via the formation of embryoid bodies. However, the system proved to be unsuitable due to large intra- and inter-sample variations. In consecutive experiments we used commercially available Cor.At cells, i.e. a pure culture of mESC derived cardiomyocytes. For the analysis of cellular and electrophysiological endpoints Cor.At cells were seeded onto chamber slides or MEA chips, respectively. Irradiation with 0.5 and 2 Gy X-rays (250 kV, 16 mA) was performed two days after seeding. At that time cardiomyocytes are electrically coupled through gap junctions and form a spontaneously beating network. Samples were examined up to four days after exposure. Analysis of the electrophysiological data revealed only minor differences between controls and X-irradiated samples indicating the functionality of cardiomyocytes is not within the dose range examined. Currently, further experiments are performed to statistically verify this finding. Additionally, the expression of Cx 43, a major

Profiling analysis of long non-coding RNAs in early postnatal mouse hearts

PubMed Central

Sun, Xiongshan; Han, Qi; Luo, Hongqin; Pan, Xiaodong; Ji, Yan; Yang, Yao; Chen, Hanying; Wang, Fangjie; Lai, Wenjing; Guan, Xiao; Zhang, Qi; Tang, Yuan; Chu, Jianhong; Yu, Jianhua; Shou, Weinian; Deng, Youcai; Li, Xiaohui

2017-01-01

Mammalian cardiomyocytes undergo a critical hyperplastic-to-hypertrophic growth transition at early postnatal age, which is important in establishing normal physiological function of postnatal hearts. In the current study, we intended to explore the role of long non-coding (lnc) RNAs in this transitional stage. We analyzed lncRNA expression profiles in mouse hearts at postnatal day (P) 1, P7 and P28 via microarray. We identified 1,146 differentially expressed lncRNAs with more than 2.0-fold change when compared the expression profiles of P1 to P7, P1 to P28, and P7 to P28. The neighboring genes of these differentially expressed lncRNAs were mainly involved in DNA replication-associated biological processes. We were particularly interested in one novel cardiac-enriched lncRNA, ENSMUST00000117266, whose expression was dramatically down-regulated from P1 to P28 and was also sensitive to hypoxia, paraquat, and myocardial infarction. Knockdown ENSMUST00000117266 led to a significant increase of neonatal mouse cardiomyocytes in G0/G1 phase and reduction in G2/M phase, suggesting that ENSMUST00000117266 is involved in regulating cardiomyocyte proliferative activity and is likely associated with hyperplastic-to-hypertrophic growth transition. In conclusion, our data have identified a large group of lncRNAs presented in the early postnatal mouse heart. Some of these lncRNAs may have important functions in cardiac hyperplastic-to-hypertrophic growth transition. PMID:28266538

Pathophysiological Responses in Rat and Mouse Models of Radiation-Induced Brain Injury.

PubMed

Yang, Lianhong; Yang, Jianhua; Li, Guoqian; Li, Yi; Wu, Rong; Cheng, Jinping; Tang, Yamei

2017-03-01

The brain is the major dose-limiting organ in patients undergoing radiotherapy for assorted conditions. Radiation-induced brain injury is common and mainly occurs in patients receiving radiotherapy for malignant head and neck tumors, arteriovenous malformations, or lung cancer-derived brain metastases. Nevertheless, the underlying mechanisms of radiation-induced brain injury are largely unknown. Although many treatment strategies are employed for affected individuals, the effects remain suboptimal. Accordingly, animal models are extremely important for elucidating pathogenic radiation-associated mechanisms and for developing more efficacious therapies. So far, models employing various animal species with different radiation dosages and fractions have been introduced to investigate the prevention, mechanisms, early detection, and management of radiation-induced brain injury. However, these models all have limitations, and none are widely accepted. This review summarizes the animal models currently set forth for studies of radiation-induced brain injury, especially rat and mouse, as well as radiation dosages, dose fractionation, and secondary pathophysiological responses.

The protein PprI provides protection against radiation injury in human and mouse cells

PubMed Central

Shi, Yi; Wu, Wei; Qiao, Huiping; Yue, Ling; Ren, Lili; Zhang, Shuyu; Yang, Wei; Yang, Zhanshan

2016-01-01

Severe acute radiation injuries are both very lethal and exceptionally difficult to treat. Though the radioresistant bacterium D. radiodurans was first characterized in 1956, genes and proteins key to its radioprotection have not yet to be applied in radiation injury therapy for humans. In this work, we express the D. radiodurans protein PprI in Pichia pastoris yeast cells transfected with the designed vector plasmid pHBM905A-pprI. We then treat human umbilical endothelial vein cells and BALB/c mouse cells with the yeast-derived PprI and elucidate the radioprotective effects the protein provides upon gamma irradiation. We see that PprI significantly increases the survival rate, antioxidant viability, and DNA-repair capacity in irradiated cells and decreases concomitant apoptosis rates and counts of damage-indicative γH2AX foci. Furthermore, we find that PprI reduces mortality and enhances bone marrow cell clone formation and white blood cell and platelet counts in irradiated mice. PprI also seems to alleviate pathological injuries to multiple organs and improve antioxidant viability in some tissues. Our results thus suggest that PprI has crucial radioprotective effects on irradiated human and mouse cells. PMID:27222438

Developing better mouse models to study cisplatin-induced kidney injury.

PubMed

Sharp, Cierra N; Siskind, Leah J

2017-10-01

Cisplatin is a potent chemotherapeutic used for the treatment of many types of cancer. However, its dose-limiting side effect is nephrotoxicity leading to acute kidney injury (AKI). Patients who develop AKI have an increased risk of mortality and are more likely to develop chronic kidney disease (CKD). Unfortunately, there are no therapeutic interventions for the treatment of AKI. It has been suggested that the lack of therapies is due in part to the fact that the established mouse model used to study cisplatin-induced AKI does not recapitulate the cisplatin dosing regimen patients receive. In recent years, work has been done to develop more clinically relevant models of cisplatin-induced kidney injury, with much work focusing on incorporation of multiple low doses of cisplatin administered over a period of weeks. These models can be used to recapitulate the development of CKD after AKI and, by doing so, increase the likelihood of identifying novel therapeutic targets for the treatment of cisplatin-induced kidney injury. Copyright © 2017 the American Physiological Society.

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout.

PubMed

Sokolova, Niina; Vendelin, Marko; Birkedal, Rikke

2009-12-17

Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20 degrees C in the absence and presence of creatine. Trout cardiomyocytes hypercontracted in the solutions used for mammalian cardiomyocytes. We developed a new solution in which they retained their shape and showed stable steady state respiration rates throughout an experiment. The apparent ADP-affinity of permeabilized cardiomyocytes was different from that of fibers. It was higher, independent of temperature and not increased by creatine. However, it was still about ten times lower than in isolated mitochondria. The differences between fibers and cardiomyocytes suggest that results from trout heart fibers were affected by incomplete separation of the cells. However, the lower ADP-affinity of cardiomyocytes compared to isolated mitochondria indicate that intracellular diffusion restrictions are still present in trout cardiomyocytes despite their lower density of intracellular membrane structures. The lack of a creatine effect indicates that trout heart lacks mitochondrial creatine kinase tightly

Mangiferin Enhanced Autophagy via Inhibiting mTORC1 Pathway to Prevent High Glucose-Induced Cardiomyocyte Injury.

PubMed

Hou, Jun; Zheng, Dezhi; Xiao, Wenjing; Li, Dandan; Ma, Jie; Hu, Yonghe

2018-01-01

Mangiferin functions as a perfect anti-oxidative compound in the diabetic heart, however, the exact mechanism remains to be elucidated. Here, we show the cardioprotective effect of mangiferin under high glucose-induced cardiotoxic condition mainly contributed to enhanced autophagy via suppressing mTORC1 downstream signal transduction. Primary neonatal rat cardiomyocytes were cultured to detect myocytes injury, autophagy, and related signal transduction under different doses of glucose and mangiferin treatment. High glucose (30 mM) reduced autophagic flux, and increased myocyte apoptosis and death compared with normal glucose (5.5 mM) as determined by variation of autophagy markers LC3-II, p62, parkin, GFP-LC3, or mRFP-LC3 fluorescence puncta, cell viability, cleaved caspase 3, cleaved PARP apoptosis indices, reactive oxygen species (ROS), MAO, and PI death indices. Conversely, mangiferin inhibited hyperglycemia associated oxidative stress by reducing ROS, MAO, cleaved caspase 3, and cleaved PARP generation, reestablishing cell viability, mitochondrial membrane potential, and enhancing autophagic flux, thereby preventing myocytes from high glucose-induced toxicity. Furthermore, cardioprotection with mangiferin was potentially related to the decreased mTOR phosphorylation and suppression of mTORC1 downstream signaling pathway. These data indicated the valuable effects of mangiferin on regulation of cardiac autophagy and pointed to the promising utilization for hyperglycemia control.

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue

PubMed Central

Welsh, David K.

2016-01-01

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a β-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+. PMID:27459195

A mouse diversity panel approach reveals the potential for clinical kidney injury due to DB289 not predicted by classical rodent models.

PubMed

Harrill, Alison H; Desmet, Kristina D; Wolf, Kristina K; Bridges, Arlene S; Eaddy, J Scott; Kurtz, C Lisa; Hall, J Ed; Paine, Mary F; Tidwell, Richard R; Watkins, Paul B

2012-12-01

DB289 is the first oral drug shown in clinical trials to have efficacy in treating African trypanosomiasis (African sleeping sickness). Mild liver toxicity was noted but was not treatment limiting. However, development of DB289 was terminated when several treated subjects developed severe kidney injury, a liability not predicted from preclinical testing. We tested the hypothesis that the kidney safety liability of DB289 would be detected in a mouse diversity panel (MDP) comprised of 34 genetically diverse inbred mouse strains. MDP mice received 10 days of oral treatment with DB289 or vehicle and classical renal biomarkers blood urea nitrogen (BUN) and serum creatinine (sCr), as well as urine biomarkers of kidney injury were measured. While BUN and sCr remained within reference ranges, marked elevations were observed for kidney injury molecule-1 (KIM-1) in the urine of sensitive mouse strains. KIM-1 elevations were not always coincident with elevations in alanine aminotransferase (ALT), suggesting that renal injury was not linked to hepatic injury. Genome-wide association analyses of KIM-1 elevations indicated that genes participating in cholesterol and lipid biosynthesis and transport, oxidative stress, and cytokine release may play a role in DB289 renal injury. Taken together, the data resulting from this study highlight the utility of using an MDP to predict clinically relevant toxicities, to identify relevant toxicity biomarkers that may translate into the clinic, and to identify potential mechanisms underlying toxicities. In addition, the sensitive mouse strains identified in this study may be useful in screening next-in-class compounds for renal injury.

Regulation of cardiomyocyte autophagy by calcium

PubMed Central

Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F. G.; Hill, Joseph A.

2016-01-01

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. PMID:26884385

Regulation of cardiomyocyte autophagy by calcium.

PubMed

Shaikh, Soni; Troncoso, Rodrigo; Criollo, Alfredo; Bravo-Sagua, Roberto; García, Lorena; Morselli, Eugenia; Cifuentes, Mariana; Quest, Andrew F G; Hill, Joseph A; Lavandero, Sergio

2016-04-15

Calcium signaling plays a crucial role in a multitude of events within the cardiomyocyte, including cell cycle control, growth, apoptosis, and autophagy. With respect to calcium-dependent regulation of autophagy, ion channels and exchangers, receptors, and intracellular mediators play fundamental roles. In this review, we discuss calcium-dependent regulation of cardiomyocyte autophagy, a lysosomal mechanism that is often cytoprotective, serving to defend against disease-related stress and nutrient insufficiency. We also highlight the importance of the subcellular distribution of calcium and related proteins, interorganelle communication, and other key signaling events that govern cardiomyocyte autophagy. Copyright © 2016 the American Physiological Society.

Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes

PubMed Central

Eng, George; Lee, Benjamin W.; Protas, Lev; Gagliardi, Mark; Brown, Kristy; Kass, Robert S.; Keller, Gordon; Robinson, Richard B.; Vunjak-Novakovic, Gordana

2016-01-01

The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration. PMID:26785135

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology.

PubMed

Rajagopal, Vijay; Bass, Gregory; Ghosh, Shouryadipta; Hunt, Hilary; Walker, Cameron; Hanssen, Eric; Crampin, Edmund; Soeller, Christian

2018-04-18

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the

Apelin and APJ orchestrate complex tissue-specific control of cardiomyocyte hypertrophy and contractility in the hypertrophy-heart failure transition.

PubMed

Parikh, Victoria Nicole; Liu, Jing; Shang, Ching; Woods, Christopher; Chang, Alex Chia Yu; Zhao, Mingming; Charo, David N; Grunwald, Zachary; Huang, Yong; Seo, Kinya; Tsao, Philip S; Bernstein, Daniel; Ruiz-Lozano, Pilar; Quertermous, Thomas; Ashley, Euan A

2018-05-18

The G protein coupled receptor APJ is a promising therapeutic target for heart failure. Constitutive deletion of APJ in the mouse is protective against the hypertrophy-heart failure transition via elimination of ligand-independent, β-arrestin dependent stretch transduction. However, the cellular origin of this stretch transduction and the details of its interaction with apelin signaling remain unknown. We generated mice with conditional elimination of APJ in the endothelium (APJ endo-/- ) and myocardium (APJ myo-/- ). No baseline difference was observed in LV function in APJ endo-/- , APJ myo-/- or controls (APJ endo+/+ , APJ myo+/+ ). After exposure to transaortic constriction (TAC), APJ endo-/- animals developed left ventricular failure while APJ myo-/- were protected. At the cellular level, carbon fiber stretch of freshly isolated single cardiomyocytes demonstrated decreased contractile response to stretch in APJ -/- cardiomyocytes compared to APJ +/+ cardiomyocytes. Calcium transient did not change with stretch in either APJ -/- or APJ +/+ cardiomyocytes. Application of apelin to APJ +/+ cardiomyocytes resulted in decreased calcium transient. Further, hearts of mice treated with apelin exhibited decreased phosphorylation at Troponin I (cTnI) N-terminal residues (Ser 22,23), consistent with increased calcium sensitivity. These data establish that APJ stretch transduction is mediated specifically by myocardial APJ, that APJ is necessary for stretch-induced increases in contractility, and that apelin opposes APJ's stretch-mediated hypertrophy signaling by lowering calcium transient while maintaining contractility through myofilament calcium sensitization. These findings underscore apelin's unique potential as a therapeutic agent that can simultaneously support cardiac function and protect against the hypertrophy-heart failure transition.

Anti-apoptotic effect of heat shock protein 90 on hypoxia-mediated cardiomyocyte damage is mediated via the phosphatidylinositol 3-kinase/AKT pathway.

PubMed

Wang, Wei; Peng, Yizhi; Wang, Yuanyuan; Zhao, Xiaohui; Yuan, Zhiqiang

2009-09-01

1. Hypoxia-induced cardiomyocyte apoptosis contributes significantly to cardiac dysfunction following trauma, shock and burn injury. There is evidence that heat shock protein (HSP) 90 is anti-apoptotic in cardiomyocytes subjected to a variety of apoptotic stimuli. Because HSP90 acts as an upstream regulator of the serine/threonine protein kinase Akt survival pathway during cellular stress, we hypothesized that HSP90 exerts a cardioprotective effect via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. 2. Neonatal rat cardiomyocytes were subjected to normoxia or hypoxia in the absence or presence of the HSP90 inhibitor geldanamycin (1 μg/mL). Cardiomyocyte apoptosis was assessed by release of lactate dehydrogenase (LDH), terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining and caspase 3 activity. Expression of HSP90, Akt, Bad and cytochrome c release was determined by western blot analysis. 3. Following exposure of cells to hypoxia, HSP90 was markedly elevated in a time-dependent manner, reaching a peak at 6 h (eightfold increase). Geldanamycin significantly increased hypoxia-induced release of LDH by 114%, the percentage of apoptotic cardiomyocytes by 102% and caspase 3 activity by 78%. Pretreatment of cells with geldanamycin also suppressed phosphorylation of both Akt and its downstream target Bad, but promoted the mitochondrial release of cytochrome c. 4. In conclusion, HSP90 activity is enhanced in cardiomyocytes following hypoxic insult. The anti-apoptotic effect of HSP90 on cardiomyocytes subjected to hypoxia is mediated, at least in part, by the PI3-K/Akt pathway. Key words: apoptosis, cardiomyocyte, heart failure, heat shock protein 90, hypoxia, phosphatidylinositol 3-kinase/Akt signalling pathway, serine/threonine protein kinase Akt.

Comparison of the protective effects of ferulic acid and its drug-containing plasma on primary cultured neonatal rat cardiomyocytes with hypoxia/reoxygenation injury

PubMed Central

Ren, Cong; Bao, Yong-rui; Meng, Xian-sheng; Diao, Yun-peng; Kang, Ting-guo

2013-01-01

Backgroud: To simulate the ischemia-reperfusion injury in vivo, hypoxia/reoxygenation injury model was established in vitro and primary cultured neonatal rat cardiomyocytes were underwent hypoxia with hydrosulfite (Na2S2O4) for 1 h followed by 1 h reoxygenation. Materials and Methods: Determination the cell viability by MTT colorimetric assay. We use kit to detect the activity of lactate dehydrogenase (LDH), Na+-K+-ATPase and Ca2+-ATPase. Do research on the effect which ferulic acid and its drug-containing plasma have to self-discipline, conductivity, action potential duration and other electrophysiological phenomena of myocardial cells by direct observation using a microscope and recording method of intracellular action potential. Results: The experimental datum showed that both can reduce the damage hydrosulfite to myocardial cell damage and improve myocardial viability, reduce the amount of LDH leak, increase activity of Na+-K+-ATPase, Ca2+-ATPase, and increase APA (Action potential amplitude), Vmax (Maximum rate of depolarization) and MPD (Maximum potential diastolic). Conclusion: Taken together, therefore, we can get the conclusion that ferulic acid drug-containing plasma has better protective effect injured myocardial cell than ferulic acid. PMID:23930002

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

PubMed

Shenje, Lincoln T; Andersen, Peter; Halushka, Marc K; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A S; Chen, Yan; Chelko, Stephen; Crosson, Jane E; Scheel, Janet; Vricella, Luca; Craig, Brian D; Marosy, Beth A; Mohr, David W; Hetrick, Kurt N; Romm, Jane M; Scott, Alan F; Valle, David; Naggert, Jürgen K; Kwon, Chulan; Doheny, Kimberly F; Judge, Daniel P

2014-03-04

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Mutations in Alström Protein Impair Terminal Differentiation of Cardiomyocytes

PubMed Central

Shenje, Lincoln T.; Andersen, Peter; Halushka, Marc K.; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B.; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A. S.; Chen, Yan; Chelko, Stephen; Crosson, Jane E.; Scheel, Janet; Vricella, Luca; Craig, Brian D.; Marosy, Beth A.; Mohr, David W.; Hetrick, Kurt N.; Romm, Jane M.; Scott, Alan F.; Valle, David; Naggert, Jürgen K.; Kwon, Chulan; Doheny, Kimberly F.; Judge, Daniel P.

2014-01-01

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognise homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at two weeks postnatal compared to wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest. PMID:24595103

Metabolites of Hypoxic Cardiomyocytes Induce the Migration of Cardiac Fibroblasts.

PubMed

Shi, Huairui; Zhang, Xuehong; He, Zekun; Wu, Zhiyong; Rao, Liya; Li, Yushu

2017-01-01

The migration of cardiac fibroblasts to the infarct region plays a major role in the repair process after myocardial necrosis or damage. However, few studies investigated whether early hypoxia in cardiomyocytes induces the migration of cardiac fibroblasts. The purpose of this study was to assess the role of metabolites of early hypoxic cardiomyocytes in the induction of cardiac fibroblast migration. Neonatal rat heart tissue was digested with a mixture of trypsin and collagenase at an appropriate ratio. Cardiomyocytes and cardiac fibroblasts were cultured via differential adhesion. The cardiomyocyte cultures were subjected to hypoxia for 2, 4, 6, 8, 10, and 12 h. The supernatants of the cardiomyocyte cultures were collected to determine the differences in cardiac fibroblast migration induced by hypoxic cardiomyocyte metabolites at various time points using a Transwell apparatus. Meanwhile, ELISA was performed to measure TNF-α, IL-1β and TGF-β expression levels in the cardiomyocyte metabolites at various time points. The metabolites of hypoxic cardiomyocytes significantly induced the migration of cardiac fibroblasts. The induction of cardiac fibroblast migration was significantly enhanced by cardiomyocyte metabolites in comparison to the control after 2, 4, and 6 h of hypoxia, and the effect was most significant after 2 h. The expression levels of TNF-α, IL-1β, IL-6, and TGF-β were substantially increased in the metabolites of cardiomyocytes, and neutralization with anti-TNF-α and anti-IL-1β antibodies markedly reduced the induction of cardiac fibroblast migration by the metabolites of hypoxic cardiomyocytes. The metabolites of early hypoxic cardiomyocytes can induce the migration of cardiac fibroblasts, and TNF-α and IL-1β may act as the initial chemotactic inducers. © 2017 The Author(s) Published by S. Karger AG, Basel.

Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy

PubMed Central

Putinski, Charis; Abdul-Ghani, Mohammad; Stiles, Rebecca; Brunette, Steve; Dick, Sarah A.; Fernando, Pasan; Megeney, Lynn A.

2013-01-01

Cardiomyocyte hypertrophy is the cellular response that mediates pathologic enlargement of the heart. This maladaptation is also characterized by cell behaviors that are typically associated with apoptosis, including cytoskeletal reorganization and disassembly, altered nuclear morphology, and enhanced protein synthesis/translation. Here, we investigated the requirement of apoptotic caspase pathways in mediating cardiomyocyte hypertrophy. Cardiomyocytes treated with hypertrophy agonists displayed rapid and transient activation of the intrinsic-mediated cell death pathway, characterized by elevated levels of caspase 9, followed by caspase 3 protease activity. Disruption of the intrinsic cell death pathway at multiple junctures led to a significant inhibition of cardiomyocyte hypertrophy during agonist stimulation, with a corresponding reduction in the expression of known hypertrophic markers (atrial natriuretic peptide) and transcription factor activity [myocyte enhancer factor-2, nuclear factor kappa B (NF-κB)]. Similarly, in vivo attenuation of caspase activity via adenoviral expression of the biologic effector caspase inhibitor p35 blunted cardiomyocyte hypertrophy in response to agonist stimulation. Treatment of cardiomyocytes with procaspase 3 activating compound 1, a small-molecule activator of caspase 3, resulted in a robust induction of the hypertrophy response in the absence of any agonist stimulation. These results suggest that caspase-dependent signaling is necessary and sufficient to promote cardiomyocyte hypertrophy. These results also confirm that cell death signal pathways behave as active remodeling agents in cardiomyocytes, independent of inducing an apoptosis response. PMID:24101493

A mouse model of alcoholic liver fibrosis-associated acute kidney injury identifies key molecular pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Furuya, Shinji; Chappell, Grace A.; Iwata, Yasuhir

Clinical data strongly indicate that acute kidney injury (AKI) is a critical complication in alcoholic hepatitis, an acute-on-chronic form of liver failure in patients with advanced alcoholic fibrosis. Development of targeted therapies for AKI in this setting is hampered by the lack of an animal model. To enable research into molecular drivers and novel therapies for fibrosis- and alcohol-associated AKI, we aimed to combine carbon tetrachloride (CCl{sub 4})-induced fibrosis with chronic intra-gastric alcohol feeding. Male C57BL/6J mice were administered a low dose of CCl{sub 4} (0.2 ml/kg 2 × week/6 weeks) followed by alcohol intragastrically (up to 25 g/kg/day formore » 3 weeks) and with continued CCl{sub 4}. We observed that combined treatment with CCl{sub 4} and alcohol resulted in severe liver injury, more pronounced than using each treatment alone. Importantly, severe kidney injury was evident only in the combined treatment group. This mouse model reproduced distinct pathological features consistent with AKI in human alcoholic hepatitis. Transcriptomic analysis of kidneys revealed profound effects in the combined treatment group, with enrichment for damage-associated pathways, such as apoptosis, inflammation, immune-response and hypoxia. Interestingly, Havcr1 and Lcn2, biomarkers of AKI, were markedly up-regulated. Overall, this study established a novel mouse model of fibrosis- and alcohol-associated AKI and identified key mechanistic pathways. - Highlights: • Acute kidney injury (AKI) is a critical complication in alcoholic hepatitis • We developed a novel mouse model of fibrosis- and alcohol-associated AKI • This model reproduces key molecular and pathological features of human AKI • This animal model can help identify new targeted therapies for alcoholic hepatitis.« less

Defining an Analytic Framework to Evaluate Quantitative MRI Markers of Traumatic Axonal Injury: Preliminary Results in a Mouse Closed Head Injury Model

PubMed Central

Sadeghi, N.; Namjoshi, D.; Irfanoglu, M. O.; Wellington, C.; Diaz-Arrastia, R.

2017-01-01

Diffuse axonal injury (DAI) is a hallmark of traumatic brain injury (TBI) pathology. Recently, the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA) was developed to generate an experimental model of DAI in a mouse. The characterization of DAI using diffusion tensor magnetic resonance imaging (MRI; diffusion tensor imaging, DTI) may provide a useful set of outcome measures for preclinical and clinical studies. The objective of this study was to identify the complex neurobiological underpinnings of DTI features following DAI using a comprehensive and quantitative evaluation of DTI and histopathology in the CHIMERA mouse model. A consistent neuroanatomical pattern of pathology in specific white matter tracts was identified across ex vivo DTI maps and photomicrographs of histology. These observations were confirmed by voxelwise and regional analysis of DTI maps, demonstrating reduced fractional anisotropy (FA) in distinct regions such as the optic tract. Similar regions were identified by quantitative histology and exhibited axonal damage as well as robust gliosis. Additional analysis using a machine-learning algorithm was performed to identify regions and metrics important for injury classification in a manner free from potential user bias. This analysis found that diffusion metrics were able to identify injured brains almost with the same degree of accuracy as the histology metrics. Good agreement between regions detected as abnormal by histology and MRI was also found. The findings of this work elucidate the complexity of cellular changes that give rise to imaging abnormalities and provide a comprehensive and quantitative evaluation of the relative importance of DTI and histological measures to detect brain injury. PMID:28966972

p53 and Mdm2 act synergistically to maintain cardiac homeostasis and mediate cardiomyocyte cell cycle arrest through a network of microRNAs.

PubMed

Stanley-Hasnain, Shanna; Hauck, Ludger; Grothe, Daniela; Aschar-Sobbi, Roozbeh; Beca, Sanja; Butany, Jagdish; Backx, Peter H; Mak, Tak W; Billia, Filio

2017-01-01

Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21 Cip1 and p27 Kip1 . Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.

MicroRNA-mediated non-viral direct conversion of embryonic fibroblasts to cardiomyocytes: comparison of commercial and synthetic non-viral vectors.

PubMed

Kim, Hyosuk; Kim, Dongkyu; Ku, Sook Hee; Kim, Kwangmeyung; Kim, Sun Hwa; Kwon, Ick Chan

Technological advances opened up new ways of directing cell fate conversion from one cell lineage to another. The direct cell conversion technique has recently attracted much attention in regenerative medicine to treat devastated organs and tissues, particularly having limited regenerative capacity such as the heart and brain. Unfortunately, its clinical application is severely limited due to a safety concern and immunogenicity of viral vectors, as human gene therapy did in the beginning stages. In this study, we examined the possibility of adopting non-viral vectors to direct cell conversion from mouse embryonic fibroblasts to induced cardiomyocytes (iCM) by transient transfection of four types of chemically synthesized micro-RNA mimics (miRNA-1, 133, 208, and 499). Herein, we tested several commercial and synthetic non-viral gene delivery carriers, which could be divided into three different categories: polymers [branched PEI (bPEI), bioreducible PEI (PEI-SS), deoxycholic acid-conjugated PEI (DA-PEI), jetPEI™, SuperFect™], lipids (Lipofectamine 2000™), and peptides (PepMute™). According to the analyses of physicochemical properties, cellular uptake, and cytotoxicity of the carrier/miRNA complexes, DA-PEI exhibited excellent miRNA delivery efficiency to mouse embryonic fibroblasts. One week after a single treatment of DA-PEI/miRNA without other adjuvants, the cells started to express cardiomyocyte-specific markers, such as α-actinin and α-MHC, indicating the formation of cardiomyocyte-like cells. Although the overall frequency of non-viral vector induced cardiomyogenic transdifferentiation was quite low (ca. 0.2%), this study can provide compelling support to develop clinically applicable transdifferentiation techniques.

Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.

PubMed

Wada, Rie; Muraoka, Naoto; Inagawa, Kohei; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Kaneda, Ruri; Suzuki, Tomoyuki; Kamiya, Kaichiro; Tohyama, Shugo; Yuasa, Shinsuke; Kokaji, Kiyokazu; Aeba, Ryo; Yozu, Ryohei; Yamagishi, Hiroyuki; Kitamura, Toshio; Fukuda, Keiichi; Ieda, Masaki

2013-07-30

Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

Molecular Signature of Mineralocorticoid Receptor Signaling in Cardiomyocytes: From Cultured Cells to Mouse Heart

PubMed Central

Latouche, Celine; Sainte-Marie, Yannis; Steenman, Marja; Castro Chaves, Paulo; Naray-Fejes-Toth, Aniko; Fejes-Toth, Geza; Farman, Nicolette; Jaisser, Frederic

2010-01-01

Excess mineralocorticoid signaling is deleterious for cardiovascular functions, as demonstrated by the beneficial effects of mineralocorticoid receptor (MR) antagonism on morbidity and mortality in patients with heart failure. However, the understanding of signaling pathways after MR activation in the heart remains limited. We performed transcriptomic analyses in the heart of double-transgenic mice with conditional, cardiomyocyte-specific, overexpression of the MR (MRcardio mice) or the glucocorticoid receptor (GR; GRcardio mice). Some of the genes induced in MRcardio mice were selected for comparative evaluation (real time PCR) in vivo in the heart of mice and ex vivo in the MR-expressing cardiomyocyte H9C2 cell line after aldosterone or corticosterone treatment. We demonstrate that chronic MR overexpression in the heart results in a limited number of induced (n = 24) and repressed (n = 22) genes compared with their control littermates. These genes are specifically modulated by MR because there is limited overlap (three induced, four repressed) with the genes that are regulated in the heart of GRcardio mice (compared with control mice: 70 induced, 73 repressed). Interestingly, some MR-induced genes that are up-regulated in vivo in mice are also induced by 24-h aldosterone treatment in H9C2 cells, such as plasminogen activator inhibitor 1 and Serpina-3 (α1-antichymotrypsin). The signaling pathways that are affected by long-term activation of MR may be of particular interest to design novel therapeutic targets in cardiac diseases. PMID:20591974

[Transcription of protein arginine N-methyltransferase genes in mouse dorsal root ganglia following peripheral nerve injury].

PubMed

Xu, Hua-Li; Xu, Shi-Yuan; Mo, Kai

2017-12-20

To investigate the changes in the transcription of protein arginine methylation enzyme family genes in the dorsal root ganglia (DRG) following peripheral nerve injury in mice. C57BL6 mouse models of neuropathic pain induced by peripheral nerve injury were established by bilateral L4 spinal nerve ligation (SNL). At 7 days after SNL or sham operation, the DRG tissue was collected for transcriptional analysis of 9 protein arginine methylation enzyme genes (Prmt1?3, Carm1, and Prmt5?9) using RNA?Seq to identify the differentially expressed genes in the injured DRGs. We also established mouse models of lateral L4 SNL and models of chronic constriction injury (CCI) of the sciatic nerve and tested the paw withdrawal frequency (PWF) in response to mechanical stimulation and paw withdrawal latency (PWL) in response to thermal stimulation on 0, 3, 7 and 14 days after SNL or CCI; the expressions of the differentially expressed genes in the injured DRGs were verified in the two models using RT?qPCR. Among the 9 protein arginine methylation enzyme family genes that were tissue?specifically expressed in the DRG, Prmt2 and Prmt3 showed the highest and Prmt6 showed the lowest basal expression. Compared with the sham?operated mice group, the mice receiving SNL exhibited upregulated Carm1 gene transcription (by 1.7 folds) but downregulated Prmt5, Prmt8 and Prmt9 transcription in the injured DRG (Prmt8 gene showed the most significant down?regulation by 16.3 folds). In mouse models of SNL and CCI, Carm1 gene expression increased progressively with time while Prmt8 transcription was obviously lowered on days 3, 7 and 14 after the injury; the transcription levels of Prmt1, Prmt5 and Prmt9 presented with no significant changes following the injuries. Both SNL and CCI induced mechanical allodynia and thermal hypersensitivities in the mice shown by increased PWF and decreased PWL on days 3, 7 and 14 after the injuries. Periphery nerve injury induces Carm1 upregulation and Prmt8

Proteome reference map and regulation network of neonatal rat cardiomyocyte

PubMed Central

Li, Zi-jian; Liu, Ning; Han, Qi-de; Zhang, You-yi

2011-01-01

Aim: To study and establish a proteome reference map and regulation network of neonatal rat cardiomyocyte. Methods: Cultured cardiomyocytes of neonatal rats were used. All proteins expressed in the cardiomyocytes were separated and identified by two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Biological networks and pathways of the neonatal rat cardiomyocytes were analyzed using the Ingenuity Pathway Analysis (IPA) program (www.ingenuity.com). A 2-DE database was made accessible on-line by Make2ddb package on a web server. Results: More than 1000 proteins were separated on 2D gels, and 148 proteins were identified. The identified proteins were used for the construction of an extensible markup language-based database. Biological networks and pathways were constructed to analyze the functions associate with cardiomyocyte proteins in the database. The 2-DE database of rat cardiomyocyte proteins can be accessed at http://2d.bjmu.edu.cn. Conclusion: A proteome reference map and regulation network of the neonatal rat cardiomyocytes have been established, which may serve as an international platform for storage, analysis and visualization of cardiomyocyte proteomic data. PMID:21841810

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

PubMed

Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

2014-11-01

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

Comparison of various iron chelators and prochelators as protective agents against cardiomyocyte oxidative injury.

PubMed

Jansová, Hana; Macháček, Miloslav; Wang, Qin; Hašková, Pavlína; Jirkovská, Anna; Potůčková, Eliška; Kielar, Filip; Franz, Katherine J; Simůnek, Tomáš

2014-09-01

Oxidative stress is a common denominator of numerous cardiovascular disorders. Free cellular iron catalyzes the formation of highly toxic hydroxyl radicals, and iron chelation may thus be an effective therapeutic approach. However, using classical iron chelators in diseases without iron overload poses risks that necessitate more advanced approaches, such as prochelators that are activated to chelate iron only under disease-specific oxidative stress conditions. In this study, three cell-membrane-permeable iron chelators (clinically used deferasirox and experimental SIH and HAPI) and five boronate-masked prochelator analogs were evaluated for their ability to protect cardiac cells against oxidative injury induced by hydrogen peroxide. Whereas the deferasirox-derived agents TIP and TRA-IMM displayed negligible protection and even considerable toxicity, the aroylhydrazone prochelators BHAPI and BSIH-PD provided significant cytoprotection and displayed lower toxicity after prolonged cellular exposure compared to their parent chelators HAPI and SIH, respectively. Overall, the most favorable properties in terms of protective efficiency and low inherent cytotoxicity were observed with the aroylhydrazone prochelator BSIH. BSIH efficiently protected both H9c2 rat cardiomyoblast-derived cells and isolated primary rat cardiomyocytes against hydrogen peroxide-induced mitochondrial and lysosomal dysregulation and cell death. At the same time, BSIH was nontoxic at concentrations up to its solubility limit (600 μM) and in 72-h incubation. Hence, BSIH merits further investigation for prevention and/or treatment of cardiovascular disorders associated with a known (or presumed) component of oxidative stress. Copyright © 2014 Elsevier Inc. All rights reserved.

Injurious effects of emodin on maturation of mouse oocytes, fertilization and fetal development via apoptosis.

PubMed

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-10-29

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20-40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process.

Early Postnatal Cardiomyocyte Proliferation Requires High Oxidative Energy Metabolism.

PubMed

de Carvalho, Ana Elisa Teófilo Saturi; Bassaneze, Vinícius; Forni, Maria Fernanda; Keusseyan, Aline Alfonso; Kowaltowski, Alicia Juliana; Krieger, José Eduardo

2017-11-13

Cardiac energy metabolism must cope with early postnatal changes in tissue oxygen tensions, hemodynamics, and cell proliferation to sustain development. Here, we tested the hypothesis that proliferating neonatal cardiomyocytes are dependent on high oxidative energy metabolism. We show that energy-related gene expression does not correlate with functional oxidative measurements in the developing heart. Gene expression analysis suggests a gradual overall upregulation of oxidative-related genes and pathways, whereas functional assessment in both cardiac tissue and cultured cardiomyocytes indicated that oxidative metabolism decreases between the first and seventh days after birth. Cardiomyocyte extracellular flux analysis indicated that the decrease in oxidative metabolism between the first and seventh days after birth was mostly related to lower rates of ATP-linked mitochondrial respiration, suggesting that overall energetic demands decrease during this period. In parallel, the proliferation rate was higher for early cardiomyocytes. Furthermore, in vitro nonlethal chemical inhibition of mitochondrial respiration reduced the proliferative capacity of early cardiomyocytes, indicating a high energy demand to sustain cardiomyocyte proliferation. Altogether, we provide evidence that early postnatal cardiomyocyte proliferative capacity correlates with high oxidative energy metabolism. The energy requirement decreases as the proliferation ceases in the following days, and both oxidative-dependent metabolism and anaerobic glycolysis subside.

Up-regulation of miR-26a promotes apoptosis of hypoxic rat neonatal cardiomyocytes by repressing GSK-3β protein expression.

PubMed

Suh, Jong Hui; Choi, Eunmi; Cha, Min-Ji; Song, Byeong-Wook; Ham, Onju; Lee, Se-Yeon; Yoon, Cheesoon; Lee, Chang-Yeon; Park, Jun-Hee; Lee, Sun Hee; Hwang, Ki-Chul

2012-06-29

Myocardial ischemia is the major cause of morbidity and mortality due to cardiovascular diseases. This disease is a severe stress condition that causes extensive biochemical changes which trigger cardiac cell death. Stress conditions such as deprivation of glucose and oxygen activate the endoplasmic reticulum in the cytoplasm of cells, including cardiomyocytes, to generate and propagate apoptotic signals in response to these conditions. microRNAs (miRNAs) are a class of small non-coding RNAs that mediate posttranscriptional gene silencing. The miRNAs play important roles in regulating cardiac physiological and pathological events such as hypertrophy, apoptosis, and heart failure. However, the roles of miRNAs in reactive oxygen species (ROS)-mediated injury on cardiomyocytes are uncertain. In this study, we identified at the apoptotic concentration of H(2)O(2), miR-26a expression was increased. To determine the potential roles of miR-26a in H(2)O(2)-mediated cardiac apoptosis, miR-26a expression was regulated by a miR-26a or an anti-miR-26a. Overexpression of miR-26a increased apoptosis as determined by upregulation of Annexin V/PI positive cell population, caspase-3 activity and expression of pro-apoptotic signal molecules, whereas inhibition of miR-26a reduced apoptosis. We identified GSK3B as a direct downstream target of miR-26a. Furthermore, miR-26a attenuated viability and increased caspase-3 activity in normal cardiomyocytes. This study demonstrates that miR-26a promotes ROS-induced apoptosis in cardiomyocytes. Thus, miR-26a affects ROS-mediated gene regulation and cellular injury response. Copyright © 2012 Elsevier Inc. All rights reserved.

Sertad1 encodes a novel transcriptional co-activator of SMAD1 in mouse embryonic hearts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Yin; Zhao, Shaomin; School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069

2013-11-29

Highlights: •SERTAD1 interacts with SMAD1. •Sertad1 is expressed in mouse embryonic hearts. •SERTAD1 is localized in both cytoplasm and nucleus of cardiomyocytes. •SERTAD1 enhances expression of BMP target cardiogenic genes as a SMAD1 co-activator. -- Abstract: Despite considerable advances in surgical repairing procedures, congenital heart diseases (CHDs) remain the leading noninfectious cause of infant morbidity and mortality. Understanding the molecular/genetic mechanisms underlying normal cardiogenesis will provide essential information for the development of novel diagnostic and therapeutic strategies against CHDs. BMP signaling plays complex roles in multiple cardiogenic processes in mammals. SMAD1 is a canonical nuclear mediator of BMP signaling, themore » activity of which is critically regulated through its interaction partners. We screened a mouse embryonic heart yeast two-hybrid library using Smad1 as bait and identified SERTAD1 as a novel interaction partner of SMAD1. SERTAD1 contains multiple potential functional domains, including two partially overlapping transactivation domains at the C terminus. The SERTAD1-SMAD1 interaction in vitro and in mammalian cells was further confirmed through biochemical assays. The expression of Sertad1 in developing hearts was demonstrated using RT-PCR, western blotting and in situ hybridization analyses. We also showed that SERTAD1 was localized in both the cytoplasm and nucleus of immortalized cardiomyocytes and primary embryonic cardiomyocyte cultures. The overexpression of SERTAD1 in cardiomyocytes not only enhanced the activity of two BMP reporters in a dose-dependent manner but also increased the expression of several known BMP/SMAD regulatory targets. Therefore, these data suggest that SERTAD1 acts as a SMAD1 transcriptional co-activator to promote the expression of BMP target genes during mouse cardiogenesis.« less

A state of reversible compensated ventricular dysfunction precedes pathological remodelling in response to cardiomyocyte-specific activity of angiotensin II type-1 receptor in mice.

PubMed

Frentzou, Georgia A; Drinkhill, Mark J; Turner, Neil A; Ball, Stephen G; Ainscough, Justin F X

2015-08-01

Cardiac dysfunction is commonly associated with high-blood-pressure-induced cardiomyocyte hypertrophy, in response to aberrant renin-angiotensin system (RAS) activity. Ensuing pathological remodelling promotes cardiomyocyte death and cardiac fibroblast activation, leading to cardiac fibrosis. The initiating cellular mechanisms that underlie this progressive disease are poorly understood. We previously reported a conditional mouse model in which a human angiotensin II type-I receptor transgene (HART) was expressed in differentiated cardiomyocytes after they had fully matured, but not during development. Twelve-month-old HART mice exhibited ventricular dysfunction and cardiomyocyte hypertrophy with interstitial fibrosis following full receptor stimulation, without affecting blood pressure. Here, we show that chronic HART activity in young adult mice causes ventricular dysfunction without hypertrophy, fibrosis or cardiomyocyte death. Dysfunction correlated with reduced expression of pro-hypertrophy markers and increased expression of pro-angiogenic markers in the cardiomyocytes experiencing increased receptor load. This stimulates responsive changes in closely associated non-myocyte cells, including the downregulation of pro-angiogenic genes, a dampened inflammatory response and upregulation of Tgfβ. Importantly, this state of compensated dysfunction was reversible. Furthermore, increased stimulation of the receptors on the cardiomyocytes caused a switch in the secondary response from the non-myocyte cells. Progressive cardiac remodelling was stimulated through hypertrophy and death of individual cardiomyocytes, with infiltration, proliferation and activation of fibroblast and inflammatory cells, leading to increased angiogenic and inflammatory signalling. Together, these data demonstrate that a state of pre-hypertrophic compensated dysfunction can exist in affected individuals before common markers of heart disease are detectable. The data also suggest that there is

Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Aibin; Liu, Jingyi; Institute of Cardiovascular Disease, General Hospital of Beijing Command, PLA, Beijing

Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, themore » roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H{sub 2}O{sub 2} led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H{sub 2}O{sub 2} and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac

Insulin-mediated inhibition of p38 mitogen-activated protein kinase protects cardiomyocytes in severe burns.

PubMed

Lv, Gen-fa; Dong, Mao-long; Hu, Da-hai; Zhang, Wan-fu; Wang, Yun-chuan; Tang, Chao-wu; Zhu, Xiong-xiang

2011-01-01

Thermal injury inhibits Akt activation and upregulates p38 mitogen-activated protein kinase, which in turn induces inflammation and increases apoptosis. This study aimed to elucidate the mechanism underlying the cytoprotective role of insulin in severe burns by examining the effects of insulin on inflammation and apoptosis mediated by p38 mitogen-activated protein kinase in burn serum-challenged cardiomyocytes. Neonatal rat cardiomyocytes were exposed to burn serum for 6 hours in the presence or absence of insulin and pretreated with inhibitors to p38 mitogen-activated protein kinase (SB203580) and Akt (LY294002). The authors examined expression of myocardial tumor necrosis factor-alpha, cardiac myofilament proteins caspase-3 and Bcl2, and apoptosis. Burn serum-induced upregulation of tumor necrosis factor was inhibited by both SB203580 and insulin. LY294002 reversed insulin-mediated downregulation of tumor necrosis factor. Both SB203580 and insulin inhibited apoptosis, resulting in fewer pyknotic nuclei and inhibition of caspase-3 activation and Bcl2 downregulation. LY294002 reversed insulin-mediated inhibition of apoptosis. Insulin decreases inflammatory cytokine expression and apoptosis via PI3K/Akt-mediated inhibition of p38 mitogen-activated protein kinase. The cytoprotective role of insulin suggests that it may have a potential role in strategies for treating thermal injuries.

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles

PubMed Central

Xu, Bing; Fu, Ying; Liu, Yan; Agvanian, Sosse; Wirka, Robert C.; Baum, Rachel; Zhou, Kang; Shaw, Robin M.

2017-01-01

Microparticles (MPs) are cell–cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

PubMed

Xu, Bing; Fu, Ying; Liu, Yan; Agvanian, Sosse; Wirka, Robert C; Baum, Rachel; Zhou, Kang; Shaw, Robin M; Hong, TingTing

2017-08-01

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT

Cardiac Arrhythmia and Injury Induced in Rats by Burst and Pulsed Mode Ultrasound with Gas Body Contrast Agent

PubMed Central

Miller, Douglas L.; Dou, Chunyan; Lucchesi, Benedict R.

2009-01-01

Objective Premature complexes (PCs) in the electrocardiogram (ECG) signal have been reported for myocardial contrast echocardiography and also for burst mode (physical therapy) ultrasound with gas body contrast agent at lower peak rarefactional pressure amplitudes (PRPAs). For contrast echocardiography, irreversibly injured cardiomyocytes have been associated with the arrhythmia. The objective was to determine if cardiomyocyte injury is associated with the PCs induced by the burst mode at lower PRPAs. Methods Anesthetized rats were exposed to focused 1.5 MHz ultrasound in a water bath. Evans blue dye was injected IP to stain injured cardiomyocytes and Definity ultrasound contrast agent was infused IV. Continuous burst mode simulated physical therapy ultrasound. Intermittent 2 ms bursts, or envelopes of pulses simulating diagnostic ultrasound, were triggered 1:4 at end systole. PCs were observed on ECG recordings and stained cardiomyocytes were counted in frozen sections. Results The continuous burst mode produced variable PCs and stained cells above 0.3 MPa PRPA. The triggered bursts above 0.3 MPa and pulse envelopes above 1.2 MPa produced statistically significant (P<0.01) PCs and stained cardiomyocytes. Conclusion Irreversible cardiomyocyte injury was associated with the development of PCs for burst mode and occurred at substantially lower PRPAs than for pulsed ultrasound. PMID:19854967

Evodiamine Inhibits Angiotensin II-Induced Rat Cardiomyocyte Hypertrophy.

PubMed

He, Na; Gong, Qi-Hai; Zhang, Feng; Zhang, Jing-Yi; Lin, Shu-Xian; Hou, Hua-Hua; Wu, Qin; Sun, An-Sheng

2018-05-01

To investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms. Cardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca 2+ ] i ) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis. Compared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca 2+ ] i ) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca 2+ ] i concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05). Evo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca 2+ ]i concentration, and inhibition of Ca

Endothelial expression of human cytochrome P450 epoxygenase CYP2C8 increases susceptibility to ischemia-reperfusion injury in isolated mouse heart

PubMed Central

Edin, Matthew L.; Wang, ZhongJing; Bradbury, J. Alyce; Graves, Joan P.; Lih, Fred B.; DeGraff, Laura M.; Foley, Julie F.; Torphy, Robert; Ronnekleiv, Oline K.; Tomer, Kenneth B.; Lee, Craig R.; Zeldin, Darryl C.

2011-01-01

Cytochrome P450 (CYP) epoxygenases CYP2C8 and CYP2J2 generate epoxyeicosatrienoic acids (EETs) from arachidonic acid. Mice with expression of CYP2J2 in cardiomyocytes (αMHC-CYP2J2 Tr) or treated with synthetic EETs have increased functional recovery after ischemia/reperfusion (I/R); however, no studies have examined the role of cardiomyocyte- vs. endothelial-derived EETs or compared the effects of different CYP epoxygenase isoforms in the ischemic heart. We generated transgenic mice with increased endothelial EET biosynthesis (Tie2-CYP2C8 Tr and Tie2-CYP2J2 Tr) or EET hydrolysis (Tie2-sEH Tr). Compared to wild-type (WT), αMHC-CYP2J2 Tr hearts showed increased recovery of left ventricular developed pressure (LVDP) and decreased infarct size after I/R. In contrast, LVDP recovery and infarct size were unchanged in Tie2-CYP2J2 Tr and Tie2-sEH Tr hearts. Surprisingly, compared to WT, Tie2-CYP2C8 Tr hearts had significantly reduced LVDP recovery (from 21 to 14%) and increased infarct size after I/R (from 51 to 61%). Tie2-CYP2C8 Tr hearts also exhibited increased reactive oxygen species (ROS) generation, dihydroxyoctadecenoic acid (DiHOME) formation, and coronary resistance after I/R. ROS scavengers and CYP2C8 inhibition reversed the detrimental effects of CYP2C8 expression in Tie2-CYP2C8 Tr hearts. Treatment of WT hearts with 250 nM 9,10-DiHOME decreased LVDP recovery compared to vehicle (16 vs. 31%, respectively) and increased coronary resistance after I/R. These data demonstrate that increased ROS generation and enhanced DiHOME synthesis by endothelial CYP2C8 impair functional recovery and mask the beneficial effects of increased EET production following I/R.—Edin, M. L., Wang, Z. J., Bradbury, J. A., Graves, J. P., Lih, F. B., DeGraff, L. M., Foley, J. F., Torphy, R., Ronnekleiv, O. K., Tomer, K. B., Lee, C. R., Zeldin, D. C. Endothelial expression of human cytochrome P450 epoxygenase CYP2C8 increases susceptibility to ischemia-reperfusion injury in isolated mouse

Biomechanical Characterization of Cardiomyocyte Using PDMS Pillar with Microgrooves

PubMed Central

Oyunbaatar, Nomin-Erdene; Lee, Deok-Hyu; Patil, Swati J.; Kim, Eung-Sam; Lee, Dong-Weon

2016-01-01

This paper describes the surface-patterned polydimethylsiloxane (PDMS) pillar arrays for enhancing cell alignment and contraction force in cardiomyocytes. The PDMS micropillar (μpillar) arrays with microgrooves (μgrooves) were fabricated using a unique micro-mold made using SU-8 double layer processes. The spring constant of the μpillar arrays was experimentally confirmed using atomic force microscopy (AFM). After culturing cardiac cells on the two different types of μpillar arrays, with and without grooves on the top of μpillar, the characteristics of the cardiomyocytes were analyzed using a custom-made image analysis system. The alignment of the cardiomyocytes on the μgrooves of the μpillars was clearly observed using a DAPI staining process. The mechanical force generated by the contraction force of the cardiomyocytes was derived from the displacement of the μpillar arrays. The contraction force of the cardiomyocytes aligned on the μgrooves was 20% higher than that of the μpillar arrays without μgrooves. The experimental results prove that applied geometrical stimulus is an effective method for aligning and improving the contraction force of cardiomyocytes. PMID:27517924

A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia–reperfusion injury

PubMed Central

Fiordaliso, Fabio; Chimenti, Stefano; Staszewsky, Lidia; Bai, Antonio; Carlo, Eleonora; Cuccovillo, Ivan; Doni, Mirko; Mengozzi, Manuela; Tonelli, Rossella; Ghezzi, Pietro; Coleman, Thomas; Brines, Michael; Cerami, Anthony; Latini, Roberto

2005-01-01

The cytokine erythropoietin (EPO) protects the heart from ischemic injury, in part by preventing apoptosis. However, EPO administration can also raise the hemoglobin concentration, which, by increasing oxygen delivery, confounds assignment of cause and effect. The availability of EPO analogs that do not bind to the dimeric EPO receptor and lack erythropoietic activity, e.g., carbamylated EPO (CEPO), provides an opportunity to determine whether EPO possesses direct cardioprotective activity. In vivo, cardiomyocyte loss after experimental myocardial infarction (MI) of rats (40 min of occlusion with reperfusion) was reduced from ≈57% in MI-control to ≈45% in animals that were administered CEPO daily for 1 week (50 μg/kg of body weight s.c.) with the first dose administered intravenously 5 min before reperfusion. CEPO did not increase the hematocrit, yet it prevented increases in left ventricular (LV) end-diastolic pressure, reduced LV wall stress in systole and diastole, and improved LV response to dobutamine infusion compared with vehicle-treated animals. In agreement with the cardioprotective effect observed in vivo, staurosporine-induced apoptosis of adult rat or mouse cardiomyocytes in vitro was also significantly attenuated (≈35%) by CEPO, which is comparable with the effect of EPO. These data indicate that prevention of cardiomyocyte apoptosis, in the absence of an increase in hemoglobin concentration, explains EPO's cardioprotection. Nonerythropoietic derivatives such as CEPO, devoid of the undesirable effects of EPO, e.g., thrombogenesis, could represent safer and more effective alternatives for treatment of cardiovascular diseases, such as MI and heart failure. Furthermore, these findings expand the activity spectrum of CEPO to tissues outside the nervous system. PMID:15671158

Injurious Effects of Emodin on Maturation of Mouse Oocytes, Fertilization and Fetal Development via Apoptosis

PubMed Central

Chang, Mei-Hui; Chang, Shao-Chung; Chan, Wen-Hsiung

2012-01-01

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Previous studies have established that emodin induces apoptosis in the inner cell mass and trophectoderm of mouse blastocysts and leads to decreased embryonic development and viability, indicating a role as an injury risk factor for normal embryonic development. However, the mechanisms underlying its hazardous effects have yet to be characterized. In the current study, we further investigated the effects of emodin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, emodin induced a significant reduction in the rates of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with emodin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments using an in vivo mouse model disclosed that consumption of drinking water containing 20–40 μM emodin led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Notably, pretreatment with a caspase-3-specific inhibitor effectively prevented emodin-triggered injury effects, suggesting that impairment of embryo development occurs via a caspase-dependent apoptotic process. PMID:23203041

Full Tensor Diffusion Imaging Is Not Required To Assess the White-Matter Integrity in Mouse Contusion Spinal Cord Injury

PubMed Central

Tu, Tsang-Wei; Kim, Joong H.; Wang, Jian

2010-01-01

Abstract In vivo diffusion tensor imaging (DTI) derived indices have been demonstrated to quantify accurately white-matter injury after contusion spinal cord injury (SCI) in rodents. In general, a full diffusion tensor analysis requires the acquisition of diffusion-weighted images (DWI) along at least six independent directions of diffusion-sensitizing gradients. Thus, DTI measurements of the rodent central nervous system are time consuming. In this study, diffusion indices derived using the two-direction DWI (parallel and perpendicular to axonal tracts) were compared with those obtained using six-direction DTI in a mouse model of SCI. It was hypothesized that the mouse spinal cord ventral-lateral white-matter (VLWM) tracts, T8–T10 in this study, aligned with the main magnet axis (z) allowing the apparent diffusion coefficient parallel and perpendicular to the axis of the spine to be derived with diffusion-weighting gradients in the z and y axes of the magnet coordinate respectively. Compared with six-direction full tensor DTI, two-direction DWI provided comparable diffusion indices in mouse spinal cords. The measured extent of spared white matter after injury, estimated by anisotropy indices, using both six-direction DTI and two-direction DWI were in close agreement and correlated well with histological staining and behavioral assessment. The results suggest that the two-direction DWI derived indices may be used, with significantly reduced imaging time, to estimate accurately spared white matter in mouse SCI. PMID:19715399

Ilexsaponin A attenuates ischemia-reperfusion-induced myocardial injury through anti-apoptotic pathway.

PubMed

Zhang, Shuang-Wei; Liu, Yu; Wang, Fang; Qiang, Jiao; Liu, Pan; Zhang, Jun; Xu, Jin-Wen

2017-01-01

The protective effects of ilexsaponin A on ischemia-reperfusion-induced myocardial injury were investigated. Myocardial ischemia/reperfusion model was established in male Sprague-Dawley rats. Myocardial injury was evaluated by TTC staining and myocardial marker enzyme leakage. The in vitro protective potential of Ilexsaponin A was assessed on hypoxia/reoxygenation cellular model in neonatal rat cardiomyocytes. Cellular viability and apoptosis were evaluated by MTT and TUNEL assay. Caspase-3, cleaved caspase-3, bax, bcl-2, p-Akt and Akt protein expression levels were detected by western-blot. Ilexsaponin A treatment was able to attenuate the myocardial injury in ischemia/reperfusion model by reducing myocardial infarct size and lower the serum levels of LDH, AST and CK-MB. The in vitro study also showed that ilexsaponin A treatment could increase cellular viability and inhibit apoptosis in hypoxia/reoxygenation cardiomyocytes. Proapoptotic proteins including caspase-3, cleaved caspase-3 and bax were significantly reduced and anti-apoptotic protein bcl-2 was significantly increased by ilexsaponin A treatment in hypoxia/reoxygenation cardiomyocytes. Moreover, Ilexsaponin A treatment was able to increase the expression levels of p-Akt in hypoxia/reoxygenation cellular model and myocardial ischemia/reperfusion animal model. Coupled results from both in vivo and in vitro experiments indicate that Ilexsaponin A attenuates ischemia-reperfusion-induced myocardial injury through anti-apoptotic pathway.

Attenuation of ischemia-reperfusion-induced alterations in intracellular Ca2+ in cardiomyocytes from hearts treated with N-acetylcysteine and N-mercaptopropionylglycine.

PubMed

Saini-Chohan, Harjot K; Dhalla, Naranjan S

2009-12-01

This study was undertaken to test whether Ca(2+)-handling abnormalities in cardiomyocytes after ischemia-reperfusion (I/R) are prevented by antioxidants such as N-acetyl L-cysteine (NAC), which is known to reduce oxidative stress by increasing the glutathione redox status, and N-(2-mercaptopropionyl)-glycine (MPG), which scavenges both peroxynitrite and hydroxyl radicals. For this purpose, isolated rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion, and cardiomyocytes were prepared to monitor changes in the intracellular concentration of free Ca(2+) ([Ca(2+)](i)). Marked depression in the left ventricular developed pressure and elevation in the left ventricular end-diastolic pressure in I/R hearts were attenuated by treatment with NAC or MPG. Cardiomyocytes obtained from I/R hearts showed an increase in the basal level of [Ca(2+)](i) as well as augmentation of the low Na(+)-induced increase in [Ca(2+)](i), with no change in the KCl-induced increase in [Ca(2+)](i). These I/R-induced alterations in Ca(2+) handling by cardiomyocytes were attenuated by treatment of hearts with NAC or MPG. Furthermore, reduction in the isoproterenol-, ATP-, ouabain-, and caffeine-induced increases in [Ca(2+)](i) in cardiomyocytes from I/R hearts were limited by treatment with NAC or MPG. The increases in the basal [Ca(2+)](i), unlike the KCl-induced increase in [Ca(2+)](i), were fully or partially prevented by both NAC and MPG upon exposing cardiomyocytes to hypoxia-reoxygenation, H(2)O(2), or a mixture of xanthine and xanthine oxidase. These results suggest that improvement in cardiac function of I/R hearts treated with NAC or MPG was associated with attenuation of changes in Ca(2+) handling by cardiomyocytes, and the results support the view that oxidative stress due to oxyradical generation and peroxynitrite formation plays an important role in the development of intracellular Ca(2+) overload in cardiomyocytes as a consequence of I/R injury.

Protease Activated Receptor-2 Contributes to Heart Failure

PubMed Central

Antoniak, Silvio; Sparkenbaugh, Erica M.; Tencati, Michael; Rojas, Mauricio; Mackman, Nigel; Pawlinski, Rafal

2013-01-01

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure. PMID:24312345

Vitamin E confers cytoprotective effects on cardiomyocytes under conditions of heat stress by increasing the expression of metallothionein.

PubMed

Wang, Xiaowu; Dong, Wenpeng; Yuan, Binbin; Yang, Yongchao; Yang, Dongpeng; Lin, Xi; Chen, Changfu; Zhang, Weida

2016-05-01

Heat stress (HS) is commonly used to refer to the heat load that an individual is subjected to due to either metabolic heat, or environmental factors, including high temperatures and high humidity levels. HS has been reported to affect and even damage the functioning of various organs; overexposure to high temperatures and high humidity may lead to accidental deaths. It has been suggested that the cardiovascular system is primarily targeted by exposure to HS conditions; the HS-induced dysfunction of cardiomyocytes, which is characterized by mitochondrial dysfunction, may result in the development of cardiovascular diseases. The excessive production of reactive oxygen species (ROS) also participates in mitochondrial dysfunction. However, effective methods for the prevention and treatment of mitochondrial and cardiovascular dysfunction induced by exposure to HS are lacking. In the present study, we hypothesized that vitamin E (VE), an antioxidant, is capable of preventing oxidative stress and mitochondrial injury in cardiomyocytes induced by exposure to HS. The results revealed that pre‑treatment with VE increased the expression of metallothionein (MT), which has previously been reported to confer cytoprotective effects, particularly on the cardiovascular system. Pre-treatment with VE restored mitochondrial function in cardiomyocytes under conditions of HS by increasing the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), and by increasing adenosine triphosphate (ATP) levels. Furthermore, pre-treatment with VE decreased the production of ROS, which was induced by exposure to HS and thus exerted antioxidant effects. In addition, pre-treatment with VE attenuated oxidative stress induced by exposure to HS, as demonstrated by the increased levels of antioxidant enzymes [superoxide dismutase (SOD) and glutathione (GSH)], and by the decreased levels

The Effect of Substrate Stiffness on Cardiomyocyte Action Potentials.

PubMed

Boothe, Sean D; Myers, Jackson D; Pok, Seokwon; Sun, Junping; Xi, Yutao; Nieto, Raymond M; Cheng, Jie; Jacot, Jeffrey G

2016-12-01

The stiffness of myocardial tissue changes significantly at birth and during neonatal development, concurrent with significant changes in contractile and electrical maturation of cardiomyocytes. Previous studies by our group have shown that cardiomyocytes generate maximum contractile force when cultured on a substrate with a stiffness approximating native cardiac tissue. However, effects of substrate stiffness on the electrophysiology and ion currents in cardiomyocytes have not been fully characterized. In this study, neonatal rat ventricular myocytes were cultured on the surface of flat polyacrylamide hydrogels with elastic moduli ranging from 1 to 25 kPa. Using whole-cell patch clamping, action potentials and L-type calcium currents were recorded. Cardiomyocytes cultured on hydrogels with a 9 kPa elastic modulus, similar to that of native myocardium, had the longest action potential duration. Additionally, the voltage at maximum calcium flux significantly decreased in cardiomyocytes on hydrogels with an elastic modulus higher than 9 kPa, and the mean inactivation voltage decreased with increasing stiffness. Interestingly, the expression of the L-type calcium channel subunit α gene and channel localization did not change with stiffness. Substrate stiffness significantly affects action potential length and calcium flux in cultured neonatal rat cardiomyocytes in a manner that may be unrelated to calcium channel expression. These results may explain functional differences in cardiomyocytes resulting from changes in the elastic modulus of the extracellular matrix, as observed during embryonic development, in ischemic regions of the heart after myocardial infarction, and during dilated cardiomyopathy.

Propofol attenuates H2O2-induced oxidative stress and apoptosis via the mitochondria- and ER-medicated pathways in neonatal rat cardiomyocytes.

PubMed

Liu, Xue-Ru; Cao, Lu; Li, Tao; Chen, Lin-Lin; Yu, Yi-Yan; Huang, Wen-Jun; Liu, Li; Tan, Xiao-Qiu

2017-05-01

Previous studies have shown that propofol, an intravenous anesthetic commonly used in clinical practice, protects the myocardium from injury. Mitochondria- and endoplasmic reticulum (ER)-mediated oxidative stress and apoptosis are two important signaling pathways involved in myocardial injury and protection. The present study aimed to test the hypothesis that propofol could exert a cardio-protective effect via the above two pathways. Cultured neonatal rat cardiomyocytes were treated with culture medium (control group), H 2 O 2 at 500 μM (H 2 O 2 group), propofol at 50 μM (propofol group), and H 2 O 2 plus propofol (H 2 O 2  + propofol group), respectively. The oxidative stress, mitochondrial membrane potential (ΔΨm) and apoptosis of the cardiomyocytes were evaluated by a series of assays including ELISA, flow cytometry, immunofluorescence microscopy and Western blotting. Propofol significantly suppressed the H 2 O 2 -induced elevations in the activities of caspases 3, 8, 9 and 12, the ratio of Bax/Bcl-2, and cell apoptosis. Propofol also inhibited the H 2 O 2 -induced reactive oxygen species (ROS) generation, lactic dehydrogenase (LDH) release and mitochondrial transmembrane potential (ΔΨm) depolarization, and restored the H 2 O 2 -induced reductions of glutathione (GSH) and superoxide dismutase (SOD). In addition, propofol decreased the expressions of glucose-regulated protein 78 kDa (Grp78) and inositol-requiring enzyme 1α (IRE1α), two important signaling molecules in the ER-mediated apoptosis pathway. Propofol protects cardiomyocytes from H 2 O 2 -induced injury by inhibiting the mitochondria- and ER-mediated apoptosis signaling pathways.

Silencing of long noncoding RNA AK139328 attenuates ischemia/reperfusion injury in mouse livers.

PubMed

Chen, Zhenzhen; Jia, Shi; Li, Danhua; Cai, Junyan; Tu, Jian; Geng, Bin; Guan, Youfei; Cui, Qinghua; Yang, Jichun

2013-01-01

Recently, increasing evidences had suggested that long noncoding RNAs (LncRNAs) are involved in a wide range of physiological and pathophysiological processes. Here we determined the LncRNA expression profile using microarray technology in mouse livers after ischemia/reperfusion treatment. Seventy one LncRNAs were upregulated, and 27 LncRNAs were downregulated in ischemia/reperfusion-treated mouse livers. Eleven of the most significantly deregulated LncRNAs were further validated by quantitative PCR assays. Among the upregulated LncRNAs confirmed by quantitative PCR assays, AK139328 exhibited the highest expression level in normal mouse livers. siRNA-mediated knockdown of hepatic AK139328 decreased plasma aminotransferase activities, and reduced necrosis area in the livers with a decrease in caspase-3 activation after ischemia/reperfusion treatment. In ischemia/reperfusion liver, knockdown of AK139328 increased survival signaling proteins including phosphorylated Akt (pAkt), glycogen synthase kinase 3 (pGSK3) and endothelial nitric oxide synthase (peNOS). Furthermore, knockdown of AK139328 also reduced macrophage infitration and inhibited NF-κB activity and inflammatory cytokines expression. In conclusion, these findings revealed that deregulated LncRNAs are involved in liver ischemia/reperfusion injury. Silencing of AK139328 ameliorated ischemia/reperfusion injury in the liver with the activation of Akt signaling pathway and inhibition of NF-κB activity. LncRNA AK139328 might be a novel target for diagnosis and treatment of liver surgery or transplantation.

Ivabradine improved left ventricular function and pressure overload-induced cardiomyocyte apoptosis in a transverse aortic constriction mouse model.

PubMed

Yu, Yihui; Hu, Zuoying; Li, Bing; Wang, Zhimei; Chen, Shaoliang

2018-05-22

This study aimed to investigate the effects and molecular mechanisms of ivabradine in preventing cardiac hypertrophy in an established transverse aortic constriction (TAC) mouse model. A total of 56 male C57BL/6 mice were randomly assigned into the following seven groups (8 mice per group): sham, TAC model, Iva-10 (10 mg/kg/day ivabradine), Iva-20 (20 mg/kg/day ivabradine), Iva-40 (40 mg/kg/day ivabradine), Iva-80 (80 mg/kg/day ivabradine), and Rap (rapamycin, a positive control). Echocardiography and left ventricular hemodynamics were performed. Hematoxylin-eosin (H&E), Masson's trichome staining, and TUNEL assays were conducted to evaluate cardiac hypertrophy, fibrosis, and apoptosis, respectively. Western blotting was performed to detect the expression of proteins related to the PI3K/Akt/mTOR/p70S6K pathway. Ivabradine could effectively improve left ventricular dysfunction and hypertrophy induced by TAC in a dose-independent manner. Moreover, no obvious change in heart rate (HR) was observed in the TAC and Rap groups, whereas a significant decrease in HR was found after ivabradine treatment (P < 0.05). Cardiac hypertrophy, fibrosis, and apoptosis induced by TAC were notably suppressed after either rapamycin or ivabradine treatment (P < 0.05). Ivabradine and rapamycin also decreased the expression of PI3K/Akt and mTOR induced by TAC. Ivabradine improved cardiac hypertrophy and fibrosis as well as reduced cardiomyocyte apoptosis via the PI3K/Akt/mTOR/p70S6K pathway in TAC model mice.

Vagal modulation of high mobility group box-1 protein mediates electroacupuncture-induced cardioprotection in ischemia-reperfusion injury.

PubMed

Zhang, Juan; Yong, Yue; Li, Xing; Hu, Yu; Wang, Jian; Wang, Yong-qiang; Song, Wei; Chen, Wen-ting; Xie, Jian; Chen, Xue-mei; Lv, Xin; Hou, Li-li; Wang, Ke; Zhou, Jia; Wang, Xiang-rui; Song, Jian-gang

2015-10-26

Excessive release of high mobility group box-1 (HMGB1) protein from ischemic cardiomyocytes activates inflammatory cascades and enhances myocardial injury after reperfusion. Here we report evidence that electroacupuncture of mice at Neiguan acupoints can inhibit the up-regulation of cardiac HMGB1 following myocardial ischemia and attenuate the associated inflammatory responses and myocardial injury during reperfusion. These benefits of electroacupuncture were partially reversed by administering recombinant HMGB1 to the mice, and further potentiated by administering anti-HMGB1 antibody. Electroacupuncture-induced inhibition of HMGB1 release was markedly reduced by unilateral vagotomy or administration of nicotinic receptor antagonist, but not by chemical sympathectomy. The cholinesterase inhibitor neostigmine mimicked the effects of electroacupuncture on HMGB1 release and myocardial ischemia reperfusion injury. Culture experiments with isolated neonatal cardiomyocytes showed that acetylcholine, but not noradrenaline, inhibited hypoxia-induced release of HMGB1 via a α7nAchR-dependent pathway. These results suggest that electroacupuncture acts via the vagal nerve and its nicotinic receptor-mediated signaling to inhibit HMGB1 release from ischemic cardiomyocytes. This helps attenuate pro-inflammatory responses and myocardial injury during reperfusion.

Vagal modulation of high mobility group box-1 protein mediates electroacupuncture-induced cardioprotection in ischemia-reperfusion injury

PubMed Central

Zhang, Juan; Yong, Yue; Li, Xing; Hu, Yu; Wang, Jian; Wang, Yong-qiang; Song, Wei; Chen, Wen-ting; Xie, Jian; Chen, Xue-mei; Lv, Xin; Hou, Li-li; Wang, Ke; Zhou, Jia; Wang, Xiang-rui; Song, Jian-gang

2015-01-01

Excessive release of high mobility group box-1 (HMGB1) protein from ischemic cardiomyocytes activates inflammatory cascades and enhances myocardial injury after reperfusion. Here we report evidence that electroacupuncture of mice at Neiguan acupoints can inhibit the up-regulation of cardiac HMGB1 following myocardial ischemia and attenuate the associated inflammatory responses and myocardial injury during reperfusion. These benefits of electroacupuncture were partially reversed by administering recombinant HMGB1 to the mice, and further potentiated by administering anti-HMGB1 antibody. Electroacupuncture-induced inhibition of HMGB1 release was markedly reduced by unilateral vagotomy or administration of nicotinic receptor antagonist, but not by chemical sympathectomy. The cholinesterase inhibitor neostigmine mimicked the effects of electroacupuncture on HMGB1 release and myocardial ischemia reperfusion injury. Culture experiments with isolated neonatal cardiomyocytes showed that acetylcholine, but not noradrenaline, inhibited hypoxia-induced release of HMGB1 via a α7nAchR-dependent pathway. These results suggest that electroacupuncture acts via the vagal nerve and its nicotinic receptor-mediated signaling to inhibit HMGB1 release from ischemic cardiomyocytes. This helps attenuate pro-inflammatory responses and myocardial injury during reperfusion. PMID:26499847

Choline Inhibits Ischemia-Reperfusion-Induced Cardiomyocyte Autophagy in Rat Myocardium by Activating Akt/mTOR Signaling.

PubMed

Hang, Pengzhou; Zhao, Jing; Su, Zhenli; Sun, Hanqi; Chen, Tingting; Zhao, Lihui; Du, Zhimin

2018-01-01

Backgroud/Aims: Growing evidence suggests that both cardiomyocyte apoptosis and excessive autophagy exacerbates cardiac dysfunction during myocardial ischemia-reperfusion (IR). As a precursor of acetylcholine, choline has been found to protect the heart by repressing ischemic cardiomyocyte apoptosis. However, the relationship between choline and cardiomyocyte autophagy is unclear. The present study aimed to investigate whether autophagy was involved in the cardioprotection of choline during IR. Rats were subjected to 30 min reversible ischemia by ligation of left anterior descending coronary artery followed by reperfusion for 2 h. Choline (5 mg/kg, i.v.) alone or along with rapamycin (5 mg/ kg, i.p.) were injected 30 min before ischemia. Transmission electron microscopy, hematoxylin and eosin (HE) and TUNEL staining were conducted to evaluate the effect of choline on cardiac apoptosis and autophagy. Protein levels of autophagic markers including LC3, beclin-1 and p62 as well as Akt and mammalian target of rapamycin (mTOR) were examined by Western blotting. Myocardial IR-induced cardiac apoptosis and accumulation of autophagosomes was attenuated by choline. Choline treatment significantly ameliorated myocardial IR-induced autophagic activity characterized by repression of beclin-1 over-activation, the reduction of autophagosomes, the LC3-II/LC3-I ratio, and p62 protein abundance. In addition, IR-induced downregulation of p-Akt/mTOR cascade was increased by choline. However, the above functions of choline were abolished by rapamycin. These findings suggest that choline plays a protective role against myocardial IR injury by inhibiting excessive autophagy, which might be associated with the activation of Akt/mTOR pathway. This study provides new mechanistic understanding of cardioprotective effect of choline and suggests novel potential therapeutic targets for cardiac IR injury. © 2018 The Author(s). Published by S. Karger AG, Basel.

Neonatal cardiomyocyte hypertrophy induced by endothelin-1 is blocked by estradiol acting on GPER.

PubMed

Goncalves, Gleisy Kelly; Scalzo, Sergio; Alves, Ana Paula; Agero, Ubirajara; Guatimosim, Silvia; Reis, Adelina M

2018-03-01

Estradiol (E 2 ) prevents cardiac hypertrophy, and these protective actions are mediated by estrogen receptor (ER)α and ERβ. The G protein-coupled estrogen receptor (GPER) mediates many estrogenic effects, and its activation in the heart has been observed in ischemia and reperfusion injury or hypertension models; however, the underlying mechanisms need to be fully elucidated. Herein, we investigated whether the protective effect of E 2 against cardiomyocyte hypertrophy induced by endothelin-1 (ET-1) is mediated by GPER and the signaling pathways involved. Isolated neonatal female rat cardiomyocytes were treated with ET-1 (100 nmol/l) for 48 h in the presence or absence of E 2 (10 nmol/l) or GPER agonist G-1 (10 nmol/l) and GPER antagonist G-15 (10 nmol/l). ET-1 increased the surface area of cardiomyocytes, and this was associated with increased expression of atrial and brain natriuretic peptides. Additionally, ET-1 increased the phosphorylation of extracellular signal-related protein kinases-1/2 (ERK1/2). Notably, E 2 or G-1 abolished the hypertrophic actions of ET-1, and that was reversed by G-15. Likewise, E 2 reversed the ET-1-mediated increase of ERK1/2 phosphorylation as well as the decrease of phosphorylated Akt and its upstream activator 3-phosphoinositide-dependent protein kinase-1 (PDK1). These effects were inhibited by G-15, indicating that they are GPER dependent. Confirming the participation of GPER, siRNA silencing of GPER inhibited the antihypertrophic effect of E 2 . In conclusion, E 2 plays a key role in antagonizing ET-1-induced hypertrophy in cultured neonatal cardiomyocytes through GPER signaling by a mechanism involving activation of the PDK1 pathway, which would prevent the increase of ERK1/2 activity and consequently the development of hypertrophy.

Pine Oil Effects on Chemical and Thermal Injury in Mice and Cultured Mouse Dorsal Root Ganglion Neurons

PubMed Central

Clark, SP; Bollag, WB; Westlund, KN; Ma, F; Falls, G; Xie, D; Johnson, M; Isales, CM; Bhattacharyya, MH

2013-01-01

A commercial resin-based pine oil derived from Pinus palustris and Pinus elliottii was the major focus of this investigation. Extracts of pine resins, needles and bark are folk medicines commonly used to treat skin ailments, including burns. The American Burn Association estimates that 500,000 people with burn injuries receive medical treatment each year; one-half of US burn victims are children, most with scald burns. This systematic study was initiated as follow-up to personal anecdotal evidence acquired over more than 10 years by MH Bhattacharyya regarding pine oil’s efficacy for treating burns. The results demonstrate that pine oil counteracted dermal inflammation in both a mouse ear model of contact irritant-induced dermal inflammation and a 2nd degree scald burn to the mouse paw. Furthermore, pine oil significantly counteracted the tactile allodynia and soft tissue injury caused by the scald burn. In mouse dorsal root ganglion (DRG) neuronal cultures, pine oil added to the medium blocked ATP-activated, but not capsaicin-activated, pain pathways, demonstrating specificity. These results together support the hypothesis that a pine-oil-based treatment can be developed to provide effective in-home care for 2nd degree burns. PMID:23595692

Modulation of cardiomyocyte activity using pulsed laser irradiated gold nanoparticles

PubMed Central

Gentemann, Lara; Kalies, Stefan; Coffee, Michelle; Meyer, Heiko; Ripken, Tammo; Heisterkamp, Alexander; Zweigerdt, Robert; Heinemann, Dag

2016-01-01

Can photothermal gold nanoparticle mediated laser manipulation be applied to induce cardiac contraction? Based on our previous work, we present a novel concept of cell stimulation. A 532 nm picosecond laser was employed to heat gold nanoparticles on cardiomyocytes. This leads to calcium oscillations in the HL-1 cardiomyocyte cell line. As calcium is connected to the contractility, we aimed to alter the contraction rate of native and stem cell derived cardiomyocytes. A contraction rate increase was particularly observed in calcium containing buffer with neonatal rat cardiomyocytes. Consequently, the study provides conceptual ideas for a light based, nanoparticle mediated stimulation system. PMID:28101410

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death

PubMed Central

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A.; Quest, Andrew F.G.; Lavandero, Sergio

2014-01-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulatenumerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. PMID:23602992

Injurious effects of curcumin on maturation of mouse oocytes, fertilization and fetal development via apoptosis.

PubMed

Chen, Chia-Chi; Chan, Wen-Hsiung

2012-01-01

Curcumin, a common dietary pigment and spice, is a hydrophobic polyphenol derived from the rhizome of the herb Curcuma longa. Previously, we reported a cytotoxic effect of curcumin on mouse embryonic stem cells and blastocysts and its association with defects in subsequent development. In the present study, we further investigated the effects of curcumin on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, curcumin induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with curcumin during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 40 μM curcumin led to decreased oocyte maturation and in vitro fertilization as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented curcumin-triggered injury effects, suggesting that embryo impairment by curcumin occurs mainly via a caspase-dependent apoptotic process.

Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes

PubMed Central

Liu, Qiaozhen; Yang, Rui; Huang, Xiuzhen; Zhang, Hui; He, Lingjuan; Zhang, Libo; Tian, Xueying; Nie, Yu; Hu, Shengshou; Yan, Yan; Zhang, Li; Qiao, Zengyong; Wang, Qing-Dong; Lui, Kathy O; Zhou, Bin

2016-01-01

Cardiac cells marked by c-Kit or Kit, dubbed cardiac stem cells (CSCs), are in clinical trials to investigate their ability to stimulate cardiac regeneration and repair. These studies were initially motivated by the purported cardiogenic activity of these cells. Recent lineage tracing studies using Kit promoter to drive expression of the inducible Cre recombinase showed that these CSCs had highly limited cardiogenic activity, inadequate to support efficient cardiac repair. Here we reassess the lineage tracing data by investigating the identity of cells immediately after Cre labeling. Our instant lineage tracing approach identifies Kit-expressing cardiomyocytes, which are labeled immediately after tamoxifen induction. In combination with long-term lineage tracing experiments, these data reveal that the large majority of long-term labeled cardiomyocytes are pre-existing Kit-expressing cardiomyocytes rather than cardiomyocytes formed de novo from CSCs. This study presents a new interpretation for the contribution of Kit+ cells to cardiomyocytes and shows that Kit genetic lineage tracing over-estimates the cardiogenic activity of Kit+ CSCs. PMID:26634606

Proteomics Unveils Fibroblast-Cardiomyocyte Lactate Shuttle and Hexokinase Paradox in Mouse Muscles.

PubMed

Rakus, Dariusz; Gizak, Agnieszka; Wiśniewski, Jacek R

2016-08-05

Quantitative mapping, given in biochemically interpretable units such as mol per mg of total protein, of tissue-specific proteomes is prerequisite for the analysis of any process in cells. We applied label- and standard-free proteomics to characterize three types of striated muscles: white, red, and cardiac muscle. The analysis presented here uncovers several unexpected and novel features of striated muscles. In addition to differences in protein expression levels, the three muscle types substantially differ in their patterns of basic metabolic pathways and isoforms of regulatory proteins. Importantly, some of the conclusions drawn on the basis of our results, such as the potential existence of a "fibroblast-cardiomyocyte lactate shuttle" and the "hexokinase paradox" point to the necessity of reinterpretation of some basic aspects of striated muscle metabolism. The data presented here constitute a powerful database and a resource for future studies of muscle physiology and for the design of pharmaceutics for the treatment of muscular disorders.

Hearts from Mice Fed a Non-Obesogenic High-Fat Diet Exhibit Changes in Their Oxidative State, Calcium and Mitochondria in Parallel with Increased Susceptibility to Reperfusion Injury

PubMed Central

Littlejohns, Ben; Pasdois, Philippe; Duggan, Simon; Bond, Andrew R.; Heesom, Kate; Jackson, Christopher L.; Angelini, Gianni D.; Halestrap, Andrew P.; Suleiman, M.-Saadeh

2014-01-01

Rationale High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown. Objectives To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury. Methods and Results Feeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet. Conclusions This study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults. PMID:24950187

Purification of Cardiomyocytes from Differentiating Pluripotent Stem Cells using Molecular Beacons Targeting Cardiomyocyte-Specific mRNA

PubMed Central

Kim, Sangsung; Park, Hun-Jun; Byun, Jaemin; Cho, Kyu-Won; Saafir, Talib; Song, Ming-Ke; Yu, Shan Ping; Wagner, Mary; Bao, Gang; Yoon, Young-Sup

2013-01-01

Background While methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) have been reported, current methods produce heterogeneous mixtures of CMs and non-CM cells. Here, we report an entirely novel system in which PSC-derived CMs are purified by CM-specific molecular beacons (MBs). MBs are nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. Method and Results Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among five MBs, a MB targeting myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 CMs, a mouse CM cell line, but < 3% of four non-CM cell types in flow cytometry analysis, indicating that MHC1-MB is specific for identifying CMs. We delivered MHC1-MB into cardiomyogenically differentiated PSCs through nucleofection. The detection rate of CMs was similar to the percentages of cardiac troponin T (TNNT2) or cardiac troponin I (TNNI3)-positive CMs, supporting the specificity of MBs. Finally, MHC1-MB-positive cells were FACS-sorted from mouse and human PSC differentiating cultures and ~97% cells expressed TNNT2- or TNNI3 determined by flow cytometry. These MB-based sorted cells maintained their CM characteristics verified by spontaneous beating, electrophysiologic studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified CMs improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. Conclusions We developed a novel CM selection system that allows production of highly purified CMs. These purified CMs and this system can be valuable for cell therapy and drug discovery. PMID:23995537

In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes

PubMed Central

Qian, Li; Huang, Yu; Spencer, C. Ian; Foley, Amy; Vedantham, Vasanth; Liu, Lei; Conway, Simon J.; Fu, Ji-dong; Srivastava, Deepak

2012-01-01

SUMMARY The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types holds great promise for regenerative medicine. We reported that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT). Here, we use genetic lineage-tracing to show that resident non-myocytes in the murine heart can be reprogrammed into cardiomyocyte-like cells in vivo by local delivery of GMT after coronary ligation. Induced cardiomyocytes became bi-nucleate, assembled sarcomeres and had cardiomyocyte-like gene expression. Analysis of single cells revealed ventricular cardiomyocyte-like action potentials, beating upon electrical stimulation, and evidence of electrical coupling. In vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast activating peptide, Thymosin β4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. PMID:22522929

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

PubMed

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

2013-08-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

Compensatory Limb Use and Behavioral Assessment of Motor Skill Learning Following Sensorimotor Cortex Injury in a Mouse Model of Ischemic Stroke

PubMed Central

Kerr, Abigail L.; Tennant, Kelly A.

2014-01-01

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models. PMID:25045916

Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model

PubMed Central

Fauconnier, Jérémy; Cellier, Laura; Tamareille, Sophie; Gharib, Abdallah; Chevrollier, Arnaud; Loufrani, Laurent; Grenier, Céline; Kamel, Rima; Sarzi, Emmanuelle; Lacampagne, Alain; Ovize, Michel; Henrion, Daniel; Reynier, Pascal; Lenaers, Guy; Mirebeau-Prunier, Delphine

2016-01-01

Background Recent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain. Objectives To investigate whether Opa1 (protein involved in mitochondrial inner-membrane fusion) deficiency affects I/R injuries. Methods and Results We examined mice exhibiting Opa1delTTAG mutations (Opa1+/-), showing 70% Opa1 protein expression in the myocardium as compared to their wild-type (WT) littermates. Cardiac left-ventricular systolic function assessed by means of echocardiography was observed to be similar in 3-month-old WT and Opa1+/- mice. After subjection to I/R, infarct size was significantly greater in Opa1+/- than in WTs both in vivo (43.2±4.1% vs. 28.4±3.5%, respectively; p<0.01) and ex vivo (71.1±3.2% vs. 59.6±8.5%, respectively; p<0.05). No difference was observed in the expression of other main fission/fusion protein, oxidative phosphorylation, apoptotic markers, or mitochondrial permeability transition pore (mPTP) function. Analysis of calcium transients in isolated ventricular cardiomyocytes demonstrated a lower sarcoplasmic reticulum Ca2+ uptake, whereas cytosolic Ca2+ removal from the Na+/Ca2+ exchanger (NCX) was increased, whilst SERCA2a, phospholamban, and NCX protein expression levels were unaffected in Opa1+/- compared to WT mice. Simultaneous whole-cell patch-clamp recordings of mitochondrial Ca2+ movements and ventricular action potential (AP) showed impairment of dynamic mitochondrial Ca2+ uptake and a marked increase in the AP late repolarization phase in conjunction with greater occurrence of arrhythmia in Opa1+/- mice. Conclusion Opa1 deficiency was associated with increased sensitivity to I/R, imbalance in dynamic mitochondrial Ca2+ uptake, and subsequent increase in NCX activity. PMID:27723783

Actin cytoskeletal remodeling with protrusion formation is essential for heart regeneration in Hippo-deficient mice

PubMed Central

Morikawa, Yuka; Zhang, Min; Heallen, Todd; Leach, John; Tao, Ge; Xiao, Yang; Bai, Yan; Li, Wei; Willerson, James T.; Martin, James F.

2015-01-01

The mammalian heart regenerates poorly, and damage commonly leads to heart failure. Hippo signaling is an evolutionarily conserved kinase cascade that regulates organ size during development and prevents adult mammalian cardiomyocyte regeneration by inhibiting the transcriptional coactivator Yap, which also responds to mechanical signaling in cultured cells to promote cell proliferation. To identify Yap target genes that are activated during cardiomyocyte renewal and regeneration, we performed Yap chromatin immunoprecipitation sequencing (ChIP-Seq) and mRNA expression profiling in Hippo signaling-deficient mouse hearts. We found that Yap directly regulated genes encoding cell cycle progression proteins, as well as genes encoding proteins that promote F-actin polymerization and that link the actin cytoskeleton to the extracellular matrix. Included in the latter group were components of the dystrophin glycoprotein complex (DGC), a large molecular complex that, when defective, results in muscular dystrophy in humans. Cardiomyocytes near scar tissue of injured Hippo signaling-deficient mouse hearts showed cellular protrusions suggestive of cytoskeletal remodeling. The hearts of mdx mutant mice, which lack functional dystrophin and are a model for muscular dystrophy, showed impaired regeneration and cytoskeleton remodeling, but normal cardiomyocyte proliferation after injury. Our data showed that, in addition to genes encoding cell cycle progression proteins, Yap regulated genes that enhance cytoskeletal remodeling Thus, blocking the Hippo pathway input to Yap may tip the balance so that Yap responds to the mechanical changes associated with heart injury to promote repair. PMID:25943351

Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oyama, Kotaro; Mizuno, Akari; Shintani, Seine A.

Highlights: Black-Right-Pointing-Pointer Infra-red laser beam generates microscopic heat pulses. Black-Right-Pointing-Pointer Heat pulses induce contraction of cardiomyocytes. Black-Right-Pointing-Pointer Ca{sup 2+} transients during the contraction were not detected. Black-Right-Pointing-Pointer Skinned cardiomyocytes in free Ca{sup 2+} solution also contracted. Black-Right-Pointing-Pointer Heat pulses regulated the contractions without Ca{sup 2+} dynamics. -- Abstract: It was recently demonstrated that laser irradiation can control the beating of cardiomyocytes and hearts, however, the precise mechanism remains to be clarified. Among the effects induced by laser irradiation on biological tissues, temperature change is one possible effect which can alter physiological functions. Therefore, we investigated the mechanism by which heatmore » pulses, produced by infra-red laser light under an optical microscope, induce contractions of cardiomyocytes. Here we show that microscopic heat pulses induce contraction of rat adult cardiomyocytes. The temperature increase, {Delta}T, required for inducing contraction of cardiomyocytes was dependent upon the ambient temperature; that is, {Delta}T at physiological temperature was lower than that at room temperature. Ca{sup 2+} transients, which are usually coupled to contraction, were not detected. We confirmed that the contractions of skinned cardiomyocytes were induced by the heat pulses even in free Ca{sup 2+} solution. This heat pulse-induced Ca{sup 2+}-decoupled contraction technique has the potential to stimulate heart and skeletal muscles in a manner different from the conventional electrical stimulations.« less

Role of cardiomyocyte circadian clock in myocardial metabolic adaptation

USDA-ARS?s Scientific Manuscript database

Marked circadian rhythmicities in cardiovascular physiology and pathophysiology exist. The cardiomyocyte circadian clock has recently been linked to circadian rhythms in myocardial gene expression, metabolism, and contractile function. For instance, the cardiomyocyte circadian clock is essential f...

Cardiac Gab1 deletion leads to dilated cardiomyopathy associated with mitochondrial damage and cardiomyocyte apoptosis

PubMed Central

Zhao, J; Yin, M; Deng, H; Jin, F Q; Xu, S; Lu, Y; Mastrangelo, M A; Luo, H; Jin, Z G

2016-01-01

A vital step in the development of heart failure is the transition from compensatory cardiac hypertrophy to decompensated dilated cardiomyopathy (DCM) during cardiac remodeling under mechanical or pathological stress. However, the molecular mechanisms underlying the development of DCM and heart failure remain incompletely understood. In the present study, we investigate whether Gab1, a scaffolding adaptor protein, protects against hemodynamic stress-induced DCM and heat failure. We first observed that the protein levels of Gab1 were markedly reduced in hearts from human patients with DCM and from mice with experimental viral myocarditis in which DCM developed. Next, we generated cardiac-specific Gab1 knockout mice (Gab1-cKO) and found that Gab-cKO mice developed DCM in hemodynamic stress-dependent and age-dependent manners. Under transverse aorta constriction (TAC), Gab1-cKO mice rapidly developed decompensated DCM and heart failure, whereas Gab1 wild-type littermates exhibited adaptive left ventricular hypertrophy without changes in cardiac function. Mechanistically, we showed that Gab1-cKO mouse hearts displayed severe mitochondrial damages and increased cardiomyocyte apoptosis. Loss of cardiac Gab1 in mice impaired Gab1 downstream MAPK signaling pathways in the heart under TAC. Gene profiles further revealed that ablation of Gab1 in heart disrupts the balance of anti- and pro-apoptotic genes in cardiomyocytes. These results demonstrate that cardiomyocyte Gab1 is a critical regulator of the compensatory cardiac response to aging and hemodynamic stress. These findings may provide new mechanistic insights and potential therapeutic target for DCM and heart failure. PMID:26517531

Hypertrophic Stimulation Increases β-actin Dynamics in Adult Feline Cardiomyocytes

PubMed Central

Balasubramanian, Sundaravadivel; Mani, Santhosh K.; Kasiganesan, Harinath; Baicu, Catalin C.; Kuppuswamy, Dhandapani

2010-01-01

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While α-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of β-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, β-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of β-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of β-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of β-actin. To determine the localization and dynamics of β-actin, we adenovirally expressed GFP-tagged β-actin in isolated adult cardiomyocytes. The ectopically expressed β-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of β-actin dynamics revealed that β-actin at the Z-discs is constantly being exchanged with β-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while β-actin overexpression improved cardiomyocyte contractility, immunoneutralization of β-actin resulted in a reduced contractility suggesting that β-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of β-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac cytoskeletal rearrangement during

Hypertrophic stimulation increases beta-actin dynamics in adult feline cardiomyocytes.

PubMed

Balasubramanian, Sundaravadivel; Mani, Santhosh K; Kasiganesan, Harinath; Baicu, Catalin C; Kuppuswamy, Dhandapani

2010-07-12

The myocardium responds to hemodynamic stress through cellular growth and organ hypertrophy. The impact of cytoskeletal elements on this process, however, is not fully understood. While alpha-actin in cardiomyocytes governs muscle contraction in combination with the myosin motor, the exact role of beta-actin has not been established. We hypothesized that in adult cardiomyocytes, as in non-myocytes, beta-actin can facilitate cytoskeletal rearrangement within cytoskeletal structures such as Z-discs. Using a feline right ventricular pressure overload (RVPO) model, we measured the level and distribution of beta-actin in normal and pressure overloaded myocardium. Resulting data demonstrated enriched levels of beta-actin and enhanced translocation to the Triton-insoluble cytoskeletal and membrane skeletal complexes. In addition, RVPO in vivo and in vitro hypertrophic stimulation with endothelin (ET) or insulin in isolated adult cardiomyocytes enhanced the content of polymerized fraction (F-actin) of beta-actin. To determine the localization and dynamics of beta-actin, we adenovirally expressed GFP-tagged beta-actin in isolated adult cardiomyocytes. The ectopically expressed beta-actin-GFP localized to the Z-discs, costameres, and cell termini. Fluorescence recovery after photobleaching (FRAP) measurements of beta-actin dynamics revealed that beta-actin at the Z-discs is constantly being exchanged with beta-actin from cytoplasmic pools and that this exchange is faster upon hypertrophic stimulation with ET or insulin. In addition, in electrically stimulated isolated adult cardiomyocytes, while beta-actin overexpression improved cardiomyocyte contractility, immunoneutralization of beta-actin resulted in a reduced contractility suggesting that beta-actin could be important for the contractile function of adult cardiomyocytes. These studies demonstrate the presence and dynamics of beta-actin in the adult cardiomyocyte and reinforce its usefulness in measuring cardiac

[The electroporation effects of high power pulse microwave and electromagnetic pulse irradiation on the membranes of cardiomyocyte cells and the mechanism therein involved].

PubMed

Deng, Hua; Wang, Dewen; Peng, Ruiyun; Wang, Shuiming; Chen, Jiankui; Zhang, Sa; Dong, Bo; Wang, Xiaomin

2005-08-01

Though there is ongoing public concern on potential hazards and risk of electromagnetic radiation, the bioeffects mechanism of electromagnetic fields remains obscure. Heart is one of the organs susceptive to electromagnetic fields (EMF). This study was designed to assess the influence of high power pulse microwave and electromagnetic pulse irradiation on cardiomyocytes, to explore the critical mechanism of electromagnetic fields, and to explain the regular course of injury caused by exposure to pulse EMF. Cultured cardiomyocytes were irradiated by high power pulse microwave and electromagnetic pulse first, then a series of apparatus including atom force microscope, laser scanning confocal microscope and flow cytometer were used to examine the changes of cell membrane conformation, structure and function. After irradiation, the cardiomyocytes pulsated slower or stop, the cells conformation was abnormal, the cells viability declined, and the percentage of apoptosis and necrosis increased significantly (P< 0.01). The cell membrane had pores unequal in size, and lost its penetration character. The concentration of Na+, K+, Ca2+, Cl-, Mg2+, Ca2+ and P3+ in cell culture medium increased significantly (P< 0.01). and the concentration of Ca2+ in cells ([Ca2+]i) decreased significantly (P<0.01). The results indicated that cardiomyocytes are susceptible to non-ionizing radiation. Pulse electromagnetic field can induce cardiomyocytes electroporation, and can do great damage to cells conformation, structure and function. Electroporation is one of the most critical mechanisms to explain the athermal effects of electromagnetic radiation.

Thymoquinone Attenuates Myocardial Ischemia/Reperfusion Injury Through Activation of SIRT1 Signaling.

PubMed

Lu, Yunyang; Feng, Yingda; Liu, Dan; Zhang, Zhiran; Gao, Kai; Zhang, Wei; Tang, Haifeng

2018-06-15

Myocardial ischemia/reperfusion (MI/R) injury is a leading factor responsible for damage in myocardial infarction, resulting in additional injury to cardiac tissues involved in oxidative stress, inflammation, and apoptosis. Thymoquinone (TQ), the main constituent of Nigella sativa L. seeds, has been reported to possess various biological activities. However, few reports regarding myocardial protection are available at present. Therefore, this study was conducted aiming to investigate the protective effect of TQ against MI/R injury and to clarify its potential mechanism. MI/R injury models of isolated rat hearts and neonatal rat cardiomyocytes were established. The Langendorff isolated perfused heart system, triphenyltetrazolium chloride staining, gene transfection, TransLaser scanning confocal microscopy, and western blotting were employed to evaluate the cardioprotection effect of TQ against MI/R injury. Compared with the MI/R group, TQ treatment could remarkably improve left ventricular function, decrease myocardial infarct size and production of lactate dehydrogenase (LDH), and attenuate mitochondrial oxidative damage by elevating superoxide dismutase (SOD) activity and reducing production of hydrogen peroxide (H2O2) and malonaldehyde (MDA). Moreover, the cardioprotective effect of TQ was accompanied by up-regulated expression of SIRT1 and inhibition of p53 acetylation. Additionally, TQ treatment could also enhance mitochondrial function and reduce the number of apoptotic cardiomyocytes. Nonetheless, the cardioprotective effect of TQ could be mitigated by SIRT1 inhibitor sirtinol and SIRT1 siRNA, respectively, which was achieved through inhibition of the SIRT1 signaling pathway. The findings in this study demonstrate that TQ is efficient in attenuating MI/R injury through activation of the SIRT1 signaling pathway, which can thus reduce mitochondrial oxidative stress damage and cardiomyocyte apoptosis. © 2018 The Author(s). Published by S. Karger AG, Basel.

Permeabilized Rat Cardiomyocyte Response Demonstrates Intracellular Origin of Diffusion Obstacles

PubMed Central

Jepihhina, Natalja; Beraud, Nathalie; Sepp, Mervi; Birkedal, Rikke; Vendelin, Marko

2011-01-01

Intracellular diffusion restrictions for ADP and other molecules have been predicted earlier based on experiments on permeabilized fibers or cardiomyocytes. However, it is possible that the effective diffusion distance is larger than the cell dimensions due to clumping of cells and incomplete separation of cells in fiber preparations. The aim of this work was to check whether diffusion restrictions exist inside rat cardiomyocytes or are caused by large effective diffusion distance. For that, we determined the response of oxidative phosphorylation (OxPhos) to exogenous ADP and ATP stimulation in permeabilized rat cardiomyocytes using fluorescence microscopy. The state of OxPhos was monitored via NADH and flavoprotein autofluorescence. By varying the ADP or ATP concentration in flow chamber, we determined that OxPhos has a low affinity in cardiomyocytes. The experiments were repeated in a fluorometer on cardiomyocyte suspensions leading to similar autofluorescence changes induced by ADP as recorded under the microscope. ATP stimulated OxPhos more in a fluorometer than under the microscope, which was attributed to accumulation of ADP in fluorometer chamber. By calculating the flow profile around the cell in the microscope chamber and comparing model solutions to measured data, we demonstrate that intracellular structures impose significant diffusion obstacles in rat cardiomyocytes. PMID:22067148

Cardiomyocyte-specific desmin rescue of desmin null cardiomyopathy excludes vascular involvement.

PubMed

Weisleder, Noah; Soumaka, Elisavet; Abbasi, Shahrzad; Taegtmeyer, Heinrich; Capetanaki, Yassemi

2004-01-01

Mice deficient in desmin, the muscle-specific member of the intermediate filament gene family, display defects in all muscle types and particularly in the myocardium. Desmin null hearts develop cardiomyocyte hypertrophy and dilated cardiomyopathy (DCM) characterized by extensive myocyte cell death, calcific fibrosis and multiple ultrastructural defects. Several lines of evidence suggest impaired vascular function in desmin null animals. To determine whether altered capillary function or an intrinsic cardiomyocyte defect is responsible for desmin null DCM, transgenic mice were generated to rescue desmin expression specifically to cardiomyocytes. Desmin rescue mice display a wild-type cardiac phenotype with no fibrosis or calcification in the myocardium and normalization of coronary flow. Cardiomyocyte ultrastructure is also restored to normal. Markers of hypertrophy upregulated in desmin null hearts return to wild-type levels in desmin rescue mice. Working hearts were perfused to assess coronary flow and cardiac power. Restoration of a wild-type cardiac phenotype in a desmin null background by expression of desmin specifically within cardiomyocyte indicates that defects in the desmin null heart are due to an intrinsic cardiomyocytes defect rather than compromised coronary circulation.

Acute over-the-counter pharmacological intervention does not adversely affect behavioral outcome following diffuse traumatic brain injury in the mouse.

PubMed

Harrison, Jordan L; Rowe, Rachel K; O'Hara, Bruce F; Adelson, P David; Lifshitz, Jonathan

2014-09-01

Following mild traumatic brain injury (TBI), patients may self-treat symptoms of concussion, including post-traumatic headache, taking over-the-counter (OTC) analgesics. Administering one dose of OTC analgesics immediately following experimental brain injury mimics the at-home treated population of concussed patients and may accelerate the understanding of the relationship between brain injury and OTC pharmacological intervention. In the current study, we investigate the effect of acute administration of OTC analgesics on neurological function and cortical cytokine levels after experimental diffuse TBI in the mouse. Adult, male C57BL/6 mice were injured using a midline fluid percussion (mFPI) injury model of concussion (6-10 min righting reflex time for brain-injured mice). Experimental groups included mFPI paired with either ibuprofen (60 mg/kg, i.p.; n = 16), acetaminophen (40 mg/kg, i.p.; n = 9), or vehicle (15% ethanol (v/v) in 0.9% saline; n = 13) and sham injury paired OTC medicine or vehicle (n = 7-10 per group). At 24 h after injury, functional outcome was assessed using the rotarod task and a modified neurological severity score. Following behavior assessment, cortical cytokine levels were measured by multiplex ELISA at 24 h post-injury. To evaluate efficacy on acute inflammation, cortical cytokine levels were measured also at 6 h post-injury. In the diffuse brain-injured mouse, immediate pharmacological intervention did not attenuate or exacerbate TBI-induced functional deficits. Cortical cytokine levels were affected by injury, time, or their interaction. However, levels were not affected by treatment at 6 or 24 h post-injury. These data indicate that acute administration of OTC analgesics did not exacerbate or attenuate brain-injury deficits which may inform clinical recommendations for the at-home treated mildly concussed patient.

Acute hyperglycaemia enhances oxidative stress and aggravates myocardial ischaemia/reperfusion injury: role of thioredoxin-interacting protein

PubMed Central

Su, Hui; Ji, Lele; Xing, Wenjuan; Zhang, Wei; Zhou, Heping; Qian, Xinhong; Wang, Xiaoming; Gao, Feng; Sun, Xin; Zhang, Haifeng

2013-01-01

Hyperglycaemia during acute myocardial infarction is common and associated with increased mortality. Thioredoxin-interacting protein (Txnip) is a modulator of cellular redox state and contributes to cell apoptosis. This study aimed to investigate whether or not hyperglycaemia enhances Txnip expression in myocardial ischaemia/reperfusion (MI/R) and consequently exacerbates MI/R injury. Rats were subjected to 30 min. of left coronary artery ligation followed by 4 hrs of reperfusion and treated with saline or high glucose (HG, 500 g/l, 4 ml/kg/h intravenously). In vitro study was performed on cultured rat cardiomyocytes subjected to simulated ischaemia/reperfusion (SI/R) and incubated with HG (25 mM) or normal glucose (5.6 mM) medium. In vivo HG infusion during MI/R significantly impaired cardiac function, aggravated myocardial injury and increased cardiac oxidative stress. Meanwhile, Txnip expression was enhanced whereas thioredoxin activity was inhibited following HG treatment in ischaemia/reperfusion (I/R) hearts. In addition, HG activated p38 MAPK and inhibited Akt in I/R hearts. In cultured cardiomyocytes subjected to SI/R, HG incubation stimulated Txnip expression and reduced thioredoxin activity. Overexpression of Txnip enhanced HG-induced superoxide generation and aggravated cardiomyocyte apoptosis, whereas Txnip RNAi significantly blunted the deleterious effects of HG. Moreover, inhibition of p38 MAPK or activation of Akt markedly blocked HG-induced Txnip expression in I/R cardiomyocytes. Most importantly, intramyocardial injection of Txnip siRNA markedly decreased Txnip expression and alleviated MI/R injury in HG-treated rats. Hyperglycaemia enhances myocardial Txnip expression, possibly through reciprocally modulating p38 MAPK and Akt activation, leading to aggravated oxidative stress and subsequently, amplification of cardiac injury following MI/R. PMID:23305039

Photothermal gold nanoparticle mediated stimulation of cardiomyocyte beating (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kalies, Stefan; Gentemann, Lara; Coffee, Michelle; Zweigerdt, Robert; Heinemann, Dag; Heisterkamp, Alexander

2017-03-01

Photothermal manipulation of cells via heating of gold nanoparticles has proven to be an efficient tool for molecular delivery into cells via cell perforation with short laser pulses. We investigated a potential extension of this technique for cell stimulation of cardiomyocytes using a 532 nm and 850 ps laser system and a surface concentration of 0.5 μg/cm2 of 200 nm gold nanoparticles. The gold nanoparticles were unspecifically attached to the cardiomyocytes after an incubation period of three hours. The laser irradiation leads to a temperature rise directly at the particles of several hundred degrees K which evokes bubble formation and membrane perforation. We examined the effect of laser based photothermal manipulation at different laser powers, with different calcium concentrations, and for a cardiomyocyte-like cell line (HL1 cells), neonatal rat cardiomyocytes and human embryonic stem cell (hESC)-derived cardiomyocytes. Fast calcium oscillations in HL1 cells were observed in the presence and absence of extracellular calcium and most pronounced in the area next to the laser spot after irradiation. Within the laser spot, in particular high laser powers led to a single rise in calcium over a time period of several seconds. These results were confirmed in stem cell-derived cardiomyocytes. In the presence of normal and high calcium concentrations, the spontaneous contraction frequency increased after laser irradiation in neonatal rat cardiomyocytes. Consequently, gold nanoparticle mediated photothermal cell manipulation via pulsed lasers may serve as a potential pacemaker-technique in regenerative approaches, next to optogenetics.

Ripk3 promotes ER stress-induced necroptosis in cardiac IR injury: A mechanism involving calcium overload/XO/ROS/mPTP pathway.

PubMed

Zhu, Pingjun; Hu, Shunying; Jin, Qinhua; Li, Dandan; Tian, Feng; Toan, Sam; Li, Yang; Zhou, Hao; Chen, Yundai

2018-06-01

Receptor-interacting protein 3 (Ripk3)-mediated necroptosis contributes to cardiac ischaemia-reperfusion (IR) injury through poorly defined mechanisms. Our results demonstrated that Ripk3 was strongly upregulated in murine hearts subjected to IR injury and cardiomyocytes treated with LPS and H 2 O 2 . The higher level of Ripk3 was positively correlated to the infarction area expansion, cardiac dysfunction and augmented cardiomyocytes necroptosis. Function study further illustrated that upregulated Ripk3 evoked the endoplasmic reticulum (ER) stress, which was accompanied with an increase in intracellular Ca 2+ level ([Ca 2+ ]c) and xanthine oxidase (XO) expression. Activated XO raised cellular reactive oxygen species (ROS) that mediated the mitochondrial permeability transition pore (mPTP) opening and cardiomyocytes necroptosis. By comparison, genetic ablation of Ripk3 abrogated the ER stress and thus blocked the [Ca 2+ ]c overload-XO-ROS-mPTP pathways, favouring a pro-survival state that ultimately resulted in the inhibition of cardiomyocytes necroptosis in the setting of cardiac IR injury. In summary, the present study helps to elucidate how necroptosis is mediated by ER stress, via the calcium overload /XO/ROS/mPTP opening axis. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+ Sensitivity in Intact Cardiomyocytes

PubMed Central

Figueiredo-Freitas, Cícero; Dulce, Raul A.; Foster, Matthew W.; Liang, Jingsheng; Yamashita, Aline M.S.; Lima-Rosa, Frederico L.; Thompson, J. Will; Moseley, M. Arthur; Hare, Joshua M.; Nogueira, Leonardo; Sorenson, Martha M.

2015-01-01

Abstract Aims: The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S-nitrosoglutathione (GSNO), a protein S-nitrosylating agent. Although S-nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S-nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments. Results: Incubation of intact mouse cardiomyocytes with S-nitrosocysteine (CysNO, a cell-permeable low-molecular-weight nitrosothiol) significantly decreased myofilament Ca2+ sensitivity. In demembranated (skinned) fibers, S-nitrosylation with 1 μM GSNO also decreased Ca2+ sensitivity of contraction and 10 μM reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥100 μM). Reducing S-nitrosylation with ascorbate partially reversed the effects on Ca2+ sensitivity and ATPase activity. In live cardiomyocytes treated with CysNO, resin-assisted capture of S-nitrosylated protein thiols was combined with label-free liquid chromatography–tandem mass spectrometry to quantify S-nitrosylation and determine the susceptible cysteine sites on myosin, actin, myosin-binding protein C, troponin C and I, tropomyosin, and titin. The ability of sarcomere proteins to form S-NO from 10–500 μM CysNO in intact cardiomyocytes was further determined by immunoblot, with actin, myosin, myosin-binding protein C, and troponin C being the more susceptible sarcomeric proteins. Innovation and Conclusions: Thus, specific physiological effects are associated with S-nitrosylation of a limited number of cysteine residues in sarcomeric proteins, which also offer potential targets for interventions in pathophysiological situations. Antioxid. Redox Signal. 23, 1017–1034. PMID:26421519

Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene

PubMed Central

Gay, Maresha S.; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela

2016-01-01

Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene. PMID:27302109

A kidney injury molecule-1 (Kim-1) gene reporter in a mouse artificial chromosome: the responsiveness to cisplatin toxicity in immortalized mouse kidney S3 cells.

PubMed

Kokura, Kenji; Kuromi, Yasushi; Endo, Takeshi; Anzai, Naohiko; Kazuki, Yasuhiro; Oshimura, Mitsuo; Ohbayashi, Tetsuya

2016-10-01

Kidney injury molecule-1 (Kim-1) has been validated as a urinary biomarker for acute and chronic renal damage. The expression of Kim-1 mRNA is also activated by acute kidney injury induced by cisplatin in rodents and humans. To date, the measurement of Kim-1 expression has not fully allowed the detection of in vitro cisplatin nephrotoxicity in immortalized culture cells, such as human kidney-2 cells and immortalized proximal tubular epithelial cells. We measured the augmentation of Kim-1 mRNA expression after the addition of cisplatin using immortalized S3 cells established from the kidneys of transgenic mice harboring temperature-sensitive large T antigen from Simian virus 40. A mouse Kim-1 gene luciferase reporter in conjunction with an Hprt gene reporter detected cisplatin-induced nephrotoxicity in S3 cells. These two reporter genes were contained in a mouse artificial chromosome, and two luciferases that emitted different wavelengths were used to monitor the respective gene expression. However, the Kim-1 reporter gene failed to respond to cisplatin in A9 fibroblast cells that contained the same reporter mouse artificial chromosome, suggesting cell type-specificity for activation of the reporter. We report the feasibility of measuring in vitro cisplatin nephrotoxicity using a Kim-1 reporter gene in S3 cells. © 2016 The Authors. The Journal of Gene Medicine Published by John Wiley & Sons, Ltd.

Recent advancements in understanding endogenous heart regeneration—insights from adult zebrafish and neonatal mice

PubMed Central

Rubin, Nicole; Harrison, Michael R.; Krainock, Michael; Kim, Richard; Lien, Ching-Ling

2016-01-01

Enhancing the endogenous regenerative capacity of the mammalian heart is a promising strategy that can lead to potential treatment of injured cardiac tissues. Studies on heart regeneration in zebrafish and neonatal mice have shown that cardiomyocyte proliferation is essential for replenishing myocardium. We will review recent advancements that have demonstrated the importance of Neuregulin 1/ErbB2 and innervation in regulating cardiomyocyte proliferation using both adult zebrafish and neonatal mouse heart regeneration models. Emerging findings suggest that different populations of macrophages and inflammation might contribute to regenerative versus fibrotic responses. Finally, we will discuss variation in the severity of the cardiac injury and size of the wound, which may explain the range of outcomes observed in different injury models. PMID:27132022

1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells

PubMed Central

Okolotowicz, Karl J.; Bushway, Paul; Lanier, Marion; Gilley, Cynthia; Cynthia, Mark; Cashman, John R.

2016-01-01

Cardiomyopathy is the leading cause of death worldwide. Despite progress in medical treatments, heart transplantation is one of the only current options for those with infarcted heart muscle. Stem cell differentiation technology may afford cell-based therapeutics that may lead to the generation of new, healthy heart muscle cells from undifferentiated stem cells. Our approach is to use small molecules to stimulate stem cell differentiation. Herein, we describe a novel class of 1,5-disubstituted benzimidazoles that induce differentiation of stem cells into cardiac cells. We report on the evaluation in vitro for cardiomyocyte differentiation and describe structure–activity relationship results that led to molecules with drug-like properties. The results of this study show the promise of small molecules to direct stem cell lineage commitment, to probe signaling pathways and to develop compounds for the stimulation of stem cells to repair damaged heart tissue. PMID:26278027

(Re-)programming of subtype specific cardiomyocytes.

PubMed

Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

2017-10-01

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Cardiomyocyte-Specific Ablation of Med1 Subunit of the Mediator Complex Causes Lethal Dilated Cardiomyopathy in Mice.

PubMed

Jia, Yuzhi; Chang, Hsiang-Chun; Schipma, Matthew J; Liu, Jing; Shete, Varsha; Liu, Ning; Sato, Tatsuya; Thorp, Edward B; Barger, Philip M; Zhu, Yi-Jun; Viswakarma, Navin; Kanwar, Yashpal S; Ardehali, Hossein; Thimmapaya, Bayar; Reddy, Janardan K

2016-01-01

Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1β that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart

Role of Extracellular RNA and TLR3‐Trif Signaling in Myocardial Ischemia–Reperfusion Injury

PubMed Central

Chen, Chan; Feng, Yan; Zou, Lin; Wang, Larry; Chen, Howard H.; Cai, Jia‐Yan; Xu, Jun‐Mei; Sosnovik, David E.; Chao, Wei

2014-01-01

Background Toll‐like receptor 3 (TLR3) was originally identified as the receptor for viral RNA and represents a major host antiviral defense mechanism. TLR3 may also recognize extracellular RNA (exRNA) released from injured tissues under certain stress conditions. However, a role for exRNA and TLR3 in the pathogenesis of myocardial ischemic injury has not been tested. This study examined the role of exRNA and TLR3 signaling in myocardial infarction (MI), apoptosis, inflammation, and cardiac dysfunction during ischemia‐reperfusion (I/R) injury. Methods and Results Wild‐type (WT), TLR3−/−, Trif−/−, and interferon (IFN) α/β receptor‐1 deficient (IFNAR1−/−) mice were subjected to 45 minutes of coronary artery occlusion and 24 hours of reperfusion. Compared with WT, TLR3−/− or Trif−/− mice had smaller MI and better preserved cardiac function. Surprisingly, unlike TLR(2/4)‐MyD88 signaling, lack of TLR3‐Trif signaling had no impact on myocardial cytokines or neutrophil recruitment after I/R, but myocardial apoptosis was significantly attenuated in Trif−/− mice. Deletion of the downstream IFNAR1 had no effect on infarct size. Importantly, hypoxia and I/R led to release of RNA including microRNA from injured cardiomyocytes and ischemic heart, respectively. Necrotic cardiomyocytes induced a robust and dose‐dependent cytokine response in cultured cardiomyocytes, which was markedly reduced by RNase but not DNase, and partially blocked in TLR3‐deficient cardiomyocytes. In vivo, RNase administration reduced serum RNA level, attenuated myocardial cytokine production, leukocytes infiltration and apoptosis, and conferred cardiac protection against I/R injury. Conclusion TLR3‐Trif signaling represents an injurious pathway during I/R. Extracellular RNA released during I/R may contribute to myocardial inflammation and infarction. PMID:24390148

Dexamethasone Induces Cardiomyocyte Terminal Differentiation via Epigenetic Repression of Cyclin D2 Gene.

PubMed

Gay, Maresha S; Dasgupta, Chiranjib; Li, Yong; Kanna, Angela; Zhang, Lubo

2016-08-01

Dexamethasone treatment of newborn rats inhibited cardiomyocyte proliferation and stimulated premature terminal differentiation of cardiomyocytes in the developing heart. Yet mechanisms remain undetermined. The present study tested the hypothesis that the direct effect of glucocorticoid receptor-mediated epigenetic repression of cyclin D2 gene in the cardiomyocyte plays a key role in the dexamethasone-mediated effects in the developing heart. Cardiomyocytes were isolated from 2-day-old rats. Cells were stained with a cardiomyocyte marker α-actinin and a proliferation marker Ki67. Cyclin D2 expression was evaluated by Western blot and quantitative real-time polymerase chain reaction. Promoter methylation of CcnD2 was determined by methylated DNA immunoprecipitation (MeDIP). Overexpression of Cyclin D2 was conducted by transfection of FlexiCcnD2 (+CcnD2) construct. Treatment of cardiomyocytes isolated from newborn rats with dexamethasone for 48 hours significantly inhibited cardiomyocyte proliferation with increased binucleation and decreased cyclin D2 protein abundance. These effects were blocked with Ru486 (mifepristone). In addition, the dexamethasone treatment significantly increased cyclin D2 gene promoter methylation in newborn rat cardiomyocytes. 5-Aza-2'-deoxycytidine inhibited dexamethasone-mediated promoter methylation, recovered dexamethasone-induced cyclin D2 gene repression, and blocked the dexamethasone-elicited effects on cardiomyocyte proliferation and binucleation. In addition, the overexpression of cyclin D2 restored the dexamethasone-mediated inhibition of proliferation and increase in binucleation in newborn rat cardiomyocytes. The results demonstrate that dexamethasone acting on glucocorticoid receptors has a direct effect and inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via epigenetic repression of cyclin D2 gene. Copyright © 2016 by The American Society for Pharmacology and

Leptin confers protection against TNF-α-induced apoptosis in rat cardiomyocytes.

PubMed

Yu, Lu; Zhao, Yanbo; Xu, Shengjie; Jin, Chongying; Wang, Min; Fu, Guosheng

2014-12-05

Leptin, an important adipose-derived hormone, is recognized as a crucial protein in energy homeostasis. Recent studies indicated that leptin is associated with cardiac pathophysiology, however, the role and mechanisms of leptin in cardiomyocytes apoptosis are poorly understood. Here we investigated whether leptin exerted protective effect on cardiomyocytes exposed to tumor necrosis factor-alpha (TNF-α) and the possible mechanisms. Neonatal rat cardiomyocytes were subjected to TNF-α in the presence or absence of leptin. By FITC/Annexin V flow cytometry and Western blot, we noticed that TNF-α increased Annexin V binding and cleaved caspase-3/PARP, which were attenuated by leptin pretreatment. Moreover, leptin protected cardiomyocytes against mitochondrial apoptosis by inhibiting cytochrome C elevation and Bcl-2 decreasing. TNF-α-induced P38 MAPK and NF-κB activation were abolished by leptin addition, and the P38 and NF-κB inhibitor, SB203580 and Bay117082, also mitigated the apoptotic effect of TNF-α, indicating that their activation might be responsible for the apoptosis in TNF-α-treated cardiomyocytes. Therefore, leptin conferred anti-apoptotic effect in cardiomyocytes exposed to TNF-α possibly by inhibiting TNF-α-activated P38 MAPK and NF-κB pathways.

Interpretation of field potentials measured on a multi electrode array in pharmacological toxicity screening on primary and human pluripotent stem cell-derived cardiomyocytes.

PubMed

Tertoolen, L G J; Braam, S R; van Meer, B J; Passier, R; Mummery, C L

2018-03-18

Multi electrode arrays (MEAs) are increasingly used to detect external field potentials in electrically active cells. Recently, in combination with cardiomyocytes derived from human (induced) pluripotent stem cells they have started to become a preferred tool to examine newly developed drugs for potential cardiac toxicity in pre-clinical safety pharmacology. The most important risk parameter is proarrhythmic activity in cardiomyocytes which can cause sudden cardiac death. Whilst MEAs can provide medium- to high- throughput noninvasive assay platform, the translation of a field potential to cardiac action potential (normally measured by low-throughput patch clamp) is complex so that accurate assessment of drug risk to the heart is in practice still challenging. To address this, we used computational simulation to study the theoretical relationship between aspects of the field potential and the underlying cardiac action potential. We then validated the model in both primary mouse- and human pluripotent (embryonic) stem cell-derived cardiomyocytes showing that field potentials measured in MEAs could be converted to action potentials that were essentially identical to those determined directly by electrophysiological patch clamp. The method significantly increased the amount of information that could be extracted from MEA measurements and thus combined the advantages of medium/high throughput with more informative readouts. We believe that this will benefit the analysis of drug toxicity screening of cardiomyocytes using in time and accuracy. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Luteolin Modulates SERCA2a Leading to Attenuation of Myocardial Ischemia/ Reperfusion Injury via Sumoylation at Lysine 585 in Mice.

PubMed

Du, Yinping; Liu, Ping; Xu, Tongda; Pan, Defeng; Zhu, Hong; Zhai, Nana; Zhang, Yanbin; Li, Dongye

2018-01-01

The myocardial sarcoplasmic reticulum calcium ATPase (SERCA2a) is a pivotal pump responsible for calcium cycling in cardiomyocytes. The present study investigated the effect of luteolin (Lut) on restoring SERCA2a protein level and stability reduced by myocardial ischemia/reperfusion (I/R) injury. We verified a hypothesis that Lut protected against myocardial I/R injury by regulating SERCA2a SUMOylation. The hemodynamic data, myocardial infarct size of intact hearts, apoptotic analysis, mitochondrial membrane potential (ΔΨm), the level of SERCA2a SUMOylation, and the activity and expression of SERCA2a were examined in vivo and in vitro to clarify the cardioprotective effects of Lut after SUMO1 was knocked down or over-expressed. The putative SUMO conjugation sites in mouse SERCA2a were investigated as the possible regulatory mechanism of Lut. Initially, we found that Lut reversed the SUMOylation and stability of SERCA2a as well as the expression of SUMO1, which were reduced by I/R injury in vitro. Furthermore, Lut increased the expression and activity of SERCA2a partly through SUMO1, thus improving ΔΨm and reducing apoptotic cells in vitro and promoting the recovery of heart function and reducing infarct size in vivo. We also demonstrated that SUMO acceptor sites in mouse SERCA2a involving lysine 585, 480 and 571. Among the three acceptor sites, Lut enhanced SERCA2a stability via lysine 585. Our results suggest that Lut regulates SERCA2a through SUMOylation at lysine 585 to attenuate myocardial I/R injury. © 2018 The Author(s). Published by S. Karger AG, Basel.

Endocrine and other physiologic modulators of perinatal cardiomyocyte endowment

PubMed Central

Jonker, S S; Louey, S

2015-01-01

Immature contractile cardiomyocytes proliferate to rapidly increase cell number, establishing cardiomyocyte endowment in the perinatal period. Developmental changes in cellular maturation, size and attrition further contribute to cardiac anatomy. These physiological processes occur concomitant with a changing hormonal environment as the fetus prepares itself for the transition to extrauterine life. There are complex interactions between endocrine, hemodynamic and nutritional regulators of cardiac development. Birth has been long assumed to be the trigger for major differences between the fetal and postnatal cardiomyocyte growth patterns, but investigations in normally growing sheep and rodents suggest this may not be entirely true; in sheep, these differences are initiated before birth, while in rodents they occur after birth. The aim of this review is to draw together our understanding of the temporal regulation of these signals and cardiomyocyte responses relative to birth. Further, we consider how these dynamics are altered in stressed and suboptimal intrauterine environments. PMID:26432905

Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

PubMed Central

Wang, Zhonghai; Qin, Wan; Shao, Yonghong; Ma, Siyu; Borg, Thomas K.; Gao, Bruce Z.

2015-01-01

Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture. PMID:25767692

Pitx2 promotes heart repair by activating the antioxidant response after cardiac injury.

PubMed

Tao, Ge; Kahr, Peter C; Morikawa, Yuka; Zhang, Min; Rahmani, Mahdis; Heallen, Todd R; Li, Lele; Sun, Zhao; Olson, Eric N; Amendt, Brad A; Martin, James F

2016-06-02

Myocardial infarction results in compromised myocardial function and heart failure owing to insufficient cardiomyocyte self-renewal. Unlike many vertebrates, mammalian hearts have only a transient neonatal renewal capacity. Reactivating primitive reparative ability in the mature mammalian heart requires knowledge of the mechanisms that promote early heart repair. By testing an established Hippo-deficient heart regeneration mouse model for factors that promote renewal, here we show that the expression of Pitx2 is induced in injured, Hippo-deficient ventricles. Pitx2-deficient neonatal mouse hearts failed to repair after apex resection, whereas adult mouse cardiomyocytes with Pitx2 gain-of-function efficiently regenerated after myocardial infarction. Genomic analyses indicated that Pitx2 activated genes encoding electron transport chain components and reactive oxygen species scavengers. A subset of Pitx2 target genes was cooperatively regulated with the Hippo pathway effector Yap. Furthermore, Nrf2, a regulator of the antioxidant response, directly regulated the expression and subcellular localization of Pitx2. Pitx2 mutant myocardium had increased levels of reactive oxygen species, while antioxidant supplementation suppressed the Pitx2 loss-of-function phenotype. These findings reveal a genetic pathway activated by tissue damage that is essential for cardiac repair.

DYRK1A is a novel negative regulator of cardiomyocyte hypertrophy.

PubMed

Kuhn, Christian; Frank, Derk; Will, Rainer; Jaschinski, Christoph; Frauen, Robert; Katus, Hugo A; Frey, Norbert

2009-06-19

Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy. Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors.

DYRK1A Is a Novel Negative Regulator of Cardiomyocyte Hypertrophy*

PubMed Central

Kuhn, Christian; Frank, Derk; Will, Rainer; Jaschinski, Christoph; Frauen, Robert; Katus, Hugo A.; Frey, Norbert

2009-01-01

Activation of the phosphatase calcineurin and its downstream targets, transcription factors of the NFAT family, results in cardiomyocyte hypertrophy. Recently, it has been shown that the dual specificity tyrosine (Y) phosphorylation-regulated kinase 1A (DYRK1A) is able to antagonize calcineurin signaling by directly phosphorylating NFATs. We thus hypothesized that DYRK1A might modulate the hypertrophic response of cardiomyocytes. In a model of phenylephrine-induced hypertrophy, adenovirus-mediated overexpression of DYKR1A completely abrogated the hypertrophic response and significantly reduced the expression of the natriuretic peptides ANF and BNP. Furthermore, DYRK1A blunted cardiomyocyte hypertrophy induced by overexpression of constitutively active calcineurin and attenuated the induction of the hypertrophic gene program. Conversely, knockdown of DYRK1A, utilizing adenoviruses encoding for a specific synthetic miRNA, resulted in an increase in cell surface area accompanied by up-regulation of ANF- mRNA. Similarly, treatment of cardiomyocytes with harmine, a specific inhibitor of DYRK1A, revealed cardiomyocyte hypertrophy on morphological and molecular level. Moreover, constitutively active calcineurin led to robust induction of an NFAT-dependent luciferase reporter, whereas DYRK1A attenuated calcineurin-induced reporter activation in cardiomyocytes. Conversely, both knockdown and pharmacological inhibition of DYRK1A significantly augmented the effect of calcineurin in this assay. In summary, we identified DYRK1A as a novel negative regulator of cardiomyocyte hypertrophy. Mechanistically, this effect appears to be mediated via inhibition of NFAT transcription factors. PMID:19372220

Cardiomyocyte Ogt limits ventricular dysfunction in mice following pressure overload without affecting hypertrophy.

PubMed

Dassanayaka, Sujith; Brainard, Robert E; Watson, Lewis J; Long, Bethany W; Brittian, Kenneth R; DeMartino, Angelica M; Aird, Allison L; Gumpert, Anna M; Audam, Timothy N; Kilfoil, Peter J; Muthusamy, Senthilkumar; Hamid, Tariq; Prabhu, Sumanth D; Jones, Steven P

2017-05-01

The myocardial response to pressure overload involves coordination of multiple transcriptional, posttranscriptional, and metabolic cues. The previous studies show that one such metabolic cue, O-GlcNAc, is elevated in the pressure-overloaded heart, and the increase in O-GlcNAcylation is required for cardiomyocyte hypertrophy in vitro. Yet, it is not clear whether and how O-GlcNAcylation participates in the hypertrophic response in vivo. Here, we addressed this question using patient samples and a preclinical model of heart failure. Protein O-GlcNAcylation levels were increased in myocardial tissue from heart failure patients compared with normal patients. To test the role of OGT in the heart, we subjected cardiomyocyte-specific, inducibly deficient Ogt (i-cmOgt -/- ) mice and Ogt competent littermate wild-type (WT) mice to transverse aortic constriction. Deletion of cardiomyocyte Ogt significantly decreased O-GlcNAcylation and exacerbated ventricular dysfunction, without producing widespread changes in metabolic transcripts. Although some changes in hypertrophic and fibrotic signaling were noted, there were no histological differences in hypertrophy or fibrosis. We next determined whether significant differences were present in i-cmOgt -/- cardiomyocytes from surgically naïve mice. Interestingly, markers of cardiomyocyte dedifferentiation were elevated in Ogt-deficient cardiomyocytes. Although no significant differences in cardiac dysfunction were apparent after recombination, it is possible that such changes in dedifferentiation markers could reflect a larger phenotypic shift within the Ogt-deficient cardiomyocytes. We conclude that cardiomyocyte Ogt is not required for cardiomyocyte hypertrophy in vivo; however, loss of Ogt may exert subtle phenotypic differences in cardiomyocytes that sensitize the heart to pressure overload-induced ventricular dysfunction.

Noninvasive evaluation of contractile behavior of cardiomyocyte monolayers based on motion vector analysis.

PubMed

Hayakawa, Tomohiro; Kunihiro, Takeshi; Dowaki, Suguru; Uno, Hatsume; Matsui, Eriko; Uchida, Masashi; Kobayashi, Seiji; Yasuda, Akio; Shimizu, Tatsuya; Okano, Teruo

2012-01-01

A noninvasive method for the characterization of cardiomyocyte contractile behavior is presented. Light microscopic video images of cardiomyocytes were captured with a high-speed camera, and motion vectors (which have a velocity dimension) were calculated with a high spatiotemporal resolution using a block-matching algorithm. This method could extract contraction and relaxation motions of cardiomyocytes separately and evaluate characteristics such as the beating rate, orientation of contraction, beating cooperativity/homogeneity in the monolayer, and wave propagation of impulses. Simultaneous phase-contrast imaging and calcium (Ca2+) fluorescence measurements confirmed that the timing of the maximum shortening velocity of cardiomyocytes correlated well with intracellular Ca2+ transients. Based on our analysis, gap junction inhibitors, 1-heptanol (2 mM) or 18-β-glycyrrhetinic acid (30 μM), resulted in clear changes in beating cooperativity and the propagation pattern of impulses in the cardiomyocyte monolayer. Additionally, the time dependence of the motion vector length indicated a prolonged relaxation process in the presence of potassium (K+) channel blockers, dl-sotalol (1 μM), E-4031 (100 nM), or terfenadine (100 nM), reflecting the prolonged QT (Q wave and T wave) interval of cardiomyocytes. Effects of autonomic agents (acetylcholine or epinephrine [EPI]) or EPI and propranolol on cardiomyocytes were clearly detected by the alterations of beating rate and the motion vector length in contraction and relaxation processes. This method was noninvasive and could sensitively evaluate the contractile behavior of cardiomyocytes; therefore, it may be used to study and/or monitor cardiomyocyte tissue during prolonged culture periods and in screens for drugs that may alter the contraction of cardiomyocytes.

Developmental alterations in centrosome integrity contribute to the post-mitotic state of mammalian cardiomyocytes

PubMed Central

Zebrowski, David C; Vergarajauregui, Silvia; Wu, Chi-Chung; Piatkowski, Tanja; Becker, Robert; Leone, Marina; Hirth, Sofia; Ricciardi, Filomena; Falk, Nathalie; Giessl, Andreas; Just, Steffen; Braun, Thomas; Weidinger, Gilbert; Engel, Felix B

2015-01-01

Mammalian cardiomyocytes become post-mitotic shortly after birth. Understanding how this occurs is highly relevant to cardiac regenerative therapy. Yet, how cardiomyocytes achieve and maintain a post-mitotic state is unknown. Here, we show that cardiomyocyte centrosome integrity is lost shortly after birth. This is coupled with relocalization of various centrosome proteins to the nuclear envelope. Consequently, postnatal cardiomyocytes are unable to undergo ciliogenesis and the nuclear envelope adopts the function as cellular microtubule organizing center. Loss of centrosome integrity is associated with, and can promote, cardiomyocyte G0/G1 cell cycle arrest suggesting that centrosome disassembly is developmentally utilized to achieve the post-mitotic state in mammalian cardiomyocytes. Adult cardiomyocytes of zebrafish and newt, which are able to proliferate, maintain centrosome integrity. Collectively, our data provide a novel mechanism underlying the post-mitotic state of mammalian cardiomyocytes as well as a potential explanation for why zebrafish and newts, but not mammals, can regenerate their heart. DOI: http://dx.doi.org/10.7554/eLife.05563.001 PMID:26247711

1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells.

PubMed

Okolotowicz, Karl J; Bushway, Paul; Lanier, Marion; Gilley, Cynthia; Mercola, Mark; Cashman, John R

2015-09-01

Cardiomyopathy is the leading cause of death worldwide. Despite progress in medical treatments, heart transplantation is one of the only current options for those with infarcted heart muscle. Stem cell differentiation technology may afford cell-based therapeutics that may lead to the generation of new, healthy heart muscle cells from undifferentiated stem cells. Our approach is to use small molecules to stimulate stem cell differentiation. Herein, we describe a novel class of 1,5-disubstituted benzimidazoles that induce differentiation of stem cells into cardiac cells. We report on the evaluation in vitro for cardiomyocyte differentiation and describe structure-activity relationship results that led to molecules with drug-like properties. The results of this study show the promise of small molecules to direct stem cell lineage commitment, to probe signaling pathways and to develop compounds for the stimulation of stem cells to repair damaged heart tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bone marrow-derived mesenchymal stem cells protect against lung injury in a mouse model of bronchopulmonary dysplasia.

PubMed

Luan, Yun; Ding, Wei; Ju, Zhi-Ye; Zhang, Zhao-Hua; Zhang, Xue; Kong, Feng

2015-03-01

The aim of the present study was to investigate the effect of bone marrow‑derived mesenchymal stem cells (BMSCs) in the treatment of lung injury in a mouse model of bronchopulmonary dysplasia (BPD) and examine the underlying mechanisms. A mouse model of BPD was created using continuous exposure to high oxygen levels for 14 days. BMSCs were isolated, cultured and then labeled with green fluorescent protein. Cells (1x106) were subsequently injected intravenously 1 h prior to high oxygen treatment. Animals were randomly divided into three groups (n=5 in each): Control group, BPD model group and BMSC injection group. At two weeks post‑treatment, the expression of transforming growth factor‑β1 (TGF‑β1), vascular endothelial growth factor (VEGF) and von Willebrand factor (vWF) was detected using immunohistochemical staining and immunofluorescence. Compared with the BPD model group, the body weight, airway structure and levels of TGF‑β1 and VEGF were significantly improved in the BMSC‑treated group. Immunofluorescence observations indicated that BMSCs were able to differentiate into cells expressing vWF and VEGF, which are markers of vascular tissues. The present study demonstrated that intravenous injection of BMSCs significantly improved lung damage in a neonatal mouse model of BPD at 14 days following hyperoxia‑induced injury. This provides novel information which may be used to guide further investigation into the use of stem cells in BPD.

Effect of a glutathione S-transferase inhibitor on oxidative stress and ischemia-reperfusion-induced apoptotic signalling of cultured cardiomyocytes

PubMed Central

Röth, E; Marczin, N; Balatonyi, B; Ghosh, S; Kovács, V; Alotti, N; Borsiczky, B; Gasz, B

2011-01-01

Oxidative stress and ischemia-reperfusion (I/R) injury are crucial in the pathogenesis of cardiovascular diseases. The antioxidant glutathione S-transferase (GST) is responsible for the high-capacity metabolic inactivation of electrophilic compounds and toxic substrates. The main objective of the present study was to examine the effect of GST inhibition (with the administration of ethacrynic acid [EA]) on the viability and apoptosis of cardiomyocytes when these cells are exposed to various stress components of I/R and mitogen-activated protein kinase (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase [ERK]) inhibitors. The primary culture of neonatal rat cardiomyocytes was divided into six experimental groups: control group of cells (group 1), cells exposed to H2O2 (group 2), I/R (group 3), I/R and EA (group 4), H2O2 coupled with EA (group 5), and EA alone (group 6). The viability of cardiomyocytes was determined using a colorimetric MTT assay. The apoptosis ratio was evaluated via fluorescein isothiocyanate-labelled annexin V and propidium iodide staining. c-Jun N-terminal kinase, p38, Akt/protein kinase B and ERK/p42-p44 transcription factors were monitored with flow cytometry. c-Jun N-terminal kinase activation increased due to GST inhibition during I/R. EA administration led to a significant increase in p38 activation following both H2O2 treatment and I/R. ERK phosphorylation increased when GST was exposed to I/R. A pronounced decrease in Akt phosphorylation was observed when cells were cotreated with EA and H2O2. GST plays an important role as a regulator of mitogen-activated protein kinase pathways in I/R injury. PMID:22065940

Mesenchymal Stem Cells and Cardiomyocytes Interplay to Prevent Myocardial Hypertrophy

PubMed Central

Tan, Xueying; Zhang, Yong; Li, Xingda; Wang, Xinyue; Zhu, Jiuxin; Wang, Yang; Yang, Fan; Wang, Baoqiu; Liu, Yanju; Xu, Chaoqian; Pan, Zhenwei; Wang, Ning; Yang, Baofeng

2015-01-01

Bone marrow-derived mesenchymal stem cells (BMSCs) have emerged as a promising therapeutic strategy for cardiovascular disease. However, there is no evidence so far that BMSCs can heal pathological myocardial hypertrophy. In this study, BMSCs were indirectly cocultured with neonatal rat ventricular cardiomyocytes (NRVCs) in vitro or intramyocardially transplanted into hypertrophic hearts in vivo. The results showed that isoproterenol (ISO)-induced typical hypertrophic characteristics of cardiomyocytes were prevented by BMSCs in the coculture model in vitro and after BMSC transplantation in vivo. Furthermore, activation of the Ca2+/calcineurin/nuclear factor of activated T cells cytoplasmic 3 (NFATc3) hypertrophic pathway in NRVCs was abrogated in the presence of BMSCs both in vitro and in vivo. Interestingly, inhibition of vascular endothelial growth factor (VEGF) release from BMSCs, but not basic fibroblast growth factor and insulin-like growth factor 1, abolished the protective effects of BMSCs on cardiomyocyte hypertrophy. Consistently, VEGF administration attenuated ISO-induced enlargement of cellular size; the upregulation of atrial natriuretic peptide, brain natriuretic peptide, and β-myosin heavy chain expression; and the activation of Ca2+/calcineurin/NFATc3 hypertrophic pathways, and these pathways can be abrogated by blocking VEGFR-1 in cardiomyocytes, indicating that VEGF receptor 1 is involved in the antihypertrophic role of VEGF. We further found that the ample VEGF secretion contributing to the antihypertrophic effects of BMSCs originates from the crosstalk of BMSCs and cardiac cells but not BMSCs or cardiomyocytes alone. Interplay of mesenchymal stem cells with cardiomyocytes produced synergistic effects on VEGF release. In summary, crosstalk between mesenchymal stem cells and cardiomyocytes contributes to the inhibition of myocardial hypertrophy via inhibiting Ca2+/calcineurin/NFATc3 hypertrophic pathways in cardiac cells. These results provide the

Differential activation of natriuretic peptide receptors modulates cardiomyocyte proliferation during development

PubMed Central

Becker, Jason R.; Chatterjee, Sneha; Robinson, Tamara Y.; Bennett, Jeffrey S.; Panáková, Daniela; Galindo, Cristi L.; Zhong, Lin; Shin, Jordan T.; Coy, Shannon M.; Kelly, Amy E.; Roden, Dan M.; Lim, Chee Chew; MacRae, Calum A.

2014-01-01

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways. PMID:24353062

Analysis of early thrombus dynamics in a humanized mouse laser injury model.

PubMed

Wang, Weiwei; Lindsey, John P; Chen, Jianchun; Diacovo, Thomas G; King, Michael R

2014-01-01

Platelet aggregation and thrombus formation at the site of injury is a dynamic process that involves the continuous addition of new platelets as well as thrombus rupture. In the early stages of hemostasis (within minutes after vessel injury) this process can be visualized by transfusing fluorescently labeled human platelets and observing their deposition and detachment. These two counterbalancing events help the developing thrombus reach a steady-state morphology, where it is large enough to cover the injured vessel surface but not too large to form a severe thrombotic occlusion. In this study, the spatial and temporal aspects of early stage thrombus dynamics which result from laser-induced injury on arterioles of cremaster muscle in the humanized mouse were visualized using fluorescent microscopy. It was found that rolling platelets show preference for the upstream region while tethering/detaching platelets were primarily found downstream. It was also determined that the platelet deposition rate is relatively steady, whereas the effective thrombus coverage area does not increase at a constant rate. By introducing a new method to graphically represent the real time in vivo physiological shear stress environment, we conclude that the thrombus continuously changes shape by regional growth and decay, and neither dominates in the high shear stress region.

Non-invasive detection of human cardiomyocyte death using methylation patterns of circulating DNA.

PubMed

Zemmour, Hai; Planer, David; Magenheim, Judith; Moss, Joshua; Neiman, Daniel; Gilon, Dan; Korach, Amit; Glaser, Benjamin; Shemer, Ruth; Landesberg, Giora; Dor, Yuval

2018-04-24

Detection of cardiomyocyte death is crucial for the diagnosis and treatment of heart disease. Here we use comparative methylome analysis to identify genomic loci that are unmethylated specifically in cardiomyocytes, and develop these as biomarkers to quantify cardiomyocyte DNA in circulating cell-free DNA (cfDNA) derived from dying cells. Plasma of healthy individuals contains essentially no cardiomyocyte cfDNA, consistent with minimal cardiac turnover. Patients with acute ST-elevation myocardial infarction show a robust cardiac cfDNA signal that correlates with levels of troponin and creatine phosphokinase (CPK), including the expected elevation-decay dynamics following coronary angioplasty. Patients with sepsis have high cardiac cfDNA concentrations that strongly predict mortality, suggesting a major role of cardiomyocyte death in mortality from sepsis. A cfDNA biomarker for cardiomyocyte death may find utility in diagnosis and monitoring of cardiac pathologies and in the study of normal human cardiac physiology and development.

BAMOS: A recording application for BAsso MOuse scale of locomotion in experimental models of spinal cord injury.

PubMed

Gómez, Alberto; Nieto-Díaz, Manuel; Del Águila, Ángela; Arias, Enrique

2018-05-01

Transparency in science is increasingly a hot topic. Scientists are required to show not only results but also evidence of how they have achieved these results. In experimental studies of spinal cord injury, there are a number of standardized tests, such as the Basso-Beattie-Bresnahan locomotor rating scale for rats and Basso Mouse Scale for mice, which researchers use to study the pathophysiology of spinal cord injury and to evaluate the effects of experimental therapies. Although the standardized data from the Basso-Beattie-Bresnahan locomotor rating scale and the Basso Mouse Scale are particularly suited for storage and sharing in databases, systems of data acquisition and repositories are still lacking. To the best of our knowledge, both tests are usually conducted manually, with the data being recorded on a paper form, which may be documented with video recordings, before the data is transferred to a spreadsheet for analysis. The data thus obtained is used to compute global scores, which is the information that usually appears in publications, with a wealth of information being omitted. This information may be relevant to understand locomotion deficits or recovery, or even important aspects of the treatment effects. Therefore, this paper presents a mobile application to record and share Basso Mouse Scale tests, meeting the following criteria: i) user-friendly; ii) few hardware requirements (only a smartphone or tablet with a camera running under Android Operating System); and iii) based on open source software such as SQLite, XML, Java, Android Studio and Android SDK. The BAMOS app can be downloaded and installed from the Google Market repository and the app code is available at the GitHub repository. The BAMOS app demonstrates that mobile technology constitutes an opportunity to develop tools for aiding spinal cord injury scientists in recording and sharing experimental data. Copyright © 2018 Elsevier Ltd. All rights reserved.

Oncostatin M (OSM) protects against cardiac ischaemia/reperfusion injury in diabetic mice by regulating apoptosis, mitochondrial biogenesis and insulin sensitivity.

PubMed

Sun, Dongdong; Li, Shuang; Wu, Hao; Zhang, Mingming; Zhang, Xiaotian; Wei, Liping; Qin, Xing; Gao, Erhe

2015-06-01

Oncostatin M (OSM) exhibits many unique biological activities by activating Oβ receptor. However, its role in myocardial I/R injury in diabetic mice remains unknown. The involvement of OSM was assessed in diabetic mice which underwent myocardial I/R injury by OSM treatment or genetic deficiency of OSM receptor Oβ. Its mechanism on cardiomyocyte apoptosis, mitochondrial biogenesis and insulin sensitivity were further studied. OSM alleviated cardiac I/R injury by inhibiting cardiomyocyte apoptosis through inhibition of inositol pyrophosphate 7 (IP7) production, thus activating PI3K/Akt/BAD pathway, decreasing Bax expression while up-regulating Bcl-2 expression and decreasing the ratio of Bax to Bcl-2 in db/db mice. OSM enhanced mitochondrial biogenesis and mitochondrial function in db/db mice subjected to cardiac I/R injury. On the contrary, OSM receptor Oβ knockout exacerbated cardiac I/R injury, increased IP7 production, enhanced cardiomyocyte apoptosis, impaired mitochondrial biogenesis, glucose homoeostasis and insulin sensitivity in cardiac I/R injured diabetic mice. Inhibition of IP7 production by TNP (IP6K inhibitor) exerted similar effects of OSM. The mechanism of OSM on cardiac I/R injury in diabetic mice is partly associated with IP7/Akt and adenine mononucleotide protein kinase/PGC-1α pathway. OSM protects against cardiac I/R Injury by regulating apoptosis, insulin sensitivity and mitochondrial biogenesis in diabetic mice through inhibition of IP7 production. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture.

PubMed

Orlandi, Alessia; Pagani, Francesca; Avitabile, Daniele; Bonanno, Giuseppina; Scambia, Giovanni; Vigna, Elisa; Grassi, Francesca; Eusebi, Fabrizio; Fucile, Sergio; Pesce, Maurizio; Capogrossi, Maurizio C

2008-04-01

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34(+) cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34(+) cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP(+) cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca(2+)] ([Ca(2+)](i)) homeostasis by [Ca(2+)](i) imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP(+) cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca(2+)](i) transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca(2+)](i) oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34(+) cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.

Inhibition of microRNA-1 attenuates hypoxia/re-oxygenation-induced apoptosis of cardiomyocytes by directly targeting Bcl-2 but not GADD45Beta

PubMed Central

Zhai, Changlin; Tang, Guanmin; Peng, Lei; Hu, Huilin; Qian, Gang; Wang, Shijun; Yao, Jiankang; Zhang, Xiaoping; Fang, Ying; Yang, Shuang; Zhang, Xiumei

2015-01-01

MicroRNAs are small non-coding RNAs that are able to regulate gene expression and play important roles in some biological and pathological processes, including the myocardial ischemia/reperfusion (I/R) injury. Recent findings demonstrated that miR-1 exacerbated I/R-induced injury. This study was to investigate theanti-apoptotic property of miR-1 inhibition and the potential regulatory mechanism. Results showed miR-1 expression reduced in the heart of rats undergoing myocardial I/R and the cardiomyocytes receiving hypoxia/reoxygenation (H/R) injury, but the serum miR-1 expression increased. The targets of miR-1 were predicted by cDNA microarray, and Bcl-2 and GADD45β were selected as candidate targets. Western blot assay and qPCR showed Bcl-2 and GADD45β protein and mRNA expressions increased after I/R injury and H/R injury. Bcl-2 was a direct target of miR-1 as shown in previous studies. Luciferase assay and Western blot assay revealed GADD45β was a direct target of miR-1, and miR-1 suppressed GADD45β expression via binding to its 3’UTR. Furthermore, miR-1 inhibition increased Bcl-2 expression and reduced IA/AAR (infarct area/area at risk) ratio and cell apoptosis in rats undergoing myocardial I/R as well as in cardiomyocytes receiving H/R injury. Importantly, Bcl-2 knockdown restored these consequences following miR-1 inhibition. However, GADD45β knockdown reduced IA/AAR ratio and cell apoptosis in vivo and in vitro, but failed torestore above consequences after miR-1 inhibition. In conclusion miR-1 inhibition protects against H/R-induced apoptosis of myocytes by directly targeting Bcl-2 but not GADD45β. PMID:26692938

Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

PubMed

Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

2016-01-01

Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid

PubMed Central

Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

2016-01-01

Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA. PMID:27057269

Low molecular weight (LMW) heparin inhibits injury-induced femoral artery remodeling in mouse via upregulating CD44 expression.

PubMed

Zhao, Gaofeng; Shaik, Rahamthulla S; Zhao, Hang; Beagle, John; Kuo, Shuennwen; Hales, Charles A

2011-05-01

The mechanism of postangioplasty restenosis remains poorly understood. Low molecular weight (LMW) heparin has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), which is the principal characteristic of restenosis. Studies have shown that LMW heparin could bind to CD44. We hypothesized that LMW heparin might modulate CD44 expression thereby decreasing vascular remodeling. Vascular remodeling was induced in CD44(+/+) and CD44(-/-) mice and treated with LMW heparin. The arteries were harvested for histologic assessment and determination of CD44 expression. Bone marrow transplantation was introduced to further explore the role and functional sites of CD44. Effects of LMW heparin on growth capacity, CD44 expression were further studied using the cultured mouse VSMCs. Transluminal injury induced remarkable remodeling in mouse femoral artery (sham wall thickness percentage [WT%]: 3.4 ± 1.2% vs injury WT%: 31.8 ± 4.7%; P < .001). LMW heparin reduced the remodeling significantly (WT%: 17.8 ± 3.5%, P < .005). CD44(-/-) mice demonstrated considerably thicker arterial wall remodeling (WT%: 46.2 ± 7.6%, P = .0035), and CD44-chimeric mice exhibited equal contributions of the local and circulating CD44 signal to the neointima formation. LMW heparin markedly upregulated CD44 expression in the injured femoral arteries. In vitro, LMW heparin decreased mouse VSMC growth capacity and upregulated its CD44 expression simultaneously in a dose-dependent and time-dependent manner, which could be partially blocked by CD44 inhibitor. LMW heparin inhibits injury-induced femoral artery remodeling, at least partially, by upregulating CD44 expression. Copyright © 2011. Published by Mosby, Inc.

Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model

PubMed Central

Tagge, Chad A; Fisher, Andrew M; Minaeva, Olga V; Gaudreau-Balderrama, Amanda; Moncaster, Juliet A; Zhang, Xiao-Lei; Wojnarowicz, Mark W; Casey, Noel; Lu, Haiyan; Kokiko-Cochran, Olga N; Saman, Sudad; Ericsson, Maria; Onos, Kristen D; Veksler, Ronel; Senatorov, Vladimir V; Kondo, Asami; Zhou, Xiao Z; Miry, Omid; Vose, Linnea R; Gopaul, Katisha R; Upreti, Chirag; Nowinski, Christopher J; Cantu, Robert C; Alvarez, Victor E; Hildebrandt, Audrey M; Franz, Erich S; Konrad, Janusz; Hamilton, James A; Hua, Ning; Tripodis, Yorghos; Anderson, Andrew T; Howell, Gareth R; Kaufer, Daniela; Hall, Garth F; Lu, Kun P; Ransohoff, Richard M; Cleveland, Robin O; Kowall, Neil W; Stein, Thor D; Lamb, Bruce T; Huber, Bertrand R; Moss, William C; Friedman, Alon; Stanton, Patric K; McKee, Ann C; Goldstein, Lee E

2018-01-01

Abstract The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood–brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood–brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or

Concussion, microvascular injury, and early tauopathy in young athletes after impact head injury and an impact concussion mouse model.

PubMed

Tagge, Chad A; Fisher, Andrew M; Minaeva, Olga V; Gaudreau-Balderrama, Amanda; Moncaster, Juliet A; Zhang, Xiao-Lei; Wojnarowicz, Mark W; Casey, Noel; Lu, Haiyan; Kokiko-Cochran, Olga N; Saman, Sudad; Ericsson, Maria; Onos, Kristen D; Veksler, Ronel; Senatorov, Vladimir V; Kondo, Asami; Zhou, Xiao Z; Miry, Omid; Vose, Linnea R; Gopaul, Katisha R; Upreti, Chirag; Nowinski, Christopher J; Cantu, Robert C; Alvarez, Victor E; Hildebrandt, Audrey M; Franz, Erich S; Konrad, Janusz; Hamilton, James A; Hua, Ning; Tripodis, Yorghos; Anderson, Andrew T; Howell, Gareth R; Kaufer, Daniela; Hall, Garth F; Lu, Kun P; Ransohoff, Richard M; Cleveland, Robin O; Kowall, Neil W; Stein, Thor D; Lamb, Bruce T; Huber, Bertrand R; Moss, William C; Friedman, Alon; Stanton, Patric K; McKee, Ann C; Goldstein, Lee E

2018-02-01

The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or

Inhibition of TGFβ signaling increases direct conversion of fibroblasts to induced cardiomyocytes.

PubMed

Ifkovits, Jamie L; Addis, Russell C; Epstein, Jonathan A; Gearhart, John D

2014-01-01

Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic.

Inhibition of TGFβ Signaling Increases Direct Conversion of Fibroblasts to Induced Cardiomyocytes

PubMed Central

Ifkovits, Jamie L.; Addis, Russell C.; Epstein, Jonathan A.; Gearhart, John D.

2014-01-01

Recent studies have been successful at utilizing ectopic expression of transcription factors to generate induced cardiomyocytes (iCMs) from fibroblasts, albeit at a low frequency in vitro. This work investigates the influence of small molecules that have been previously reported to improve differentiation to cardiomyocytes as well as reprogramming to iPSCs in conjunction with ectopic expression of the transcription factors Hand2, Nkx2.5, Gata4, Mef2C, and Tbx5 on the conversion to functional iCMs. We utilized a reporter system in which the calcium indicator GCaMP is driven by the cardiac Troponin T promoter to quantify iCM yield. The TGFβ inhibitor, SB431542 (SB), was identified as a small molecule capable of increasing the conversion of both mouse embryonic fibroblasts and adult cardiac fibroblasts to iCMs up to ∼5 fold. Further characterization revealed that inhibition of TGFβ by SB early in the reprogramming process led to the greatest increase in conversion of fibroblasts to iCMs in a dose-responsive manner. Global transcriptional analysis at Day 3 post-induction of the transcription factors revealed an increased expression of genes associated with the development of cardiac muscle in the presence of SB compared to the vehicle control. Incorporation of SB in the reprogramming process increases the efficiency of iCM generation, one of the major goals necessary to enable the use of iCMs for discovery-based applications and for the clinic. PMID:24586958

IL-21 promotes myocardial ischaemia/reperfusion injury through the modulation of neutrophil infiltration.

PubMed

Wang, Kejing; Wen, Shuang; Jiao, Jiao; Tang, Tingting; Zhao, Xin; Zhang, Min; Lv, Bingjie; Lu, Yuzhi; Zhou, Xingdi; Li, Jingyong; Nie, Shaofang; Liao, Yuhua; Wang, Qing; Tu, Xin; Mallat, Ziad; Xia, Ni; Cheng, Xiang

2018-04-01

The immune system plays an important role in driving the acute inflammatory response following myocardial ischaemia/reperfusion injury (MIRI). IL-21 is a pleiotropic cytokine with multiple immunomodulatory effects, but its role in MIRI is not known. Myocardial injury, neutrophil infiltration and the expression of neutrophil chemokines KC (CXCL1) and MIP-2 (CXCL2) were studied in a mouse model of MIRI. Effects of IL-21 on the expression of KC and MIP-2 in neonatal mouse cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were determined by real-time PCR and ELISA. The signalling mechanisms underlying these effects were explored by western blot analysis. IL-21 was elevated within the acute phase of murine MIRI. Neutralization of IL-21 attenuated myocardial injury, as illustrated by reduced infarct size, decreased cardiac troponin T levels and improved cardiac function, whereas exogenous IL-21 administration exerted opposite effects. IL-21 increased the infiltration of neutrophils and increased the expression of KC and MIP-2 in myocardial tissue following MIRI. Moreover, neutrophil depletion attenuated the IL-21-induced myocardial injury. Mechanistically, IL-21 increased the production of KC and MIP-2 in neonatal CMs and CFs, and enhanced neutrophil migration, as revealed by the migration assay. Furthermore, we demonstrated that this IL-21-mediated increase in chemokine expression involved the activation of Akt/NF-κB signalling in CMs and p38 MAPK/NF-κB signalling in CFs. Our data provide novel evidence that IL-21 plays a pathogenic role in MIRI, most likely by promoting cardiac neutrophil infiltration. Therefore, targeting IL-21 may have therapeutic potential as a treatment for MIRI. This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc. © 2017 The British Pharmacological Society.

[Over-expression of BDNF inhibits angiotensin II-induced apoptosis of cardiomyocytes in SD rats].

PubMed

Cao, Jingli; Wu, Yingfeng; Liu, Geming; Li, Zhenlong

2018-03-01

Objective To investigate the role and molecular mechanism of brain-derived neurotrophic factor (BDNF) against the process of cardiomyocyte hypertrophy and apoptosis. Methods Cardiomyocyte hypertrophy were estabolished by angiotensin II (Ang II) in neonatal cardiomyocytes in vitro and incomplete ligature of abdominal aorta of SD rats in vivo. BDNF over-expressing recombinant vector pcDNA5-BDNF was transfected into cardiomyocytes by liposomes. Immunofluorescence staining was used to detect the effect of BDNF transfection on the surface area of myocardial cells. The effect of BDNF transfection on the apoptosis of cardiomyocytes was assayed by flow cytometry. Real-time fluorescent quantitative PCR was performed to detect the effect of over-expression of BDNF on the expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNAs in cardiomyocytes. Western blot assay was used to observe the changes of BDNF, ANP and BNP, calmodulin kinase 2 (CaMK2) and phosphorylated calmodulin kinase 2 (p-CaMK2), calcineurin (CaN), p-CaN, nuclear factor of activated T cells 3 (NFATC3) and p-NFATC3 protein expressions in the myocardial tissues and cardiomyocytes. Results The expression of BDNF protein increased significantly in cardiac hypertrophy animal and cell models in a time-dependent manner. Compared with the untransfected control cardiomyocytes, the surface area of cardiomyocytes, the rate of apoptosis, the levels of ANP and BNP mRNA and protein expression, the levels of p-CaMK2 and CaN protein in the BDNF over-expressed cardiomyocytes were remarkably reduced, while the level of p-NFATC3 protein rose significantly. Conclusion BDNF inhibits the apoptosis of cardiomyocytes induced by Ang II, and it plays the role by inhibiting CaMK2 and CaN signaling pathways.

Mapping of Redox State of Mitochondrial Cytochromes in Live Cardiomyocytes Using Raman Microspectroscopy

PubMed Central

Brazhe, Nadezda A.; Treiman, Marek; Brazhe, Alexey R.; Find, Ninett L.; Maksimov, Georgy V.; Sosnovtseva, Olga V.

2012-01-01

This paper presents a nonivasive approach to study redox state of reduced cytochromes , and of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we perform studies of rod- and round-shaped cardiomyocytes, representing different morphological and functional states. Raman mapping and cluster analysis reveal that these cardiomyocytes differ in the amounts of reduced cytochromes , and . The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes , and in cell center and periphery. Moreover, by means of Raman spectroscopy we demonstrated the decrease in the relative amounts of reduced cytochromes , and in the rod-shaped cardiomyocytes caused by H2O2-induced oxidative stress before any visible changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data. PMID:22957018

Effect of electroacupuncture on brain-derived neurotrophic factor mRNA expression in mouse hippocampus following cerebral ischemia-reperfusion injury.

PubMed

Zhao, Jianxin; Xu, Huazhou; Tian, Yuanxiang; Hu, Manxiang; Xiao, Hongling

2013-04-01

This work aims to observe the effects of electroacupuncture on brain-derived neurotrophic factor (BDNF) mRNA expression in mouse hippocampus following cerebral ischemia-reperfusion injury. The models of mouse cerebral ischemia-reperfusion injury were established. A total of 96 healthy mice were randomly assigned into 4 groups, namely, the sham surgery, model, model + electroacupuncture, and mode + hydergine groups. Mice in the model + electroacupuncture group were treated through electroacupuncture at the Shenshu (BL 23), Geshu (BL 17), and Baihui (GV 20) acupoints. Mice in the model+hydergine group were intragastrically administered with hydergine (0.77 mg/kg(-1) x day(-1)). The levels of BDNF mRNA expressions in the hippocampus were ana lyzed through a semi-quantitative reverse transcription-polymerase chain reaction assay on days 1 and 7 after the surgeries. BDNF mRNA expressions in the mouse hippocampus of the model group on days 1 and 7 after the surgery were higher than those of the sham surgery group (both P < 0.01). On days 1 and 7 of the electroacupuncture treatment, BDNF mRNA expression in the mouse hippocampus of the model + electroacupuncture group was significantly elevated compared with the model group (both P < 0.01) or the model + hydergine group (both P < 0.01). On days 1 and 7 of the hydergine treatment, BDNF mRNA expression in the mouse hippocampus of the model + hydergine group tended to increase compared with the model group; however, statistical significance was not achieved (both P > 0.05). Electroacupuncture treatment enhances endogenous BDNF expression, which may improve the survival environment for intracerebral neurons and inhibit the apoptosis of hippocampal cells.

Pim-2 protects H9c2 cardiomyocytes from hypoxia/reoxygenation-induced apoptosis via downregulation of Bim expression.

PubMed

Xu, Yan; Xing, Yawei; Xu, Yanjie; Huang, Chahua; Bao, Huihui; Hong, Kui; Cheng, Xiaoshu

2016-12-01

We know that silencing Bim, a pro-apoptosis protein, significantly attenuates glucose and oxygen-deprived induced apoptosis in cardiomyocytes. However, the mechanisms underlying the regulation of the Bim activation in the heart have remained unknown. Pim-2 is one of three Pim serine/threonine kinase family members thought to be involved in cell survival and proliferation. H9c2 cardiomyocytes were subjected to a hypoxia/reoxygenation (H/R) condition in vitro, mimicking ischemic/reperfusion injury in vivo. H/R augmented the expression of Bim, Cyt C, and Pim-2 and induced H9c2 cell apoptosis. Overexpression of Pim-2 attenuated apoptosis which induced by H/R in H9c2 cells, via downregulation of Bim and Cyt C expression. Silencing of Pim-2 promoted H/R-induced apoptosis via upregulation of Bim and Cyt C expression. Co-IP revealed the interaction between Pim-2 and Bim protein, with Bim Ser 65 phosphorylated by Pim-2. Furthermore, blocking proteasome activity by MG132 prevented Bim degradation, and Bim S65A mutation could reverse the anti-apoptotic role of Pim-2 which induced by H/R. These data demonstrated that Pim-2 is a novel Bim-interacting protein, which negatively regulates Bim degradation and protects H9c2 cardiomyocytes from H/R-induced apoptosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Lipocalin-2 Induces Cardiomyocyte Apoptosis by Increasing Intracellular Iron Accumulation*

PubMed Central

Xu, Guoxiong; Ahn, JinHee; Chang, SoYoung; Eguchi, Megumi; Ogier, Arnaud; Han, SungJun; Park, YoungSam; Shim, ChiYoung; Jang, YangSoo; Yang, Bo; Xu, Aimin; Wang, Yu; Sweeney, Gary

2012-01-01

Our objective was to determine whether lipocalin-2 (Lcn2) regulates cardiomyocyte apoptosis, the mechanisms involved, and the functional significance. Emerging evidence suggests that Lcn2 is a proinflammatory adipokine associated with insulin resistance and obesity-related complications, such as heart failure. Here, we used both primary neonatal rat cardiomyocytes and H9c2 cells and demonstrated for the first time that Lcn2 directly induced cardiomyocyte apoptosis, an important component of cardiac remodeling leading to heart failure. This was shown by detection of DNA fragmentation using TUNEL assay, phosphatidylserine exposure using flow cytometry to detect annexin V-positive cells, caspase-3 activity using enzymatic assay and immunofluorescence, and Western blotting for the detection of cleaved caspase-3. We also observed that Lcn2 caused translocation of the proapoptotic protein Bax to mitochondria and disruption of mitochondrial membrane potential. Using transient transfection of GFP-Bax, we confirmed that Lcn2 induced co-localization of Bax with MitoTracker® dye. Importantly, we used the fluorescent probe Phen Green SK to demonstrate an increase in intracellular iron in response to Lcn2, and depleting intracellular iron using an iron chelator prevented Lcn2-induced cardiomyocyte apoptosis. Administration of recombinant Lcn2 to mice for 14 days increased cardiomyocyte apoptosis as well as an acute inflammatory response with compensatory changes in cardiac functional parameters. In conclusion, Lcn2-induced cardiomyocyte apoptosis is of physiological significance and occurs via a mechanism involving elevated intracellular iron levels and Bax translocation. PMID:22117066

Effects of Acanthopanax senticosus on Brain Injury Induced by Simulated Spatial Radiation in Mouse Model Based on Pharmacokinetics and Comparative Proteomics

PubMed Central

Cheng, Cuilin; Baranenko, Denis; Wang, Jiaping; Li, Yongzhi; Lu, Weihong

2018-01-01

The active compounds in Acanthopanax senticosus (AS) have different pharmacokinetic characteristics in mouse models. Cmax and AUC of Acanthopanax senticosus polysaccharides (ASPS) were significantly reduced in radiation-injured mice, suggesting that the blood flow of mouse was blocked or slowed, due to the pathological state of ischemia and hypoxia, which are caused by radiation. In contrast, the ability of various metabolizing enzymes to inactivate, capacity of biofilm transport decrease, and lessening of renal blood flow accounts for radiation, resulting in the accumulation of syringin and eleutheroside E in the irradiated mouse. Therefore, there were higher pharmacokinetic parameters—AUC, MRT, and t1/2 of the two compounds in radiation-injured mouse, when compared with normal mouse. In order to investigate the intrinsic mechanism of AS on radiation injury, AS extract’s protective effects on brain, the main part of mouse that suffered from radiation, were explored. The function of AS extract in repressing expression changes of radiation response proteins in prefrontal cortex (PFC) of mouse brain included tubulin protein family (α-, β-tubulin subunits), dihydropyrimidinase-related protein 2 (CRMP2), γ-actin, 14-3-3 protein family (14-3-3ζ, ε), heat shock protein 90β (HSP90β), and enolase 2. The results demonstrated the AS extract had positive effects on nerve cells’ structure, adhesion, locomotion, fission, and phagocytosis, through regulating various action pathways, such as Hippo, phagosome, PI3K/Akt (phosphatidylinositol 3 kinase/protein kinase B), Neurotrophin, Rap1 (Ras-related protein RAP-1A), gap junction glycolysis/gluconeogenesis, and HIF-1 (Hypoxia-inducible factor 1) signaling pathways to maintain normal mouse neurological activity. All of the results indicated that AS may be a promising alternative medicine for the treatment of radiation injury in mouse brain. It would be tested that whether the bioactive ingredients of AS could be

Mitochondrial Cardiomyopathy Caused by Elevated Reactive Oxygen Species and Impaired Cardiomyocyte Proliferation.

PubMed

Zhang, Donghui; Li, Yifei; Heims-Waldron, Danielle; Bezzerides, Vassilios; Guatimosim, Silvia; Guo, Yuxuan; Gu, Fei; Zhou, Pingzhu; Lin, Zhiqiang; Ma, Qing; Liu, Jianming; Wang, Da-Zhi; Pu, William T

2018-01-05

Although mitochondrial diseases often cause abnormal myocardial development, the mechanisms by which mitochondria influence heart growth and function are poorly understood. To investigate these disease mechanisms, we studied a genetic model of mitochondrial dysfunction caused by inactivation of Tfam (transcription factor A, mitochondrial), a nuclear-encoded gene that is essential for mitochondrial gene transcription and mitochondrial DNA replication. Tfam inactivation by Nkx2.5 Cre caused mitochondrial dysfunction and embryonic lethal myocardial hypoplasia. Tfam inactivation was accompanied by elevated production of reactive oxygen species (ROS) and reduced cardiomyocyte proliferation. Mosaic embryonic Tfam inactivation confirmed that the block to cardiomyocyte proliferation was cell autonomous. Transcriptional profiling by RNA-seq demonstrated the activation of the DNA damage pathway. Pharmacological inhibition of ROS or the DNA damage response pathway restored cardiomyocyte proliferation in cultured fetal cardiomyocytes. Neonatal Tfam inactivation by AAV9-cTnT-Cre caused progressive, lethal dilated cardiomyopathy. Remarkably, postnatal Tfam inactivation and disruption of mitochondrial function did not impair cardiomyocyte maturation. Rather, it elevated ROS production, activated the DNA damage response pathway, and decreased cardiomyocyte proliferation. We identified a transient window during the first postnatal week when inhibition of ROS or the DNA damage response pathway ameliorated the detrimental effect of Tfam inactivation. Mitochondrial dysfunction caused by Tfam inactivation induced ROS production, activated the DNA damage response, and caused cardiomyocyte cell cycle arrest, ultimately resulting in lethal cardiomyopathy. Normal mitochondrial function was not required for cardiomyocyte maturation. Pharmacological inhibition of ROS or DNA damage response pathways is a potential strategy to prevent cardiac dysfunction caused by some forms of mitochondrial

Engineering adolescence: maturation of human pluripotent stem cell-derived cardiomyocytes.

PubMed

Yang, Xiulan; Pabon, Lil; Murry, Charles E

2014-01-31

The discovery of human pluripotent stem cells (hPSCs), including both human embryonic stem cells and human-induced pluripotent stem cells, has opened up novel paths for a wide range of scientific studies. The capability to direct the differentiation of hPSCs into functional cardiomyocytes has provided a platform for regenerative medicine, development, tissue engineering, disease modeling, and drug toxicity testing. Despite exciting progress, achieving the optimal benefits has been hampered by the immature nature of these cardiomyocytes. Cardiac maturation has long been studied in vivo using animal models; however, finding ways to mature hPSC cardiomyocytes is only in its initial stages. In this review, we discuss progress in promoting the maturation of the hPSC cardiomyocytes, in the context of our current knowledge of developmental cardiac maturation and in relation to in vitro model systems such as rodent ventricular myocytes. Promising approaches that have begun to be examined in hPSC cardiomyocytes include long-term culturing, 3-dimensional tissue engineering, mechanical loading, electric stimulation, modulation of substrate stiffness, and treatment with neurohormonal factors. Future studies will benefit from the combinatorial use of different approaches that more closely mimic nature's diverse cues, which may result in broader changes in structure, function, and therapeutic applicability.

Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.

PubMed

Bergmann, Olaf; Zdunek, Sofia; Alkass, Kanar; Druid, Henrik; Bernard, Samuel; Frisén, Jonas

2011-01-15

Assays to quantify myocardial renewal rely on the accurate identification of cardiomyocyte nuclei. We previously ¹⁴C birth dated human cardiomyocytes based on the nuclear localization of cTroponins T and I. A recent report by Kajstura et al. suggested that cTroponin I is only localized to the nucleus in a senescent subpopulation of cardiomyocytes, implying that ¹⁴C birth dating of cTroponin T and I positive cell populations underestimates cardiomyocyte renewal in humans. We show here that the isolation of cell nuclei from the heart by flow cytometry with antibodies against cardiac Troponins T and I, as well as pericentriolar material 1 (PCM-1), allows for isolation of close to all cardiomyocyte nuclei, based on ploidy and marker expression. We also present a reassessment of cardiomyocyte ploidy, which has important implications for the analysis of cell turnover, and iododeoxyuridine (IdU) incorporation data. These data provide the foundation for reliable analysis of cardiomyocyte turnover in humans. Copyright © 2010 Elsevier Inc. All rights reserved.

Solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation: piece by piece.

PubMed

Lundy, David J; Lee, Desy S; Hsieh, Patrick C H

2017-03-01

There is a growing need for in vitro models which can serve as platforms for drug screening and basic research. Human adult cardiomyocytes cannot be readily obtained or cultured, and so pluripotent stem cell-derived cardiomyocytes appear to be an attractive option. Unfortunately, these cells are structurally and functionally immature-more comparable to foetal cardiomyocytes than adult. A recent study by Ruan et al ., provides new insights into accelerating the maturation process and takes us a step closer to solving the puzzle of pluripotent stem cell-derived cardiomyocyte maturation.

Electromechanical integration of cardiomyocytes derived from human embryonic stem cells.

PubMed

Kehat, Izhak; Khimovich, Leonid; Caspi, Oren; Gepstein, Amira; Shofti, Rona; Arbel, Gil; Huber, Irit; Satin, Jonathan; Itskovitz-Eldor, Joseph; Gepstein, Lior

2004-10-01

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

CXCL12 Gene Therapy Ameliorates Ischemia-Induced White Matter Injury in Mouse Brain.

PubMed

Li, Yaning; Tang, Guanghui; Liu, Yanqun; He, Xiaosong; Huang, Jun; Lin, Xiaojie; Zhang, Zhijun; Yang, Guo-Yuan; Wang, Yongting

2015-10-01

Remyelination is an important repair process after ischemic stroke-induced white matter injury. It often fails because of the insufficient recruitment of oligodendrocyte progenitor cells (OPCs) to the demyelinated site or the inefficient differentiation of OPCs to oligodendrocytes. We investigated whether CXCL12 gene therapy promoted remyelination after middle cerebral artery occlusion in adult mice. The results showed that CXCL12 gene therapy at 1 week after ischemia could protect myelin sheath integrity in the perifocal region, increase the number of platelet-derived growth factor receptor-α (PDGFRα)-positive and PDGFRα/bromodeoxyuridine-double positive OPCs in the subventricular zone, and further enhance their migration to the ischemic lesion area. Coadministration of AMD3100, the antagonist for CXCL12 receptor CXCR4, eliminated the beneficial effect of CXCL12 on myelin sheath integrity and negatively influenced OPC proliferation and migration. At 5 weeks after ischemia, CXCR4 was found on the PDGFRα- and/or neuron/glia type 2 (NG2)-positive OPCs but not on the myelin basic protein-positive mature myelin sheaths, and CXCR7 was only expressed on the mature myelin sheath in the ischemic mouse brain. Our data indicated that CXCL12 gene therapy effectively protected white matter and promoted its repair after ischemic injury. The treatment at 1 week after ischemia is effective, suggesting that this strategy has a longer therapeutic time window than the treatments currently available. This study has demonstrated for the first time that CXCL12 gene therapy significantly ameliorates brain ischemia-induced white matter injury and promotes oligodendrocyte progenitor cell proliferation in the subventricular zone and migration to the perifocal area in the ischemic mouse brain. Additional data showed that CXCR4 receptor plays an important role during the proliferation and migration of oligodendrocyte progenitor cells, and CXCR7 might play a role during maturation. In

[Rhythmic beating cardiomyocytes derived from human embryonic germ (EG) cells in vitro].

PubMed

Hua, Jinlian; Xu, Xiaoming; Dou, Zhongying

2006-10-01

Embryonic germ (EG) cells are pluripotent cells derived from primordial germ cells (PGCs) of gonads, gonadal ridges and mesenteries, analogies of fetuses,with the ability to undergo both highly self-renewal and multiple differentiation. These cells in vitro can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cells of the body. The aim of this study is to investigate the potentiality of human EG cells differentiation into cardiomyocytes. Inducing human EG cells with the method of murine ES cells differentiation into cardiomyocytes, supplemented with 0.75%-1% DMSO, 20% NBS, 10(-7) mM RA and 20% cardiomyocytes conditioned medium. 20 heart-like (rhythmic beating cell masses were observed in vitro culture and delayed human EG cells, which beat spontaneously from 20-120 times per minute and maintained beating for 2-15 days, periodic acid's staining (PAS), Myoglobin and a-actin immunological histology positive were all positive and reacted with K+, Ca2+ and adrenalin. Relatively unorganized myofibrillar bundles or more organized sarcomeres, z-bands or a gap junction, the presence of desmosomes in a few cells of the cell masses was observed with transmision electron microscope, which initially demonstrated that these cells were cardiomyocytes. We could not get rhythmly beating cardiomyocytes with 0.75%-1% DMSO, 10-7 mM RA and 20% cardiomyocytes conditioned medium,but in which the percentage of cardiac alpha-actin immunostaining positive cells were increased. The results first demonstrated that human EG cells can differentiate into rhythmic beating cardiomyocytes in vitro and suggests that human EG cells may represent a new potent resource for cardiomyocytes transplantation therapy for myocardium infarction.

[Effect of hydrogen-rich saline on cardiomyocyte autophagy during myocardial ischemia-reperfusion in aged rats].

PubMed

Pan, Zhenhua; Zhao, Yue; Yu, Hongying; Liu, Dayi; Xu, Hua

2015-07-07

To investigate the effects of hydrogen-rich saline on cardiomyocyte autophagy during myocardial ischemia-reperfusion in aged rats. One hundred and fifty healthy male Sprague Dawley rats, 18 months old, weighing 400-540 g were selected. The rats were then randomly divided into 5 groups (n = 30): Normal control group (group I); Sham operation group (group II); Myocardial ischemia-reperfusion group (group III); Hydrogen-rich saline group (group IV); Normal saline group (group V). No any processing in group I. In group II, the anterior descending branch was only exposed but not ligated. Myocardial I/R was induced by occlusion of anterior descending branch of left coronary artery for 30 min followed by 12 h and 24 h of reperfusion with Bimbaum. Hydrogen-rich saline 1 ml/100 g were injected intraperitoneally 5 min before reperfusion in group IV. Normal saline 1 ml/100 g were injected intraperitoneally 5 min before reperfusion in group V. The rats were sacrificed at 12 h and 24 h of reperfusion and hearts were removed. The pathological changes of myocardial tissue were detected by HE staining. The rate of cardiomyocyte autophagy were detected by the MDC fluorescent dye and flow cytometry instrument. The expression of AMPK, mTOR, Beclin1, LC3 in myocardial tissue was investigated by Western blot. Compared with groups I and II, the rate of cardiomyocyte autophagy, the expression of AMPK, mTOR, Beclin1, LC3 in myocardial tissue were significantly increased at 12 h, 24 h in groups III, IV and V (F = 23.45, 26.65, 25.58; F = 23.16, 25.15, 27.85; F = 21.04, 24.83, 27.43; F = 22.15, 25.79, 29.05; F = 22.58, 27.25, 28.46), P < 0.05. Compared with group III and V, the rate of cardiomyocyte autophagy, the expression of AMPK, mTOR, Beclin1, LC3 were significantly decreased at 12 h, 24 h in group IV (F = 21.29, 24.71; F = 22.37, 25.84; F = 20.48, 22.38; F = 21.76, 28.43; F = 22.54, 27.21), P < 0.05. Hydrogen-rich saline can attenuate myocardial reperfusion injury through inhibiting

Excitation model of pacemaker cardiomyocytes of cardiac conduction system

NASA Astrophysics Data System (ADS)

Grigoriev, M.; Babich, L.

2015-11-01

Myocardium includes typical and atypical cardiomyocytes - pacemakers, which form the cardiac conduction system. Excitation from the atrioventricular node in normal conditions is possible only in one direction. Retrograde direction of pulses is impossible. The most important prerequisite for the work of cardiomyocytes is the anatomical integrity of the conduction system. Changes in contractile force of the cardiomyocytes, which appear periodically, are due to two mechanisms of self-regulation - heterometric and homeometric. Graphic course of the excitation pulse propagation along the heart muscle more accurately reveals the understanding of the arrhythmia mechanism. These models have the ability to visualize the essence of excitation dynamics. However, they do not have the proper forecasting function for result estimation. Integrative mathematical model enables further investigation of general laws of the myocardium active behavior, allows for determination of the violation mechanism of electrical and contractile function of cardiomyocytes. Currently, there is no full understanding of the topography of pacemakers and ionic mechanisms. There is a need for the development of direction of mathematical modeling and comparative studies of the electrophysiological arrangement of cells of atrioventricular connection and ventricular conduction system.

Induction of heme oxygenase-1 protects mouse liver from apoptotic ischemia/reperfusion injury

PubMed Central

Issan, Y.; Katz, Y.; Sultan, M.; Safran, M.; Michal, Laniado-Schwartzman; Nader, G. Abraham; Kornowski, R.; Grief, F.; Pappo, O.; Hochhauser, E.

2017-01-01

Ischemia/reperfusion (I/R) injury is the main cause of primary graft dysfunction of liver allografts. Cobalt-protoporphyrin (CoPP)–dependent induction of heme oxygenase (HO)-1 has been shown to protect the liver from I/R injury. This study analyzes the apoptotic mechanisms of HO-1-mediated cytoprotection in mouse liver exposed to I/R injury. HO-1 induction was achieved by the administration of CoPP (1.5 mg/kg body weight i.p.). Mice were studied in in vivo model of hepatic segmental (70 %) ischemia for 60 min and reperfusion injury. Mice were randomly allocated to four main experimental groups (n = 10 each): (1) A control group undergoing sham operation. (2) Similar to group 1 but with the administration of CoPP 72 h before the operation. (3) Mice undergoing in vivo hepatic I/R. (4) Similar to group 3 but with the administration of CoPP 72 h before ischemia induction. When compared with the I/R mice group, in the I/R+CoPP mice group, the increased hepatic expression of HO-1 was associated with a significant reduction in liver enzyme levels, fewer apoptotic hepatocytes cells were identified by morphological criteria and by immunohistochemistry for caspase-3, there was a decreased mean number of proliferating cells (positively stained for Ki67), and a reduced hepatic expression of: C/EBP homologous protein (an index of endoplasmic reticulum stress), the NF-κB’s regulated genes (CIAP2, MCP-1 and IL-6), and increased hepatic expression of IκBa (the inhibitory protein of NF-κB). HO-1 over-expression plays a pivotal role in reducing the hepatic apoptotic IR injury. HO-1 may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation. PMID:23435964

Induction of heme oxygenase-1 protects mouse liver from apoptotic ischemia/reperfusion injury.

PubMed

Ben-Ari, Z; Issan, Y; Katz, Y; Sultan, M; Safran, M; Michal, Laniado-Schwartzman; Nader, G Abraham; Kornowski, R; Grief, F; Pappo, O; Hochhauser, E

2013-05-01

Ischemia/reperfusion (I/R) injury is the main cause of primary graft dysfunction of liver allografts. Cobalt-protoporphyrin (CoPP)-dependent induction of heme oxygenase (HO)-1 has been shown to protect the liver from I/R injury. This study analyzes the apoptotic mechanisms of HO-1-mediated cytoprotection in mouse liver exposed to I/R injury. HO-1 induction was achieved by the administration of CoPP (1.5 mg/kg body weight i.p.). Mice were studied in in vivo model of hepatic segmental (70 %) ischemia for 60 min and reperfusion injury. Mice were randomly allocated to four main experimental groups (n = 10 each): (1) A control group undergoing sham operation. (2) Similar to group 1 but with the administration of CoPP 72 h before the operation. (3) Mice undergoing in vivo hepatic I/R. (4) Similar to group 3 but with the administration of CoPP 72 h before ischemia induction. When compared with the I/R mice group, in the I/R+CoPP mice group, the increased hepatic expression of HO-1 was associated with a significant reduction in liver enzyme levels, fewer apoptotic hepatocytes cells were identified by morphological criteria and by immunohistochemistry for caspase-3, there was a decreased mean number of proliferating cells (positively stained for Ki67), and a reduced hepatic expression of: C/EBP homologous protein (an index of endoplasmic reticulum stress), the NF-κB's regulated genes (CIAP2, MCP-1 and IL-6), and increased hepatic expression of IκBa (the inhibitory protein of NF-κB). HO-1 over-expression plays a pivotal role in reducing the hepatic apoptotic IR injury. HO-1 may serve as a potential target for therapeutic intervention in hepatic I/R injury during liver transplantation.

MicroRNA-1 overexpression blunts cardiomyocyte hypertrophy elicited by thyroid hormone.

PubMed

Diniz, Gabriela Placoná; Lino, Caroline Antunes; Moreno, Camila Rodrigues; Senger, Nathalia; Barreto-Chaves, Maria Luiza Morais

2017-12-01

It is well-known that increased thyroid hormone (TH) levels induce cardiomyocyte growth. MicroRNAs (miRNAs) have been identified as key players in cardiomyocyte hypertrophy, which is associated with increased risk of heart failure. In this study, we evaluated the miR-1 expression in TH-induced cardiac hypertrophy, as well as the potential involvement of miR-1 in cardiomyocyte hypertrophy elicited by TH in vitro. The possible role of type 1 angiotensin II receptor (AT1R) in the effect promoted by TH in miR-1 expression was also evaluated. Neonatal rat cardiac myocytes (NRCMs) were treated with T 3 for 24 hr and Wistar rats were subjected to hyperthyroidism for 14 days combined or not with AT1R blocker. Real Time RT-PCR analysis indicated that miR-1 expression was decreased in cardiac hypertrophy in response to TH in vitro and in vivo, and this effect was unchanged by AT1R blocker. In addition, HDAC4, which is target of miR-1, was increased in NRCMs after T 3 treatment. A gain-of-function study revealed that overexpression of miR-1 prevented T 3 -induced cardiomyocyte hypertrophy and reduced HADC4 mRNA levels in NRCMs. In vivo experiments confirmed the downregulation of miR-1 in cardiac tissue from hyperthyroid animals, which was accompanied by increased HDAC4 mRNA levels. In addition, HDAC inhibitor prevented T 3 -induced cardiomyocyte hypertrophy. Our data reveal a new mechanistic insight into cardiomyocyte growth in response to TH, suggesting that miR-1 plays a role in cardiomyocyte hypertrophy induced by TH potentially via targeting HADC4. © 2017 Wiley Periodicals, Inc.

Automated Grouping of Action Potentials of Human Embryonic Stem Cell-Derived Cardiomyocytes

PubMed Central

Gorospe, Giann; Zhu, Renjun; Millrod, Michal A.; Zambidis, Elias T.; Tung, Leslie; Vidal, René

2015-01-01

Methods for obtaining cardiomyocytes from human embryonic stem cells (hESCs) are improving at a significant rate. However, the characterization of these cardiomyocytes is evolving at a relatively slower rate. In particular, there is still uncertainty in classifying the phenotype (ventricular-like, atrial-like, nodal-like, etc.) of an hESC-derived cardiomyocyte (hESC-CM). While previous studies identified the phenotype of a cardiomyocyte based on electrophysiological features of its action potential, the criteria for classification were typically subjective and differed across studies. In this paper, we use techniques from signal processing and machine learning to develop an automated approach to discriminate the electrophysiological differences between hESC-CMs. Specifically, we propose a spectral grouping-based algorithm to separate a population of cardiomyocytes into distinct groups based on the similarity of their action potential shapes. We applied this method to a dataset of optical maps of cardiac cell clusters dissected from human embryoid bodies (hEBs). While some of the 9 cell clusters in the dataset presented with just one phenotype, the majority of the cell clusters presented with multiple phenotypes. The proposed algorithm is generally applicable to other action potential datasets and could prove useful in investigating the purification of specific types of cardiomyocytes from an electrophysiological perspective. PMID:25148658

Cardiomyocyte A Disintegrin And Metalloproteinase 17 (ADAM17) Is Essential in Post-Myocardial Infarction Repair by Regulating Angiogenesis.

PubMed

Fan, Dong; Takawale, Abhijit; Shen, Mengcheng; Wang, Wang; Wang, Xiuhua; Basu, Ratnadeep; Oudit, Gavin Y; Kassiri, Zamaneh

2015-09-01

A disintegrin and metalloproteinase 17 (ADAM17) is a membrane-bound enzyme that mediates shedding of many membrane-bound molecules, thereby regulating multiple cellular responses. We investigated the role of cardiomyocyte ADAM17 in myocardial infarction (MI). Cardiomyocyte-specific ADAM17 knockdown mice (ADAM17(flox/flox)/α-MHC-Cre; f/f/Cre) and parallel controls (ADAM17(flox/flox); f/f) were subjected to MI by ligation of the left anterior descending artery. Post MI, f/f/Cre mice showed compromised survival, higher rates of cardiac rupture, more severe left ventricular dilation, and suppressed ejection fraction compared with parallel f/f-MI mice. Ex vivo ischemic injury (isolated hearts) resulted in comparable recovery in both genotypes. Myocardial vascular density (fluorescent-labeled lectin perfusion and CD31 immunofluorescence staining) was significantly lower in the infarct areas of f/f/Cre-MI compared with f/f-MI mice. Activation of vascular endothelial growth factor receptor 2 (VEGFR2), its mRNA, and total protein levels were reduced in infarcted myocardium in ADAM17 knockdown mice. Transcriptional regulation of VEGFR2 by ADAM17 was confirmed in cocultured cardiomyocyte-fibroblast as ischemia-induced VEGFR2 expression was blocked by ADAM17-siRNA. Meanwhile, ADAM17-siRNA did not alter VEGFA bioavailability in the conditioned media. ADAM17 knockdown mice (f/f/Cre-MI) exhibited reduced nuclear factor-κB activation (DNA binding) in the infarcted myocardium, which could underlie the suppressed VEGFR2 expression in these hearts. Post MI, inflammatory response was not altered by ADAM17 downregulation. This study highlights the key role of cardiomyocyte ADAM17 in post-MI recovery by regulating VEGFR2 transcription and angiogenesis, thereby limiting left ventricular dilation and dysfunction. Therefore, ADAM17 upregulation, within the physiological range, could provide protective effects in ischemic cardiomyopathy. © 2015 American Heart Association, Inc.

Targeted myocardial delivery of GDF11 gene rejuvenates the aged mouse heart and enhances myocardial regeneration after ischemia-reperfusion injury.

PubMed

Du, Guo-Qing; Shao, Zheng-Bo; Wu, Jie; Yin, Wen-Juan; Li, Shu-Hong; Wu, Jun; Weisel, Richard D; Tian, Jia-Wei; Li, Ren-Ke

2017-01-01

Ischemic cardiac injury is the main contributor to heart failure, and the regenerative capacity of intrinsic stem cells plays an important role in tissue repair after injury. However, stem cells in aged individuals have reduced regenerative potential and aged tissues lack the capacity to renew. Growth differentiation factor 11 (GDF11), from the activin-transforming growth factor β superfamily, has been shown to promote stem cell activity and rejuvenation. We carried out non-invasive targeted delivery of the GDF11 gene to the heart using ultrasound-targeted microbubble destruction (UTMD) and cationic microbubble (CMB) to investigate the ability of GDF11 to rejuvenate the aged heart and improve tissue regeneration after injury. Young (3 months) and old (21 months) mice were used to evaluate the expression of GDF11 mRNA in the myocardium at baseline and after ischemia/reperfusion (I/R) and myocardial infarction. GDF11 expression decreased with age and following myocardial injury. UTMD-mediated delivery of the GDF11 plasmid to the aged heart after I/R injury effectively and selectively increased GDF11 expression in the heart, and improved cardiac function and reduced infarct size. Over-expression of GDF11 decreased senescence markers, p16 and p53, as well as the number of p16 + cells in old mouse hearts. Furthermore, increased proliferation of cardiac stem cell antigen 1 (Sca-1 + ) cells and increased homing of endothelial progenitor cells and angiogenesis in old ischemic hearts occurred after GDF11 over-expression. Repetitive targeted delivery of the GDF11 gene via UTMD can rejuvenate the aged mouse heart and protect it from I/R injury.

Rapamycin Protects against Myocardial Ischemia-Reperfusion Injury through JAK2-STAT3 Signaling Pathway

PubMed Central

Das, Anindita; Salloum, Fadi N.; Durrant, David; Ockaili, Ramzi; Kukreja, Rakesh C

2012-01-01

Rapamycin (Sirolimus®) is used to prevent rejection of transplanted organs and coronary restenosis. We reported that rapamycin induced cardioprotection against ischemia-reperfusion (I/R) injury through opening of mitochondrial KATP channels. However, signaling mechanisms in rapamycin-induced cardioprotection are currently unknown. Considering that STAT3 is protective in the heart, we investigated the potential role of this transcription factor in rapamycin-induced protection against (I/R) injury. Adult male ICR mice were treated with rapamycin (0.25 mg/kg, i.p.) or vehicle (DMSO) with/without inhibitor of JAK2 (AG-490) or STAT3 (stattic). One hour later, the hearts were subjected to I/R either in Langendorf mode or in situ ligation of left coronary artery. Additionally, primary murine cardiomyocytes were subjected to simulated ischemia/reoxygenation (SI-RO) injury in vitro. For in situ targeted knockdown of STAT3, lentiviral vector containing short hairpin RNA was injected into left ventricle 3 weeks prior to initiating I/R injury. Infarct size, cardiac function, cardiomyocyte necrosis and apoptosis were assessed. Rapamycin reduced infarct size, improved cardiac function following I/R, limited cardiomyocytes necrosis as well as apoptosis following SI-RO which were blocked by AG-490 and stattic. In situ knock-down of STAT3 attenuated rapamycin-induced protection against I/R injury. Rapamycin triggered unique cardioprotecive signaling including phosphorylation of ERK, STAT3, eNOS and glycogen synthase kinase-3β in concert with increased prosurvival Bcl-2 to Bax ratio. Our data suggest that JAK2-STAT3 signaling plays an essential role in rapamycin-induced cardioprotection. We propose that rapamycin is a novel and clinically relevant pharmacological strategy to target STAT3 activation for treatment of myocardial infarction. PMID:22999860

A fetal human heart cardiac-inducing RNA (CIR) promotes the differentiation of stem cells into cardiomyocytes.

PubMed

Kochegarov, Andrei; Moses-Arms, Ashley; Lemanski, Larry F

2015-08-01

A specific human fetal heart RNA has been discovered, which has the ability to induce myocardial cell formation from mouse embryonic and human-induced pluripotent stem cells in culture. In this study, commercially obtained RNA from human fetal heart was cloned, sequenced, and synthesized using standard laboratory approaches. Molecular analyses of the specific fetal cardiac-inducing RNA (CIR), revealed that it is a fragment of N-sulfoglucosaminesulfohydrolase and the caspase recruitment domain family member 14 precursor. Stem cells transfected with CIRs often form into spindle-shaped cells characteristic of cardiomyocytes,and express the cardiac-specific contractile protein marker, troponin-T, in addition to tropomyosin and α-actinin as detected by immunohistochemical staining. Expression of these contractile proteins showed organization into sarcomeric myofibrils characteristic of striated cardiac muscle cells. Computer analyses of the RNA secondary structures of the active CIR show significant similarities to a RNA from salamander or myofibril-inducing RNA (MIR), which also promotes non-muscle cells to differentiate into cardiac muscle. Thus, these two RNAs, salamander MIR and the newly discovered human-cloned CIR reported here, appear to have evolutionarily conserved secondary structures suggesting that both play major roles in vertebrate heart development and, particularly, in the differentiation of cardiomyocytes from non-muscle cells during development.

Importance of Thickness in Human Cardiomyocyte Network for Effective Electrophysiological Stimulation Using On-Chip Extracellular Microelectrodes

NASA Astrophysics Data System (ADS)

Hamada, Tomoyo; Nomura, Fumimasa; Kaneko, Tomoyuki; Yasuda, Kenji

2012-06-01

We have developed a three-dimensionally controlled in vitro human cardiomyocyte network assay for the measurements of drug-induced conductivity changes and the appearance of fatal arrhythmia such as ventricular tachycardia/fibrillation for more precise in vitro predictive cardiotoxicity. To construct an artificial conductance propagation model of a human cardiomyocyte network, first, we examined the cell concentration dependence of the cell network heights and found the existence of a height limit of cell networks, which was double-layer height, whereas the cardiomyocytes were effectively and homogeneously cultivated within the microchamber maintaining their spatial distribution constant and their electrophysiological conductance and propagation were successfully recorded using a microelectrode array set on the bottom of the microchamber. The pacing ability of a cardiomyocyte's electrophysiological response has been evaluated using microelectrode extracellular stimulation, and the stimulation for pacing also successfully regulated the beating frequencies of two-layered cardiomyocyte networks, whereas monolayered cardiomyocyte networks were hardly stimulated by the external electrodes using the two-layered cardiomyocyte stimulation condition. The stability of the lined-up shape of human cardiomyocytes within the rectangularly arranged agarose microchambers was limited for a two-layered cardiomyocyte network because their stronger force generation shrunk those cells after peeling off the substrate. The results indicate the importance of fabrication technology of thickness control of cellular networks for effective extracellular stimulation and the potential concerning thick cardiomyocyte networks for long-term cultivation.

Inhibition of miR-128-3p by Tongxinluo Protects Human Cardiomyocytes from Ischemia/reperfusion Injury via Upregulation of p70s6k1/p-p70s6k1

PubMed Central

Chen, Gui-hao; Xu, Chuan-sheng; Zhang, Jie; Li, Qing; Cui, He-he; Li, Xiang-dong; Chang, Li-ping; Tang, Rui-jie; Xu, Jun-yan; Tian, Xia-qiu; Huang, Pei-sen; Xu, Jun; Jin, Chen; Yang, Yue-jin

2017-01-01

Background and Aims: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine that has been widely used to treat cardiovascular and cerebrovascular diseases. However, no studies have explored whether TXL can protect human cardiomyocytes (HCMs) from ischemia/reperfusion (I/R) injury. Reperfusion Injury Salvage Kinase (RISK) pathway activation was previously demonstrated to protect the hearts against I/R injury and it is generally activated via Akt or (and) Erk 1/2, and their common downstream protein, ribosomal protein S6 kinase (p70s6k). In addition, prior studies proved that TXL treatment of cells promoted secretion of VEGF, which could be stimulated by the increased phosphorylation of one p70s6k subtype, p70s6k1. Consequently, we hypothesized TXL could protect HCMs from I/R injury by activating p70s6k1 and investigated the underlying mechanism. Methods and Results: HCMs were exposed to hypoxia (18 h) and reoxygenation (2 h) (H/R), with or without TXL pretreatment. H/R reduced mitochondrial membrane potential, increased bax/bcl-2 ratios and cytochrome C levels and induced HCM apoptosis. TXL preconditioning reversed these H/R-induced changes in a dose-dependent manner and was most effective at 400 μg/mL. The anti-apoptotic effect of TXL was abrogated by rapamycin, an inhibitor of p70s6k. However, inhibitors of Erk1/2 (U0126) or Akt (LY294002) failed to inhibit the protective effect of TXL. TXL increased p70s6k1 expression and, thus, enhanced its phosphorylation. Furthermore, transfection of cardiomyocytes with siRNA to p70s6k1 abolished the protective effects of TXL. Among the micro-RNAs (miR-145-5p, miR-128-3p and miR-497-5p) previously reported to target p70s6k1, TXL downregulated miR-128-3p in HCMs during H/R, but had no effects on miR-145-5p and miR-497-5p. An in vivo study confirmed the role of the p70s6k1 pathway in the infarct-sparing effect of TXL, demonstrating that TXL decreased miR-128-3p levels in the rat myocardium during I/R. Transfection

C1q-targeted inhibition of the classical complement pathway prevents injury in a novel mouse model of acute motor axonal neuropathy.

PubMed

McGonigal, Rhona; Cunningham, Madeleine E; Yao, Denggao; Barrie, Jennifer A; Sankaranarayanan, Sethu; Fewou, Simon N; Furukawa, Koichi; Yednock, Ted A; Willison, Hugh J

2016-03-02

Guillain-Barré syndrome (GBS) is an autoimmune disease that results in acute paralysis through inflammatory attack on peripheral nerves, and currently has limited, non-specific treatment options. The pathogenesis of the acute motor axonal neuropathy (AMAN) variant is mediated by complement-fixing anti-ganglioside antibodies that directly bind and injure the axon at sites of vulnerability such as nodes of Ranvier and nerve terminals. Consequently, the complement cascade is an attractive target to reduce disease severity. Recently, C5 complement component inhibitors that block the formation of the membrane attack complex and subsequent downstream injury have been shown to be efficacious in an in vivo anti-GQ1b antibody-mediated mouse model of the GBS variant Miller Fisher syndrome (MFS). However, since gangliosides are widely expressed in neurons and glial cells, injury in this model was not targeted exclusively to the axon and there are currently no pure mouse models for AMAN. Additionally, C5 inhibition does not prevent the production of early complement fragments such as C3a and C3b that can be deleterious via their known role in immune cell and macrophage recruitment to sites of neuronal damage. In this study, we first developed a new in vivo transgenic mouse model of AMAN using mice that express complex gangliosides exclusively in neurons, thereby enabling specific targeting of axons with anti-ganglioside antibodies. Secondly, we have evaluated the efficacy of a novel anti-C1q antibody (M1) that blocks initiation of the classical complement cascade, in both the newly developed anti-GM1 antibody-mediated AMAN model and our established MFS model in vivo. Anti-C1q monoclonal antibody treatment attenuated complement cascade activation and deposition, reduced immune cell recruitment and axonal injury, in both mouse models of GBS, along with improvement in respiratory function. These results demonstrate that neutralising C1q function attenuates injury with a

Oxidative stress and cardiomyocyte necrosis with elevated serum troponins: pathophysiologic mechanisms.

PubMed

Robinson, Antwon D; Ramanathan, Kodangudi B; McGee, Jesse E; Newman, Kevin P; Weber, Karl T

2011-08-01

The progressive nature of heart failure is linked to multiple factors, including an ongoing loss of cardiomyocytes and necrosis. Necrotic cardiomyocytes leave behind several footprints: the spillage of their contents leading to elevations in serum troponins; and morphologic evidence of tissue repair with scarring. The pathophysiologic origins of cardiomyocyte necrosis relates to neurohormonal activation, including the adrenergic nervous system. Catecholamine-initiated excessive intracellular Ca accumulation and mitochondria Ca overloading in particular initiate a mitochondriocentric signal-transducer-effector pathway to necrosis and which includes the induction of oxidative stress and opening of their inner membrane permeability transition pore. Hypokalemia, ionized hypocalcemia and hypomagnesemia, where consequent elevations in parathyroid hormone further account for excessive intracellular Ca accumulation, hypozincemia and hyposelenemia each compromise metalloenzyme-based antioxidant defenses. The necrotic loss of cardiomyocytes and adverse structural remodeling of myocardium is related to the central role played by a mitochondriocentric pathway initiated by neurohormonal activation.

Endolysosomal two‐pore channels regulate autophagy in cardiomyocytes

PubMed Central

García‐Rúa, Vanessa; Feijóo‐Bandín, Sandra; Rodríguez‐Penas, Diego; Mosquera‐Leal, Ana; Abu‐Assi, Emad; Beiras, Andrés; María Seoane, Luisa; Lear, Pamela; Parrington, John; Portolés, Manuel; Roselló‐Lletí, Esther; Rivera, Miguel; Gualillo, Oreste; Parra, Valentina; Hill, Joseph A.; Rothermel, Beverly; González‐Juanatey, José Ramón

2016-01-01

Key points Two‐pore channels (TPCs) were identified as a novel family of endolysosome‐targeted calcium release channels gated by nicotinic acid adenine dinucleotide phosphate, as also as intracellular Na+ channels able to control endolysosomal fusion, a key process in autophagic flux.Autophagy, an evolutionarily ancient response to cellular stress, has been implicated in the pathogenesis of a wide range of cardiovascular pathologies, including heart failure.We report direct evidence indicating that TPCs are involved in regulating autophagy in cardiomyocytes, and that TPC knockout mice show alterations in the cardiac lysosomal system. TPC downregulation implies a decrease in the viability of cardiomyocytes under starvation conditions. In cardiac tissues from both humans and rats, TPC transcripts and protein levels were higher in females than in males, and correlated negatively with markers of autophagy.We conclude that the endolysosomal channels TPC1 and TPC2 are essential for appropriate basal and induced autophagic flux in cardiomyocytes, and also that they are differentially expressed in male and female hearts. Abstract Autophagy participates in physiological and pathological remodelling of the heart. The endolysosomal two‐pore channels (TPCs), TPC1 and TPC2, have been implicated in the regulation of autophagy. The present study aimed to investigate the role of TPC1 and TPC2 in basal and induced cardiac autophagic activity. In cultured cardiomyocytes, starvation induced a significant increase in TPC1 and TPC2 transcripts and protein levels that paralleled the increase in autophagy identified by increased LC3‐II and decreased p62 levels. Small interfering RNA depletion of TPC2 alone or together with TPC1 increased both LC3II and p62 levels under basal conditions and in response to serum starvation, suggesting that, under conditions of severe energy depletion (serum plus glucose starvation), changes in the autophagic flux (as assessed by use of bafilomycin

Activation of IGF-2R stimulates cardiomyocyte hypertrophy in the late gestation sheep fetus

PubMed Central

Wang, Kimberley C W; Brooks, Doug A; Thornburg, Kent L; Morrison, Janna L

2012-01-01

In vitro studies using rat and fetal sheep cardiomyocytes indicate that, in addition to its role as a clearance receptor, the insulin-like growth factor 2 receptor (IGF-2R) can induce cardiomyocyte hypertrophy. In the present study, we have determined the effect of specific activation of the IGF-2R in the heart of the late gestation fetus on cardiomyocyte development. Leu27IGF-2, an IGF-2R agonist, was infused into the fetal left circumflex coronary artery for 4 days beginning at 128.1 ± 0.4 days gestation. Ewes were humanely killed at 132.2 ± 1.2 days gestation (term, 150 days). Fetuses were delivered and hearts dissected to isolate the cardiomyocytes and to collect and snap-freeze tissue. Leu27IGF-2 infusion into the left circumflex coronary artery of fetal sheep increased the area of binucleated cardiomyocytes in the left, but not the right, ventricle. However, this infusion of Leu27IGF-2 did not change fetal weight, heart weight, blood pressure, blood gases or cardiomyocyte proliferation/binucleation. The increase in cardiomyocyte size in the Leu27IGF-2-infused group was associated with increased expression of proteins in the Gαs, but not the Gαq, signalling pathway. We concluded that infusion of Leu27IGF-2 into the left circumflex coronary artery causes cardiac IGF-2R activation in the left ventricle of the heart, and this stimulates cardiomyocyte hypertrophy in a Gαs-dependent manner. PMID:22930271

Alteration in mitochondrial Ca(2+) uptake disrupts insulin signaling in hypertrophic cardiomyocytes.

PubMed

Gutiérrez, Tomás; Parra, Valentina; Troncoso, Rodrigo; Pennanen, Christian; Contreras-Ferrat, Ariel; Vasquez-Trincado, César; Morales, Pablo E; Lopez-Crisosto, Camila; Sotomayor-Flores, Cristian; Chiong, Mario; Rothermel, Beverly A; Lavandero, Sergio

2014-11-07

Cardiac hypertrophy is characterized by alterations in both cardiac bioenergetics and insulin sensitivity. Insulin promotes glucose uptake by cardiomyocytes and its use as a substrate for glycolysis and mitochondrial oxidation in order to maintain the high cardiac energy demands. Insulin stimulates Ca(2+) release from the endoplasmic reticulum, however, how this translates to changes in mitochondrial metabolism in either healthy or hypertrophic cardiomyocytes is not fully understood. In the present study we investigated insulin-dependent mitochondrial Ca(2+) signaling in normal and norepinephrine or insulin like growth factor-1-induced hypertrophic cardiomyocytes. Using mitochondrion-selective Ca(2+)-fluorescent probes we showed that insulin increases mitochondrial Ca(2+) levels. This signal was inhibited by the pharmacological blockade of either the inositol 1,4,5-triphosphate receptor or the mitochondrial Ca(2+) uniporter, as well as by siRNA-dependent mitochondrial Ca(2+) uniporter knockdown. Norepinephrine-stimulated cardiomyocytes showed a significant decrease in endoplasmic reticulum-mitochondrial contacts compared to either control or insulin like growth factor-1-stimulated cells. This resulted in a reduction in mitochondrial Ca(2+) uptake, Akt activation, glucose uptake and oxygen consumption in response to insulin. Blocking mitochondrial Ca(2+) uptake was sufficient to mimic the effect of norepinephrine-induced cardiomyocyte hypertrophy on insulin signaling. Mitochondrial Ca(2+) uptake is a key event in insulin signaling and metabolism in cardiomyocytes.

Interleukin-13 protects mouse intestine from ischemia and reperfusion injury through regulation of innate and adaptive immunity.

PubMed

Farmer, Douglas G; Ke, Bibo; Shen, Xiu-Da; Kaldas, Fady M; Gao, Feng; Watson, Melissa J; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W

2011-04-15

Ischemia-reperfusion (I/R) injury is a major factor leading to intestinal dysfunction or graft loss after intestinal surgery or transplantation. This study investigated the cytoprotective effects and putative mechanisms of interleukin (IL)-13 after intestinal I/R injury in the mouse. Mouse warm intestinal I/R injury induced by clamping the superior mesenteric artery for 100 min with tissue analysis at 4 and 24 hr after reperfusion. Treated animals received intravenous recombinant murine IL-13 (rIL-13) and anti-IL-13 antibody, whereas controls received saline. rIL-13 administration markedly prolonged animal survival (100% vs. 50% in saline controls) and resulted in near normal histopathological architecture. rIL-13 treatment also significantly decreased myeloperoxidase activity. Mice conditioned with rIL-13 had a markedly depressed Toll-like receptor-4 expression and increased the expression of Stat6, antioxidant hemeoxygenase-1, and antiapoptotic A20, Bcl-2/Bcl-xl, compared with that of controls. Unlike in controls, the expression of mRNA coding for IL-2/interferon-γ, and interferon-γ-inducible protein (IP)-10/monocyte chemotactic protein-1 remained depressed, whereas that of IL-13/IL-4 reciprocally increased in the mice treated with rIL-13. Administration of anti-IL13 antibody alone or in combination with rIL-13 resulted in outcomes similar to that seen in controls. This study demonstrates for the first time that IL-13 plays a protective role in intestinal warm I/R injury and a critical role in the regulation of Stat6 and Toll-like receptor-4 signaling. The administration of IL-13 exerts cytoprotective effects in this model by regulating innate and adaptive immunity while the removal of IL-13 using antibody therapy abrogates this effect.

Stretch-Induced Hypertrophy Activates NFkB-Mediated VEGF Secretion in Adult Cardiomyocytes

PubMed Central

Leychenko, Anna; Konorev, Eugene; Jijiwa, Mayumi; Matter, Michelle L.

2011-01-01

Hypertension and myocardial infarction are associated with the onset of hypertrophy. Hypertrophy is a compensatory response mechanism to increases in mechanical load due to pressure or volume overload. It is characterized by extracellular matrix remodeling and hypertrophic growth of adult cardiomyocytes. Production of Vascular Endothelial Growth Factor (VEGF), which acts as an angiogenic factor and a modulator of cardiomyocyte function, is regulated by mechanical stretch. Mechanical stretch promotes VEGF secretion in neonatal cardiomyocytes. Whether this effect is retained in adult cells and the molecular mechanism mediating stretch-induced VEGF secretion has not been elucidated. Our objective was to investigate whether cyclic mechanical stretch induces VEGF secretion in adult cardiomyocytes and to identify the molecular mechanism mediating VEGF secretion in these cells. Isolated primary adult rat cardiomyocytes (ARCMs) were subjected to cyclic mechanical stretch at an extension level of 10% at 30 cycles/min that induces hypertrophic responses. Cyclic mechanical stretch induced a 3-fold increase in VEGF secretion in ARCMs compared to non-stretch controls. This increase in stretch-induced VEGF secretion correlated with NFkB activation. Cyclic mechanical stretch-mediated VEGF secretion was blocked by an NFkB peptide inhibitor and expression of a dominant negative mutant IkBα, but not by inhibitors of the MAPK/ERK1/2 or PI3K pathways. Chromatin immunoprecipitation assays demonstrated an interaction of NFkB with the VEGF promoter in stretched primary cardiomyocytes. Moreover, VEGF secretion is increased in the stretched myocardium during pressure overload-induced hypertrophy. These findings are the first to demonstrate that NFkB activation plays a role in mediating VEGF secretion upon cyclic mechanical stretch in adult cardiomyocytes. Signaling by NFkB initiated in response to cyclic mechanical stretch may therefore coordinate the hypertrophic response in adult

Troglitazone stimulates {beta}-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1{sub A} receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tilley, Douglas G., E-mail: douglas.tilley@jefferson.edu; Center for Translational Medicine, Thomas Jefferson University; Nguyen, Anny D.

2010-06-11

Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPAR{gamma}-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPAR{gamma} activity, thus we hypothesized that a PPAR{gamma} agonist may exert physiologic effects via the angiotensin II type 1{sub A} receptor (AT1{sub A}R). In AT1{sub A}R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPAR{gamma} agonist troglitazone (Trog) enhanced AT1{sub A}R internalization and recruitment of endogenous {beta}-arrestin1/2 ({beta}arr1/2) to the AT1{sub A}R. A fluorescence assay to measure diacylglycerolmore » (DAG) accumulation showed that although Ang II induced AT1{sub A}R-G{sub q} protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of {beta}arr1/2 was selective to AT1{sub A}R as the response was prevented by an ARB- and Trog-mediated {beta}arr1/2 recruitment to {beta}1-adrenergic receptor ({beta}1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be {beta}arr2-dependent, as cardiomyocytes isolated from {beta}arr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPAR{gamma} agonist Trog acts at the AT1{sub A}R to simultaneously block G{sub q} protein activation and induce the recruitment of {beta}arr1/2, which leads to an increase in cardiomyocyte contractility.« less

Structural phenotyping of stem cell-derived cardiomyocytes.

PubMed

Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

2015-03-10

Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

An Optimized and Simplified System of Mouse Embryonic Stem Cell Cardiac Differentiation for the Assessment of Differentiation Modifiers

PubMed Central

Hartman, Matthew E.; Librande, Jason R.; Medvedev, Ivan O.; Ahmad, Rabiah N.; Moussavi-Harami, Farid; Gupta, Pritha P.; Chien, Wei-Ming; Chin, Michael T.

2014-01-01

Generating cardiomyocytes from embryonic stem cells is an important technique for understanding cardiovascular development, the origins of cardiovascular diseases and also for providing potential reagents for cardiac repair. Numerous methods have been published but often are technically challenging, complex, and are not easily adapted to assessment of specific gene contributions to cardiac myocyte differentiation. Here we report the development of an optimized protocol to induce the differentiation of mouse embryonic stem cells to cardiac myocytes that is simplified and easily adapted for genetic studies. Specifically, we made four critical findings that distinguish our protocol: 1) mouse embryonic stem cells cultured in media containing CHIR99021 and PD0325901 to maintain pluripotency will efficiently form embryoid bodies containing precardiac mesoderm when cultured in these factors at a reduced dosage, 2) low serum conditions promote cardiomyocyte differentiation and can be used in place of commercially prepared StemPro nutrient supplement, 3) the Wnt inhibitor Dkk-1 is dispensable for efficient cardiac differentiation and 4) tracking differentiation efficiency may be done with surface expression of PDGFRα alone. In addition, cardiac mesodermal precursors generated by this system can undergo lentiviral infection to manipulate the expression of specific target molecules to assess effects on cardiac myocyte differentiation and maturation. Using this approach, we assessed the effects of CHF1/Hey2 on cardiac myocyte differentiation, using both gain and loss of function. Overexpression of CHF1/Hey2 at the cardiac mesoderm stage had no apparent effect on cardiac differentiation, while knockdown of CHF1/Hey2 resulted in increased expression of atrial natriuretic factor and connexin 43, suggesting an alteration in the phenotype of the cardiomyocytes. In summary we have generated a detailed and simplified protocol for generating cardiomyocytes from mES cells that is

Adenosine kinase regulation of cardiomyocyte hypertrophy

PubMed Central

Fassett, John T.; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie

2011-01-01

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKTSer473 phosphorylation but did attenuate sustained phosphorylation of RafSer338 (24–48 h), mTORSer2448 (24–48 h), p70S6kThr389 (2.5–48 h), and ERKThr202/Tyr204 (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6kThr389 phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERKThr202/Tyr204 and AKTSer473. Reduction of Raf-induced p70S6kThr389 phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte hypertrophy, which acts, at least in part, through inhibition of

Adenosine kinase regulation of cardiomyocyte hypertrophy.

PubMed

Fassett, John T; Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Chen, Yingjie; Bache, Robert J

2011-05-01

There is evidence that extracellular adenosine can attenuate cardiac hypertrophy, but the mechanism by which this occurs is not clear. Here we investigated the role of adenosine receptors and adenosine metabolism in attenuation of cardiomyocyte hypertrophy. Phenylephrine (PE) caused hypertrophy of neonatal rat cardiomyocytes with increases of cell surface area, protein synthesis, and atrial natriuretic peptide (ANP) expression. These responses were attenuated by 5 μM 2-chloroadenosine (CADO; adenosine deaminase resistant adenosine analog) or 10 μM adenosine. While antagonism of adenosine receptors partially blocked the reduction of ANP expression produced by CADO, it did not restore cell size or protein synthesis. In support of a role for intracellular adenosine metabolism in regulating hypertrophy, the adenosine kinase (AK) inhibitors iodotubercidin and ABT-702 completely reversed the attenuation of cell size, protein synthesis, and expression of ANP by CADO or ADO. Examination of PE-induced phosphosignaling pathways revealed that CADO treatment did not reduce AKT(Ser⁴⁷³) phosphorylation but did attenuate sustained phosphorylation of Raf(Ser³³⁸) (24-48 h), mTOR(Ser²⁴⁴⁸) (24-48 h), p70S6k(Thr³⁸⁹) (2.5-48 h), and ERK(Thr²⁰²/Tyr²⁰⁴) (48 h). Inhibition of AK restored activation of these enzymes in the presence of CADO. Using dominant negative and constitutively active Raf adenoviruses, we found that Raf activation is necessary and sufficient for PE-induced mTORC1 signaling and cardiomyocyte hypertrophy. CADO treatment still blocked p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy downstream of constitutively active Raf, however, despite a high level phosphorylation of ERK(Thr202/Tyr204) and AKT(Ser⁴⁷³). Reduction of Raf-induced p70S6k(Thr³⁸⁹) phosphorylation and hypertrophy by CADO was reversed by inhibiting AK. Together, these results identify AK as an important mediator of adenosine attenuation of cardiomyocyte

The role of mAKAPβ in the process of cardiomyocyte hypertrophy induced by angiotensin II

PubMed Central

GUO, HUIXIN; LIU, BAOXIN; HOU, LEI; THE, ERLINDA; LI, GANG; WANG, DONGZHI; JIE, QIQIANG; CHE, WENLIANG; WEI, YIDONG

2015-01-01

Angiotensin II (AngII) is the central product of the renin-angiotensin system (RAS) and this octapeptide contributes to the pathophysiology of cardiac hypertrophy and remodeling. mAKAPβ is an A-kinase anchoring protein (AKAP) that has the function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. In this study, we aimed to investigate the role of mAKAPβ in AngII-induced cardiomyocyte hypertrophy and the possible mechanisms involved. Cultured cardiomyocytes from neonatal rats were treated with AngII. Subsequently, the morphology of the cardiomyocytes was observed and the expression of mAKAPβ and cardiomyocyte hypertrophic markers was measured. mAKAPβ-shRNA was constructed for RNA interference; the expression of mAKAPβ and hypertrophic markers, the cell surface area and the [3H]Leucine incorporation rate in the AngII-treated rat cardiomyocytes were detected following RNA interference. Simultaneously, changes in the expression levels of phosphorylated extracellular signal-regulated kinase (p-ERK)2 in the cardiomyocytes were assessed. The cell size of the AngII-treated cardiaomyocytes was significantly larger than that of the untreated cardiomyocytes. The expression of hypertrophic markers and p-ERK2, the cell surface area and the [3H]Leucine incorporation rate were all significantly increased in the AngII-treated cells. However, the expression of mAKAPβ remained unaltered in this process. RNA interference simultaneously inhibited the protein expression of mAKAPβ and p-ERK2, and the hypertrophy of the cardiomyocytes induced by AngII was attenuated. These results demonstrate that AngII induces hypertrophy in cardiomyocytes, and mAKAPβ is possibly involved in this process. The effects of mAKAPβ on AngII-induced cardiomyocyte hypertrophy may be associated with p-ERK2 expression. PMID:25739102

Neuronal Ca2+ sensor-1 contributes to stress tolerance in cardiomyocytes via activation of mitochondrial detoxification pathways.

PubMed

Nakamura, Tomoe Y; Nakao, Shu; Wakabayashi, Shigeo

2016-10-01

Identification of the molecules involved in cell death/survival pathways is important for understanding the mechanisms of cell loss in cardiac disease, and thus is clinically relevant. Ca 2+ -dependent signals are often involved in these pathways. Here, we found that neuronal Ca 2+ -sensor-1 (NCS-1), a Ca 2+ -binding protein, has an important role in cardiac survival during stress. Cardiomyocytes derived from NCS-1-deficient (Ncs1 -/- ) mice were more susceptible to oxidative and metabolic stress than wild-type (WT) myocytes. Cellular ATP levels and mitochondrial respiration rates, as well as the levels of mitochondrial marker proteins, were lower in Ncs1 -/- myocytes. Although oxidative stress elevated mitochondrial proton leak, which exerts a protective effect by inhibiting the production of reactive oxygen species in WT myocytes, this response was considerably diminished in Ncs1 -/- cardiomyocytes, and this would be a major reason for cell death. Consistently, H 2 O 2 -induced loss of mitochondrial membrane potential, a critical early event in cell death, was accelerated in Ncs1 -/- myocytes. Furthermore, NCS-1 was upregulated in hearts subjected to ischemia-reperfusion, and ischemia-reperfusion injury was more severe in Ncs1 -/- hearts. Activation of stress-induced Ca 2+ -dependent survival pathways, such as Akt and PGC-1α (which promotes mitochondrial biogenesis and function), was diminished in Ncs1 -/- hearts. Overall, these data demonstrate that NCS-1 contributes to stress tolerance in cardiomyocytes at least in part by activating certain Ca 2+ -dependent survival pathways that promote mitochondrial biosynthesis/function and detoxification pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mammalian target of rapamycin is essential for cardiomyocyte survival and heart development in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Pengpeng; Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070; Department of Animal Sciences, Purdue University, West Lafayette, IN 47907

Highlights: • mTOR is a critical regulator of many biological processes yet its function in heart is not well understood. • MCK-Cre/Mtor{sup flox/flox} mice were established to delete Mtor in cardiomyocytes. • The mTOR-mKO mice developed normally but die prematurely within 5 weeks after birth due to heart disease. • The mTOR-mKO mice had dilated myocardium and increased cell death. • mTOR-mKO hearts had reduced expression of metabolic genes and activation of mTOR target proteins. - Abstract: Mammalian target of rapamycin (mTOR) is a critical regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive knockout of Mtor leadsmore » to embryonic lethality, the in vivo function of mTOR in perinatal development and postnatal growth of heart is not well defined. In this study, we established a muscle-specific mTOR conditional knockout mouse model (mTOR-mKO) by crossing MCK-Cre and Mtor{sup flox/flox} mice. Although the mTOR-mKO mice survived embryonic and perinatal development, they exhibited severe postnatal growth retardation, cardiac muscle pathology and premature death. At the cellular level, the cardiac muscle of mTOR-mKO mice had fewer cardiomyocytes due to apoptosis and necrosis, leading to dilated cardiomyopathy. At the molecular level, the cardiac muscle of mTOR-mKO mice expressed lower levels of fatty acid oxidation and glycolysis related genes compared to the WT littermates. In addition, the mTOR-mKO cardiac muscle had reduced Myh6 but elevated Myh7 expression, indicating cardiac muscle degeneration. Furthermore, deletion of Mtor dramatically decreased the phosphorylation of S6 and AKT, two key targets downstream of mTORC1 and mTORC2 mediating the normal function of mTOR. These results demonstrate that mTOR is essential for cardiomyocyte survival and cardiac muscle function.« less

Role of Nodal-PITX2C signaling pathway in glucose-induced cardiomyocyte hypertrophy.

PubMed

Su, Dongmei; Jing, Sun; Guan, Lina; Li, Qian; Zhang, Huiling; Gao, Xiaobo; Ma, Xu

2014-06-01

Pathological cardiac hypertrophy is a major cause of morbidity and mortality in cardiovascular disease. Recent studies have shown that cardiomyocytes, in response to high glucose (HG) stimuli, undergo hypertrophic growth. While much work still needs to be done to elucidate this important mechanism of hypertrophy, previous works have showed that some pathways or genes play important roles in hypertrophy. In this study, we showed that sublethal concentrations of glucose (25 mmol/L) could induce cardiomyocyte hypertrophy with an increase in the cellular surface area and the upregulation of the atrial natriuretic peptide (ANP) gene, a hypertrophic marker. High glucose (HG) treatments resulted in the upregulation of the Nodal gene, which is under-expressed in cardiomyocytes. We also determined that the knockdown of the Nodal gene resisted HG-induced cardiomyocyte hypertrophy. The overexpression of Nodal was able to induce hypertrophy in cardiomyocytes, which was associated with the upregulation of the PITX2C gene. We also showed that increases in the PITX2C expression, in response to Nodal, were mediated by the Smad4 signaling pathway. This study is highly relevant to the understanding of the effects of the Nodal-PITX2C pathway on HG-induced cardiomyocyte hypertrophy, as well as the related molecular mechanisms.

Lipopolysaccharide-induced endotoxemia in corn oil-preloaded mice causes an extended course of lung injury and repair and pulmonary fibrosis: A translational mouse model of acute respiratory distress syndrome.

PubMed

Wu, Chaomin; Evans, Colin E; Dai, Zhiyu; Huang, Xiaojia; Zhang, Xianming; Jin, Hua; Hu, Guochang; Song, Yuanlin; Zhao, You-Yang

2017-01-01

Acute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia respiratory failure, bilateral pulmonary infiltrates, and pulmonary edema of non-cardiac origin. Effective treatments for ARDS patients may arise from experimental studies with translational mouse models of this disease that aim to delineate the mechanisms underlying the disease pathogenesis. Mouse models of ARDS, however, can be limited by their rapid progression from injured to recovery state, which is in contrast to the course of ARDS in humans. Furthermore, current mouse models of ARDS do not recapitulate certain prominent aspects of the pathogenesis of ARDS in humans. In this study, we developed an improved endotoxemic mouse model of ARDS resembling many features of clinical ARDS including extended courses of injury and recovery as well as development of fibrosis following i.p. injection of lipopolysaccharide (LPS) to corn oil-preloaded mice. Compared with mice receiving LPS alone, those receiving corn oil and LPS exhibited extended course of lung injury and repair that occurred over a period of >2 weeks instead of 3-5days. Importantly, LPS challenge of corn oil-preloaded mice resulted in pulmonary fibrosis during the repair phase as often seen in ARDS patients. In summary, this simple novel mouse model of ARDS could represent a valuable experimental tool to elucidate mechanisms that regulate lung injury and repair in ARDS patients.

Baicalin Attenuates IL-17-Mediated Acetaminophen-Induced Liver Injury in a Mouse Model

PubMed Central

Liao, Chia-Chih; Day, Yuan-Ji; Lee, Hung-Chen; Liou, Jiin-Tarng; Chou, An-Hsun; Liu, Fu-Chao

2016-01-01

Background IL-17 has been shown to be involved in liver inflammatory disorders in both mice and humans. Baicalin (BA), a major compound extracted from traditional herb medicine (Scutellariae radix), has potent hepatoprotective properties. Previous study showed that BA inhibits IL-17-mediated lymphocyte adhesion and downregulates joint inflammation. The aim of this study is to investigate the role of IL-17 in the hepatoprotective effects of BA in an acetaminophen (APAP)-induced liver injury mouse model. Methods Eight weeks male C57BL/6 (B6) mice were used for this study. Mice received intraperitoneal hepatotoxic injection of APAP (300 mg/kg) and after 30 min of injection, the mice were treated with BA at a concentration of 30 mg/kg. After 16 h of treatment, mice were killed. Blood samples and liver tissues were harvested for analysis of liver injury parameters. Results APAP overdose significantly increased the serum alanine transferase (ALT) levels, hepatic activities of myeloperoxidase (MPO), expression of cytokines (TNF-α, IL-6, and IL-17), and malondialdehyde (MDA) activity when compared with the control animals. BA treatment after APAP administration significantly attenuated the elevation of these parameters in APAP-induced liver injury mice. Furthermore, BA treatment could also decrease hepatic IL-17-producing γδT cells recruitment, which was induced after APAP overdose. Conclusion Our data suggested that baicalin treatment could effectively decrease APAP-induced liver injury in part through attenuation of hepatic IL-17 expression. These results indicate that baicalin is a potential hepatoprotective agent. PMID:27855209

17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model

PubMed Central

Chen, Yan; Guo, Wenjie; Li, Wenjuan; Cheng, Meng; Hu, Ying; Xu, Wenming

2016-01-01

Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation. PMID:27872858

Cardiomyocyte-Specific Telomere Shortening is a Distinct Signature of Heart Failure in Humans.

PubMed

Sharifi-Sanjani, Maryam; Oyster, Nicholas M; Tichy, Elisia D; Bedi, Kenneth C; Harel, Ofer; Margulies, Kenneth B; Mourkioti, Foteini

2017-09-07

Telomere defects are thought to play a role in cardiomyopathies, but the specific cell type affected by the disease in human hearts is not yet identified. The aim of this study was to systematically evaluate the cell type specificity of telomere shortening in patients with heart failure in relation to their cardiac disease, age, and sex. We studied cardiac tissues from patients with heart failure by utilizing telomere quantitative fluorescence in situ hybridization, a highly sensitive method with single-cell resolution. In this study, total of 63 human left ventricular samples, including 37 diseased and 26 nonfailing donor hearts, were stained for telomeres in combination with cardiomyocyte- or α-smooth muscle cell-specific markers, cardiac troponin T, and smooth muscle actin, respectively, and assessed for telomere length. Patients with heart failure demonstrate shorter cardiomyocyte telomeres compared with nonfailing donors, which is specific only to cardiomyocytes within diseased human hearts and is associated with cardiomyocyte DNA damage. Our data further reveal that hypertrophic hearts with reduced ejection fraction exhibit the shortest telomeres. In contrast to other reported cell types, no difference in cardiomyocyte telomere length is evident with age. However, under the disease state, telomere attrition manifests in both young and older patients with cardiac hypertrophy. Finally, we demonstrate that cardiomyocyte-telomere length is better sustained in women than men under diseased conditions. This study provides the first evidence of cardiomyocyte-specific telomere shortening in heart failure. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

[Protection and its mechanism of catechin morphon on hypoxia-reoxygenation [corrected] induced injury in myocardial cells].

PubMed

Ye, Jin-Xia; Wang, Lan; Liang, Ri-Xin; Yang, Bin

2008-04-01

To observe the protective effects of catechin morphon (GCG and EGCG) on hypoxia-reoxygenation induced injury in myocardial cells and to explore the mechanisms. In cultured neonatal rat cardiomyocytes, we investigated the preconditioning protection by GCG and EGCG on the spontaneous beating, the survival rate, the release of LDH, MDA, SOD, GSH-Px and the ATP enzyme activity of cardiomyocyte cellular membrane in cultured rat cardiomyocytes treated during the reoxygenation 1h following hypoxia 3 h. The blocking agent of protien kinase C staurosporine (10 nmol x L(-1)) or the deactivator of Gi/o protein pertussis toxin (PTX, 200 microg x mL(-1)) were added before the catechin treatment. Preconditioning by GCG and EGCG increased the spontaneous beating and the survival rate, and decreased the release of LDH and MDA with the rise of SOD and ATP enzyme activity. Inhibition of PKC by staurosporine and Gi/o protein by PTX abolished the protection by catechin with the reduction of the beating, survival rate and activity of SOD, and the increase of the release of LDH and MDA. The results indicated that the activation of signal transduction pathway from PKC and Gi/o protein seemed to be involved in the cardioprotection of preconditioning by GCG and EGCG. The protection by GCG and EGCG on hypoxia-reoxygenation injury in cultured neonatal rat cardiomyocytes is found, which is related with scavenging of free radicals, and PKC Gi/o signal transduction pathway.

Progression in attenuating myocardial reperfusion injury: an overview.

PubMed

Bernink, F J P; Timmers, L; Beek, A M; Diamant, M; Roos, S T; Van Rossum, A C; Appelman, Y

2014-01-01

Reperfusion by means of percutaneous coronary intervention or thrombolytic therapy is the most effective treatment for acute myocardial infarction, markedly reducing mortality and morbidity. Reperfusion however induces necrotic and apoptotic damages to cardiomyocytes, that were viable prior to reperfusion, a process called lethal reperfusion injury. This process, consisting of many single processes, may be responsible of up to half of the final infarct size. A myriad of therapies as an adjunct to reperfusion have been studied with the purpose to attenuate reperfusion injury. The majority of these studies have been disappointing or contradicting, but recent proof-of-concept trials show that reperfusion injury still is a legitimate target. This overview will discuss these trials, the progression in attenuating myocardial reperfusion injury, promising therapies, and future perspectives. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeng, Xiang Jun; Department of Anesthesiology, 101 Woodruff Circle, Suite 617, Emory University School of Medicine, Atlanta, GA 30322; Yu, Shan Ping

2010-07-01

The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release andmore » activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca{sup 2+} accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.« less

Early Administration of Glutamine Protects Cardiomyocytes from Post-Cardiac Arrest Acidosis.

PubMed

Lin, Yan-Ren; Li, Chao-Jui; Syu, Shih-Han; Wen, Cheng-Hao; Buddhakosai, Waradee; Wu, Han-Ping; Hsu Chen, Cheng; Lu, Huai-En; Chen, Wen-Liang

2016-01-01

Postcardiac arrest acidosis can decrease survival. Effective medications without adverse side effects are still not well characterized. We aimed to analyze whether early administration of glutamine could improve survival and protect cardiomyocytes from postcardiac arrest acidosis using animal and cell models. Forty Wistar rats with postcardiac arrest acidosis (blood pH < 7.2) were included. They were divided into study (500 mg/kg L-alanyl-L-glutamine, n = 20) and control (normal saline, n = 20) groups. Each of the rats received resuscitation. The outcomes were compared between the two groups. In addition, cardiomyocytes derived from human induced pluripotent stem cells were exposed to HBSS with different pH levels (7.3 or 6.5) or to culture medium (control). Apoptosis-related markers and beating function were analyzed. We found that the duration of survival was significantly longer in the study group ( p < 0.05). In addition, in pH 6.5 or pH 7.3 HBSS buffer, the expression levels of cell stress (p53) and apoptosis (caspase-3, Bcl-xL) markers were significantly lower in cardiomyocytes treated with 50 mM L-glutamine than those without L-glutamine (RT-PCR). L-glutamine also increased the beating function of cardiomyocytes, especially at the lower pH level (6.5). More importantly, glutamine decreased cardiomyocyte apoptosis and increased these cells' beating function at a low pH level.

Extracellular matrix protein laminin enhances mesenchymal stem cell (MSC) paracrine function through αvβ3/CD61 integrin to reduce cardiomyocyte apoptosis.

PubMed

Peng, Kai-Yen; Liu, Yuan-Hung; Li, Yu-Wei; Yen, Betty Linju; Yen, Men-Luh

2017-08-01

Myocardial ischaemia (MI) results in extensive cardiomyocyte death and reactive oxygen species (ROS)-induced damage in an organ with little or no regenerative capacity. Although the use of adult bone marrow mesenchymal stem cells (BMMSCs) has been proposed as a treatment option, the high cell numbers required for clinical use are difficult to achieve with this source of MSCs, and animal studies have produced inconsistent data. We recently demonstrated in small and large animal models of acute MI that the application of human term placenta-derived multipotent cells (PDMCs), a foetal-stage MSC, resulted in reversal of cardiac injury with therapeutic efficacy. However, the mechanisms involved are unclear, making it difficult to strategize for therapeutic improvements. We found that PDMCs significantly reduced cardiomyocyte apoptosis and ROS production through the paracrine factors GRO-α, HGF and IL-8. Moreover, culturing PDMCs on plates coated with laminin, an extracellular matrix (ECM) protein, resulted in significantly enhanced secretion of all three paracrine factors, which further reduced cardiomyocyte apoptosis. The enhancement of PDMC paracrine function by laminin was mediated through αvβ3 integrin, with involvement of the signalling pathways of JNK, for GRO-α and IL-8 secretion, and PI3K/AKT, for HGF secretion. Our results demonstrated the utility of PDMC therapy to reduce cardiomyocyte apoptosis through modulation of ECM proteins in in vitro culture systems as a strategy to enhance the therapeutic functions of stem cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashok, Ajay; Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288; Kanwar, Jagat Rakesh

injury. - Highlights: • Protection/regeneration of dying myocardium post myocardial infarction is important. • Downregulation of survivin induces apoptosis in hypoxic human cardiomyocytes (HCM). • Bio-replenishment with SurR9-C84A reinstates HCM survival, recovery and growth. • SurR9-C84A targets mitochondrial depolarization, fcTnT and ROS generation in HCM. • SurR9-C84A upregulates survivin, PCNA, PI3K/Akt pathway, VEGF and HSP levels. • SurR9-C84A holds promise as a treatment and preventive agent to replenish survivin.« less

Cardiomyocyte architectural plasticity in fetal, neonatal, and adult pig hearts delineated with diffusion tensor MRI.

PubMed

Zhang, Lei; Allen, John; Hu, Lingzhi; Caruthers, Shelton D; Wickline, Samuel A; Chen, Junjie

2013-01-15

Cardiomyocyte organization is a critical determinant of coordinated cardiac contractile function. Because of the acute opening of the pulmonary circulation, the relative workload of the left ventricle (LV) and right ventricle (RV) changes substantially immediately after birth. We hypothesized that three-dimensional cardiomyocyte architecture might be required to adapt rapidly to accommodate programmed perinatal changes of cardiac function. Isolated fixed hearts from pig fetuses or pigs at midgestation, preborn, postnatal day 1 (P1), postnatal day 5, postnatal day 14 (P14), and adulthood (n = 5 for each group) were acquired for diffusion-weighted magnetic resonance imaging. Cardiomyocyte architecture was visualized by three-dimensional fiber tracking and was quantitatively evaluated by the measured helix angle (α(h)). Upon the completion of MRI, hearts were sectioned and stained with hematoxylin/eosin (H&E) to evaluate cardiomyocyte alignment, with picrosirius red to evaluate collagen content, and with anti-Ki67 to evaluate postnatal cell proliferation. The helical architecture of cardiomyocyte was observed as early as the midgestational period. Postnatal changes of cardiomyocyte architecture were observed from P1 to P14, which primary occurred in the septum and RV free wall (RVFW). In the septum, the volume ratio of LV- vs. RV-associated cardiomyocytes rapidly changed from RV-LV balanced pattern at birth to LV dominant pattern by P14. In the RVFW, subendocardial α(h) decreased by ~30° from P1 to P14. These findings indicate that the helical architecture of cardiomyocyte is developed as early as the midgestation period. Substantial and rapid adaptive changes in cardiac microarchitecture suggested considerable developmental plasticity of cardiomyocyte form and function in the postnatal period in response to altered cardiac mechanical function.

Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Li; Wu, Zhou; Baba, Masashi

Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, themore » understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in

Protective effect of 4,4'-diaminodiphenylsulfone against paraquat-induced mouse lung injury

PubMed Central

Cho, Sung Chun; Rhim, Ji Heon; Choi, Hae Ri; Son, Young Hoon; Lee, Seok Jin; Song, Kye-Yong

2011-01-01

Although 4,4'-diaminodiphenylsulfone (DDS, dapsone) has been used to treat several dermatologic conditions, including Hansen disease, for the past several decades, its mode of action has remained a topic of debate. We recently reported that DDS treatment significantly extends the lifespan of the nematode C. elegans by decreasing the generation of reactive oxygen species. Additionally, in in vitro experiments using non-phagocytic human fibroblasts, we found that DDS effectively counteracted the toxicity of paraquat (PQ). In the present study, we extended our work to test the protective effect of DDS against PQ in vivo using a mouse lung injury model. Oral administration of DDS to mice significantly attenuated the lung tissue damage caused by subsequent administration of PQ. Moreover, DDS reduced the local expression of mRNA transcripts encoding inflammation-related molecules, including endothelin-1 (ET-1), macrophage inflammatory protein-1α (MIP-1α), and transforming growth factor-β (TGF-β). In addition, DDS decreased the PQ-induced expression of NADPH oxidase mRNA and activation of protein kinase Cµ (PKCµ). DDS treatment also decreased the PQ-induced generation of superoxide anions in mouse lung fibroblasts. Taken together, these data suggest the novel efficacy of DDS as an effective protective agent against oxidative stress-induced tissue damages. PMID:21765237

Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification.

PubMed

Li, Dan L; Wang, Zhao V; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W; Gillette, Thomas G; Hill, Joseph A

2016-04-26

The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. © 2016 American Heart Association, Inc.

Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

PubMed Central

Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

2013-01-01

Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

Functional expression and pharmaceutical efficacy of cardiac-specific ion channels in human embryonic stem cell-derived cardiomyocytes.

PubMed

Kim, Han Sol; Yoon, Jung Won; Li, Hongliang; Jeong, Geun Ok; Park, Jin Ju; Shin, Sung Eun; Jang, Il Ho; Kim, Jae Ho; Park, Won Sun

2017-10-23

Cardiomyocytes differentiated from human pluripotent stem cells provide promising tools for screening of cardiotoxic drugs. For evaluation of human pluripotent stem cell-derived cardiomyocytes for cardiotoxicity test, in the present study, human embryonic stem cells (hESCs) were differentiated to cardiomyocytes, followed by metabolic selection to enrich the differentiated cardiomyocytes. The highly purified hESC-derived cardiomyocytes (hESC-CMs) expressed several cardiomyocyte-specific markers including cTnT, MLC2a, and α-SA, but not pluripotency markers, such as OCT4 and NANOG. Patch clamp technique and RT-PCR revealed the expression of cardiomyocyte-specific Na + , Ca 2+ , and K + channels and cardiac action potential in hESC-CMs. To explore the potential use of hESC-CMs as functional cardiomyocytes for drug discovery and cardiotoxicity screening, we examined the effects of bisindolylmaleimide (BIM) (I), which inhibits native cardiac Ca 2+ channels, on the Ca 2+ channel activity of hESC-CMs. We observed a similar response for the BIM (I)-induced modulation of Ca 2+ channels between hESC-CMs and native cardiomyocytes through L-type Ca 2+ channel current. These results suggest that hESC-CMs can be useful for evaluation of pharmaceutical efficacy and safety of novel drug candidate in cardiac research.

Lumican Deficiency Results In Cardiomyocyte Hypertrophy With Altered Collagen Assembly

PubMed Central

Dupuis, Loren E.; Berger, Matthew G.; Feldman, Samuel; Doucette, Lorna; Fowlkes, Vennece; Chakravarti, Shukti; Thibaudeau, Sarah; Alcala, Nicolas E.; Bradshaw, Amy D.; Kern, Christine B.

2015-01-01

The ability of the heart to adapt to increased stress is dependent on modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest LUM deficient mice (lum−/−) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum−/− hearts have not been evaluated. These studies show LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum−/− neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum−/− hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum−/− hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum−/− hearts. There was also a reduction in the β and γ forms of collagenα1(I) in lum−/− hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum−/− hearts, indicating LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate

Localization of sarcomeric proteins during myofibril assembly in cultured mouse primary skeletal myotubes

PubMed Central

White, Jennifer; Barro, Marietta V.; Makarenkova, Helen P.; Sanger, Joseph W.; Sanger, Jean M.

2014-01-01

It is important to understand how muscle forms normally in order to understand muscle diseases that result in abnormal muscle formation. Although the structure of myofibrils is well understood, the process through which the myofibril components form organized contractile units is not clear. Based on the staining of muscle proteins in avian embryonic cardiomyocytes, we previously proposed that myofibrils formation occurred in steps that began with premyofibrils followed by nascent myofibrils and ending with mature myofibrils. The purpose of this study was to determine whether the premyofibril model of myofibrillogenesis developed from studies developed from studies in avian cardiomyocytes was supported by our current studies of myofibril assembly in mouse skeletal muscle. Emphasis was on establishing how the key sarcomeric proteins, F-actin, non-muscle myosin II, muscle myosin II, and α-actinin were organized in the three stages of myofibril assembly. The results also test previous reports that non-muscle myosins II A and B are components of the Z-Bands of mature myofibrils, data that are inconsistent with the premyofibril model. We have also determined that in mouse muscle cells, telethonin is a late assembling protein that is present only in the Z-Bands of mature myofibrils. This result of using specific telethonin antibodies supports the approach of using YFP-tagged proteins to determine where and when these YFP-sarcomeric fusion proteins are localized. The data presented in this study on cultures of primary mouse skeletal myocytes are consistent with the premyofibril model of myofibrillogenesis previously proposed for both avian cardiac and skeletal muscle cells. PMID:25125171

Derivation of highly purified cardiomyocytes from human induced pluripotent stem cells using small molecule-modulated differentiation and subsequent glucose starvation.

PubMed

Sharma, Arun; Li, Guang; Rajarajan, Kuppusamy; Hamaguchi, Ryoko; Burridge, Paul W; Wu, Sean M

2015-03-18

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/β-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Micro pumping with cardiomyocyte-polymer hybrid.

PubMed

Park, Jungyul; Kim, Il Chaek; Baek, Jeongeun; Cha, Misun; Kim, Jinseok; Park, Sukho; Lee, Junghoon; Kim, Byungkyu

2007-10-01

This paper presents a hybrid micropump actuated by the up-down motion of a dome shaped cell-polymer membrane composite. The contractile force induced from self-beating cardiomyocytes cultured on the membrane causes shrinkage and relaxation of a microchamber, leading to a flow in a microchannel. Flow direction is controlled by the geometry of diffuser/nozzle in the microchannel. The fabrication process is noninvasive to cells, thus, cardiomyocytes can robustly maintain their activity for a long time. The fluid motion in the microchannel was monitored by tracking 2 microm polystyrene beads. A net flow rate of 0.226 nl min(-1) was obtained in our microscale device. Our device demonstrates a unique performance of a cell-microdevice hybrid lab-on-a-chip that does not require any external power source, preventing electrical or heat shock to analytes.

MicroRNA-297 promotes cardiomyocyte hypertrophy via targeting sigma-1 receptor.

PubMed

Bao, Qinxue; Zhao, Mingyue; Chen, Li; Wang, Yu; Wu, Siyuan; Wu, Wenchao; Liu, Xiaojing

2017-04-15

Sigma-1 receptor (Sig-1R) is a ligand-regulated endoplasmic reticulum (ER) chaperone involved in cardiac hypertrophy, but it is not known whether Sig-1R is regulated by microRNAs (miRNAs). According to bioinformatic analysis, miR-297 was suggested as a potential target miRNA for Sig-1R. Therefore, we verified whether miR-297 could target Sig-1R and investigated the possible mechanisms underlying the role of miR-297 in cardiac hypertrophy. Bioinformatic analysis combined with laboratory experiments, including quantitative RT-PCR, Western blotting, and luciferase assay, were performed to identify the target miRNA of Sig-1R. Transverse aortic constriction (TAC) model and neonatal rat cardiomyocytes (NCMs) stimulated with angiotensin II (AngII) were used to explore the relationship between miR-297 and Sig-1R. Additionally, the function of miR-297 in cardiomyocyte hypertrophy and ER stress/unfolded protein response (UPR) signaling pathway was investigated by transfecting miR-297 mimics/inhibitor. miR-297 levels were increased in both TAC-induced hypertrophic heart tissue and AngII-induced cardiomyocyte hypertrophy. Up-regulation of miR-297 by specific mimics exacerbated AngII-induced cardiomyocyte hypertrophy, whereas inhibition of miR-297 suppressed the process. During cardiomyocyte hypertrophy, Sig-1R expression, which was negatively regulated by miR-297 by directly targeting its 3'untranslated region (UTR), was decreased. Furthermore, attenuation of miR-297 inhibited the activation of X-box binding protein 1 (Xbp1) and activating transcriptional factor 4 (ATF4) signaling pathways in NCMs. Our data demonstrate that miR-297 promotes cardiomyocyte hypertrophy by inhibiting the expression of Sig-1R and activation of ER stress signaling, which provides a novel interpretation for cardiac hypertrophy. Copyright © 2017 Elsevier Inc. All rights reserved.

Glycosylation and Processing of Pro-B-type Natriuretic Peptide in Cardiomyocytes

PubMed Central

Peng, Jianhao; Jiang, Jingjing; Wang, Wei; Qi, Xiaofei; Sun, Xue-Long; Wu, Qingyu

2011-01-01

B-type natriuretic peptide (BNP) and its related peptides are biomarkers for the diagnosis of heart failure. Recent studies identified several O-glycosylation sites, including Thr-71, on human pro-BNP but the functional significance was unclear. In this study, we analyzed glycosylation and proteolytic processing of pro-BNP in cardiomyocytes. Human pro-BNP wild-type (WT) and mutants were expressed in HEK 293 cells and murine HL-1 cardiomyocytes. Pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting. Glycosidases and glycosylation inhibitors were used to examine carbohydrates on pro-BNP. The effects of furin and corin expression on pro-BNP processing in cells also were examined. We found that in HEK 293 cells, recombinant pro-BNP contained significant amounts of O-glycans with terminal oligosialic acids. Mutation at Thr-71 reduced O-glycans on pro-BNP and increased pro-BNP processing. In HL-1 cardiomyocytes, residue Thr-71 contained little O-glycans, and pro-BNP WT and T71A mutant were processed similarly. In HEK 293 cells, pro-BNP was processed by furin. Mutations at Arg-73 and Arg-76, but not Lys-79, prevented pro-BNP processing. In HL-1 cardiomyocytes, which express furin and corin, single or double mutations at Arg-73, Arg-76 and Lys-79 did not prevent pro-BNP processing. Only when all these three residues were mutated, was pro-BNP processing completely blocked. Our data indicate that pro-BNP glycosylation in cardiomyocytes differed significantly from that in HEK 293 cells. In HEK 293 cells, furin cleaved pro-BNP at Arg-76 whereas in cardiomyocytes corin cleaved pro-BNP at multiple residues including Arg-73, Arg-76 and Lys-79. PMID:21763278

Blockade of the swelling-induced chloride current attenuates the mouse neonatal hypoxic-ischemic brain injury in vivo.

PubMed

Wong, Raymond; Abussaud, Ahmed; Leung, Joseph Wh; Xu, Bao-Feng; Li, Fei-Ya; Huang, Sammen; Chen, Nai-Hong; Wang, Guan-Lei; Feng, Zhong-Ping; Sun, Hong-Shuo

2018-05-01

Activation of swelling-induced Cl - current (I Cl,swell ) during neonatal hypoxia-ischemia (HI) may induce brain damage. Hypoxic-ischemic brain injury causes chronic neurological morbidity in neonates as well as acute mortality. In this study, we investigated the role of I Cl,swell in hypoxic-ischemic brain injury using a selective blocker, 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB). In primary cultured cortical neurons perfusion of a 30% hypotonic solution activated I Cl,swell , which was completely blocked by the application of DCPIB (10 μmol/L). The role of I Cl,swell in neonatal hypoxic-ischemic brain injury in vivo was evaluated in a modified neonatal hypoxic-ischemic brain injury model. Before receiving the ischemic insult, the mouse pups were injected with DCPIB (10 mg/kg, ip). We found that pretreatment with DCPIB significantly reduced the brain damage assessed using TTC staining, Nissl staining and whole brain imaging, and improved the sensorimotor and vestibular recovery outcomes evaluated in neurobehavioural tests (i.e. geotaxis reflex, and cliff avoidance reflex). These results show that DCPIB has neuroprotective effects on neonatal hypoxic-ischemic brain injury, and that the I Cl,swell may serve as a therapeutic target for treatment of hypoxic-ischemic encephalopathy.

Febuxostat pretreatment attenuates myocardial ischemia/reperfusion injury via mitochondrial apoptosis.

PubMed

Wang, Shulin; Li, Yunpeng; Song, Xudong; Wang, Xianbao; Zhao, Cong; Chen, Aihua; Yang, Pingzhen

2015-07-02

Febuxostat is a selective inhibitor of xanthine oxidase (XO). XO is a critical source of reactive oxygen species (ROS) during myocardial ischemia/reperfusion (I/R) injury. Inhibition of XO is therapeutically effective in I/R injury. Evidence suggests that febuxostat exerts antioxidant effects by directly scavenging ROS. The present study was performed to investigate the effects of febuxostat on myocardial I/R injury and its underlying mechanisms. We utilized an in vivo mouse model of myocardial I/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of hypoxia/reoxygenation (H/R) injury. Mice were randomized into five groups: Sham, I/R (I/R + Vehicle), I/R + FEB (I/R + febuxostat), AL + I/R (I/R + allopurinol) and FEB (febuxostat), respectively. The I/R + FEB mice were pretreated with febuxostat (5 mg/kg; i.p.) 24 and 1 h prior to I/R. NRCs received febuxostat (1 and 10 µM) at 24 and 1 h before exposure to hypoxia for 3 h followed by reoxygenation for 3 h. Cardiac function, myocardial infarct size, serum levels of creatine kinase (CK) and lactate dehydrogenase (LDH), and myocardial apoptotic index (AI) were measured in order to ascertain the effects of febuxostat on myocardial I/R injury. Hypoxia/reperfusion (H/R) injury in NRCs was examined using MTT, LDH leakage assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The underlying mechanisms were determined by measuring ROS production, mitochondrial membrane potential (ΔΨm), and expression of cytochrome c, cleaved caspases as well as Bcl-2 protein levels. Myocardial I/R led to an elevation in the myocardial infarct size, serum levels of CK and LDH, cell death and AI. Furthermore, I/R reduced cardiac function. These changes were significantly attenuated by pretreatment with febuxostat and allopurinol, especially by febuxostat. Febuxostat also protected the mitochondrial structure following myocardial I/R, inhibited H/R-induced ROS generation, stabilized the �

Expression analysis of Baf60c during heart regeneration in axolotls and neonatal mice.

PubMed

Nakamura, Ryo; Koshiba-Takeuchi, Kazuko; Tsuchiya, Megumi; Kojima, Mizuyo; Miyazawa, Asuka; Ito, Kohei; Ogawa, Hidesato; Takeuchi, Jun K

2016-05-01

Some organisms, such as zebrafish, urodele amphibians, and newborn mice, have a capacity for heart regeneration following injury. However, adult mammals fail to regenerate their hearts. To know why newborn mice can regenerate their hearts, we focused on epigenetic factors, which are involved in cell differentiation in many tissues. Baf60c (BRG1/BRM-associated factor 60c), a component of ATP-dependent chromatin-remodeling complexes, has an essential role for cardiomyocyte differentiation at the early heart development. To address the function of Baf60c in postnatal heart homeostasis and regeneration, we examined the detailed expression/localization patterns of Baf60c in both mice and axolotls. In the mouse heart development, Baf60c was highly expressed in the entire heart at the early stages, but gradually downregulated at the postnatal stages. During heart regeneration in neonatal mice and axolotls, Baf60c expression was strongly upregulated after resection. Interestingly, the timing of Baf60c upregulation after resection was consistent with the temporal dynamics of cardiomyocyte proliferation. Moreover, knockdown of Baf60c downregulated proliferation of neonatal mouse cardiomyocytes. These data suggested that Baf60c plays an important role in cardiomyocyte proliferation in heart development and regeneration. This is the first study indicating that Baf60c contributes to the heart regeneration in vertebrates. © 2016 Japanese Society of Developmental Biologists.

Cardiomyocyte architectural plasticity in fetal, neonatal, and adult pig hearts delineated with diffusion tensor MRI

PubMed Central

Zhang, Lei; Allen, John; Hu, Lingzhi; Caruthers, Shelton D.; Wickline, Samuel A.

2013-01-01

Cardiomyocyte organization is a critical determinant of coordinated cardiac contractile function. Because of the acute opening of the pulmonary circulation, the relative workload of the left ventricle (LV) and right ventricle (RV) changes substantially immediately after birth. We hypothesized that three-dimensional cardiomyocyte architecture might be required to adapt rapidly to accommodate programmed perinatal changes of cardiac function. Isolated fixed hearts from pig fetuses or pigs at midgestation, preborn, postnatal day 1 (P1), postnatal day 5, postnatal day 14 (P14), and adulthood (n = 5 for each group) were acquired for diffusion-weighted magnetic resonance imaging. Cardiomyocyte architecture was visualized by three-dimensional fiber tracking and was quantitatively evaluated by the measured helix angle (αh). Upon the completion of MRI, hearts were sectioned and stained with hematoxylin/eosin (H&E) to evaluate cardiomyocyte alignment, with picrosirius red to evaluate collagen content, and with anti-Ki67 to evaluate postnatal cell proliferation. The helical architecture of cardiomyocyte was observed as early as the midgestational period. Postnatal changes of cardiomyocyte architecture were observed from P1 to P14, which primary occurred in the septum and RV free wall (RVFW). In the septum, the volume ratio of LV- vs. RV-associated cardiomyocytes rapidly changed from RV-LV balanced pattern at birth to LV dominant pattern by P14. In the RVFW, subendocardial αh decreased by ∼30° from P1 to P14. These findings indicate that the helical architecture of cardiomyocyte is developed as early as the midgestation period. Substantial and rapid adaptive changes in cardiac microarchitecture suggested considerable developmental plasticity of cardiomyocyte form and function in the postnatal period in response to altered cardiac mechanical function. PMID:23161881

Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes.

PubMed

Björk, Susann; Ojala, Elina A; Nordström, Tommy; Ahola, Antti; Liljeström, Mikko; Hyttinen, Jari; Kankuri, Esko; Mervaala, Eero

2017-01-01

Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating

Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

PubMed Central

Björk, Susann; Ojala, Elina A.; Nordström, Tommy; Ahola, Antti; Liljeström, Mikko; Hyttinen, Jari; Kankuri, Esko; Mervaala, Eero

2017-01-01

Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP) waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD) parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging) we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs), cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and paced beating

Schwann cells use TAM receptor-mediated phagocytosis in addition to autophagy to clear myelin in a mouse model of nerve injury.

PubMed

Brosius Lutz, Amanda; Chung, Won-Suk; Sloan, Steven A; Carson, Glenn A; Zhou, Lu; Lovelett, Emilie; Posada, Sean; Zuchero, J Bradley; Barres, Ben A

2017-09-19

Ineffective myelin debris clearance is a major factor contributing to the poor regenerative ability of the central nervous system. In stark contrast, rapid clearance of myelin debris from the injured peripheral nervous system (PNS) is one of the keys to this system's remarkable regenerative capacity, but the molecular mechanisms driving PNS myelin clearance are incompletely understood. We set out to discover new pathways of PNS myelin clearance to identify novel strategies for activating myelin clearance in the injured central nervous system, where myelin debris is not cleared efficiently. Here we show that Schwann cells, the myelinating glia of the PNS, collaborate with hematogenous macrophages to clear myelin debris using TAM (Tyro3, Axl, Mer) receptor-mediated phagocytosis as well as autophagy. In a mouse model of PNS nerve crush injury, Schwann cells up-regulate TAM phagocytic receptors Axl and Mertk following PNS injury, and Schwann cells lacking both of these phagocytic receptors exhibit significantly impaired myelin phagocytosis both in vitro and in vivo. Autophagy-deficient Schwann cells also display reductions in myelin clearance after mouse nerve crush injury, as has been recently shown following nerve transection. These findings add a mechanism, Axl/Mertk-mediated myelin clearance, to the repertoire of cellular machinery used to clear myelin in the injured PNS. Given recent evidence that astrocytes express Axl and Mertk and have previously unrecognized phagocytic potential, this pathway may be a promising avenue for activating myelin clearance after CNS injury.

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

PubMed Central

Laugwitz, Karl-Ludwig; Moretti, Alessandra; Lam, Jason; Gruber, Peter; Chen, Yinhong; Woodard, Sarah; Lin, Li-Zhu; Cai, Chen-Leng; Lu, Min Min; Reth, Michael; Platoshyn, Oleksandr; Yuan, Jason X.-J.; Evans, Sylvia; Chien, Kenneth R.

2017-01-01

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases1–3. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart4. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease. PMID:15703750

Cardiomyocyte Regulation of Systemic Lipid Metabolism by the Apolipoprotein B-Containing Lipoproteins in Drosophila

PubMed Central

Ishikawa, Zachary

2017-01-01

The heart has emerged as an important organ in the regulation of systemic lipid homeostasis; however, the underlying mechanism remains poorly understood. Here, we show that Drosophila cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body. In a Drosophila genetic screen, we discovered that when haplo-insufficient, microsomal triglyceride transfer protein (mtp), required for the biosynthesis of apoB-lipoproteins, suppressed the development of diet-induced obesity. Tissue-specific inhibition of Mtp revealed that whereas knockdown of mtp only in the fat body decreases systemic triglyceride (TG) content on normal food diet (NFD) as expected, knockdown of mtp only in the cardiomyocytes also equally decreases systemic TG content on NFD, suggesting that the cardiomyocyte- and fat body-derived apoB-lipoproteins serve similarly important roles in regulating whole-body lipid metabolism. Unexpectedly, on high fat diet (HFD), knockdown of mtp in the cardiomyocytes, but not in fat body, protects against the gain in systemic TG levels. We further showed that inhibition of the Drosophila apoB homologue, apolipophorin or apoLpp, another gene essential for apoB-lipoprotein biosynthesis, affects systemic TG levels similarly to that of Mtp inhibition in the cardiomyocytes on NFD or HFD. Finally, we determined that HFD differentially alters Mtp and apoLpp expression in the cardiomyocytes versus the fat body, culminating in higher Mtp and apoLpp levels in the cardiomyocytes than in fat body and possibly underlying the predominant role of cardiomyocyte-derived apoB-lipoproteins in lipid metabolic regulation. Our findings reveal a novel and significant function of heart-mediated apoB-lipoproteins in controlling lipid homeostasis. PMID:28095410

Joint loads resulting in ACL rupture: Effects of age, sex, and body mass on injury load and mode of failure in a mouse model.

PubMed

Blaker, Carina L; Little, Christopher B; Clarke, Elizabeth C

2017-08-01

Anterior cruciate ligament (ACL) tears are a common knee injury with a known but poorly understood association with secondary joint injuries and post-traumatic osteoarthritis (OA). Female sex and age are known risk factors for ACL injury but these variables are rarely explored in mouse models of injury. This study aimed to further characterize a non-surgical ACL injury model to determine its clinical relevance across a wider range of mouse specifications. Cadaveric and anesthetized C57BL/6 mice (9-52 weeks of age) underwent joint loading to investigate the effects of age, sex, and body mass on ACL injury mechanisms. The ACL injury load (whole joint load required to rupture the ACL) was measured from force-displacement data, and mode of failure was assessed using micro-dissection and histology. ACL injury load was found to increase with body mass and age (p < 0.001) but age was not significant when controlling for mass. Sex had no effect. In contrast, the mode of ACL failure varied with both age and sex groups. Avulsion fractures (complete or mixed with mid-substance tears) were common in all age groups but the proportion of mixed and mid-substance failures increased with age. Females were more likely than males to have a major avulsion relative to a mid-substance tear (p < 0.01). This data compliments studies in human cadaveric knees, and provides a basis for determining the severity of joint injury relative to a major ACL tear in mice, and for selecting joint loading conditions in future experiments using this model. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1754-1763, 2017. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

PubMed

Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

2016-12-01

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na +  current (I Na ), nifedipine, a blocker of L-type Ca 2+  current (I CaL ), and E4031, a blocker of the rapid component of delayed rectifier K +  current (I Kr ). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K +  current (I Ks ). In hiPSC-derived cardiomyocytes of cardiac origin, I Na , I CaL , I Kr , and I Ks were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic

Ischaemia-reperfusion injury in orthotopic mouse lung transplants - a scanning electron microscopy study.

PubMed

Draenert, Alice; Marquardt, Klaus; Inci, Ilhan; Soltermann, Alex; Weder, Walter; Jungraithmayr, Wolfgang

2011-02-01

Lung ischaemia-reperfusion (I/R) injury remains a major cause of graft failure in lung transplantation (Tx). With the implementation of orthotopic lung Tx in mice, a physiological model on the base of a perfused and ventilated graft became available for the investigation of I/R injury. Using the scanning electron microscopy (SEM) technique, we here present an analysis of early and late morphological changes of pulmonary I/R injury. Syngeneic lungs were orthotopically transplanted between C57BL/6 mice. Grafts were exposed to 2 h of cold ischaemia. Transplants and right lungs were examined by SEM with corresponding haematoxylin-eosin histology 30 min and 4 h after reperfusion. Thirty minutes after reperfusion, the alveolar surface of transplants showed a discontinued lining of surfactant, while the lining of the non-transplanted lung was normal. Within the graft, leucocytes displayed an irregular surface with development of pseudopodia, and microvilli were detected on the membrane of pneumocytes. At 4 h after reperfusion, leucocytes significantly increased in numbers within the alveolar space. Also, the number of microvilli on pneumocytes increased significantly. Similar to these, the endothelium of vessels increasingly developed microvilli from 30 min towards 4 h after reperfusion. The airways of transplanted grafts showed mild changes with thickening of the bronchial epithelium and a destruction of kinocilia. Taken together, SEM detects pathological events of I/R that are previously not described in normal histology. These findings may influence the interpretation of studies investigating the I/R injury in the mouse model of lung Tx. © 2011 The Authors. International Journal of Experimental Pathology © 2011 International Journal of Experimental Pathology.

Global transcriptomic analysis of induced cardiomyocytes predicts novel regulators for direct cardiac reprogramming.

PubMed

Talkhabi, Mahmood; Razavi, Seyed Morteza; Salari, Ali

2017-06-01

Heart diseases are the most significant cause of morbidity and mortality in the world. De novo generated cardiomyocytes (CMs) are a great cellular source for cell-based therapy and other potential applications. Direct cardiac reprogramming is the newest method to produce CMs, known as induced cardiomyocytes (iCMs). During a direct cardiac reprogramming, also known as transdifferentiation, non-cardiac differentiated adult cells are reprogrammed to cardiac identity by forced expression of cardiac-specific transcription factors (TFs) or microRNAs. To this end, many different combinations of TFs (±microRNAs) have been reported for direct reprogramming of mouse or human fibroblasts to iCMs, although their efficiencies remain very low. It seems that the investigated TFs and microRNAs are not sufficient for efficient direct cardiac reprogramming and other cardiac specific factors may be required for increasing iCM production efficiency, as well as the quality of iCMs. Here, we analyzed gene expression data of cardiac fibroblast (CFs), iCMs and adult cardiomyocytes (aCMs). The up-regulated and down-regulated genes in CMs (aCMs and iCMs) were determined as CM and CF specific genes, respectively. Among CM specific genes, we found 153 transcriptional activators including some cardiac and non-cardiac TFs that potentially activate the expression of CM specific genes. We also identified that 85 protein kinases such as protein kinase D1 (PKD1), protein kinase A (PRKA), calcium/calmodulin-dependent protein kinase (CAMK), protein kinase C (PRKC), and insulin like growth factor 1 receptor (IGF1R) that are strongly involved in establishing CM identity. CM gene regulatory network constructed using protein kinases, transcriptional activators and intermediate proteins predicted some new transcriptional activators such as myocyte enhancer factor 2A (MEF2A) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A), which may be required for qualitatively and

Essential role of STIM1 in the development of cardiomyocyte hypertrophy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohba, Takayoshi; Watanabe, Hiroyuki; Murakami, Manabu

2009-11-06

Store-operated Ca{sup 2+} entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48 h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential rolemore » in the development of cardiomyocyte hypertrophy.« less

Carbamoyl phosphate synthetase-1 is a rapid turnover biomarker in mouse and human acute liver injury.

PubMed

Weerasinghe, Sujith V W; Jang, You-Jin; Fontana, Robert J; Omary, M Bishr

2014-08-01

Several serum markers are used to assess hepatocyte damage, but they have limitations related to etiology specificity and prognostication. Identification of novel hepatocyte-specific biomarkers could provide important prognostic information and better pathogenesis classification. We tested the hypothesis that hepatocyte-selective biomarkers are released after subjecting isolated mouse hepatocytes to Fas-ligand-mediated apoptosis. Proteomic analysis of hepatocyte culture medium identified the mitochondrial matrix protein carbamoyl phosphate synthetase-1 (CPS1) among the most readily detected proteins that are released by apoptotic hepatocytes. CPS1 was also detected in mouse serum upon acute challenge with Fas-ligand or acetaminophen and in hepatocytes upon hypoosmotic stress, independent of hepatocyte caspase activation. Furthermore, CPS1 was observed in sera of mice chronically fed the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mouse CPS1 detectability was similar in serum and plasma, and its half-life was 126 ± 9 min. Immune staining showed that CPS1 localized to mouse hepatocytes but not ductal cells. Analysis of a few serum samples from patients with acute liver failure (ALF) due to acetaminophen, Wilson disease, or ischemia showed readily detectable CPS1 that was not observed in several patients with chronic viral hepatitis or in control donors. Notably, CPS1 rapidly decreased to undetectable levels in sera of patients with acetaminophen-related ALF who ultimately recovered, while alanine aminotransferase levels remained elevated. Therefore, CPS1 becomes readily detectable upon hepatocyte apoptotic and necrotic death in culture or in vivo. Its abundance and short serum half-life, compared with alanine aminotransferase, suggest that it may be a useful prognostic biomarker in human and mouse liver injury. Copyright © 2014 the American Physiological Society.

Ischaemia-reperfusion injury in orthotopic mouse lung transplants – a scanning electron microscopy study

PubMed Central

Draenert, Alice; Marquardt, Klaus; Inci, Ilhan; Soltermann, Alex; Weder, Walter; Jungraithmayr, Wolfgang

2011-01-01

Lung ischaemia-reperfusion (I/R) injury remains a major cause of graft failure in lung transplantation (Tx). With the implementation of orthotopic lung Tx in mice, a physiological model on the base of a perfused and ventilated graft became available for the investigation of I/R injury. Using the scanning electron microscopy (SEM) technique, we here present an analysis of early and late morphological changes of pulmonary I/R injury. Syngeneic lungs were orthotopically transplanted between C57BL/6 mice. Grafts were exposed to 2 h of cold ischaemia. Transplants and right lungs were examined by SEM with corresponding haematoxylin–eosin histology 30 min and 4 h after reperfusion. Thirty minutes after reperfusion, the alveolar surface of transplants showed a discontinued lining of surfactant, while the lining of the non-transplanted lung was normal. Within the graft, leucocytes displayed an irregular surface with development of pseudopodia, and microvilli were detected on the membrane of pneumocytes. At 4 h after reperfusion, leucocytes significantly increased in numbers within the alveolar space. Also, the number of microvilli on pneumocytes increased significantly. Similar to these, the endothelium of vessels increasingly developed microvilli from 30 min towards 4 h after reperfusion. The airways of transplanted grafts showed mild changes with thickening of the bronchial epithelium and a destruction of kinocilia. Taken together, SEM detects pathological events of I/R that are previously not described in normal histology. These findings may influence the interpretation of studies investigating the I/R injury in the mouse model of lung Tx. PMID:21272104

Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications

PubMed Central

Gay, Maresha S.; Li, Yong; Xiong, Fuxia; Lin, Thant; Zhang, Lubo

2015-01-01

The potential adverse effect of synthetic glucocorticoid, dexamethasone therapy on the developing heart remains unknown. The present study investigated the effects of dexamethasone on cardiomyocyte proliferation and binucleation in the developing heart of newborn rats and evaluated DNA methylation as a potential mechanism. Dexamethasone was administered intraperitoneally in a three day tapered dose on postnatal day 1 (P1), 2 and 3 to rat pups in the absence or presence of a glucocorticoid receptor antagonist Ru486, given 30 minutes prior to dexamethasone. Cardiomyocytes from P4, P7 or P14 animals were analyzed for proliferation, binucleation and cell number. Dexamethasone treatment significantly increased the percentage of binucleated cardiomyocytes in the hearts of P4 pups, decreased myocyte proliferation in P4 and P7 pups, reduced cardiomyocyte number and increased the heart to body weight ratio in P14 pups. Ru486 abrogated the effects of dexamethasone. In addition, 5-aza-2'-deoxycytidine (5-AZA) blocked the effects of dexamethasone on binucleation in P4 animals and proliferation at P7, leading to recovered cardiomyocyte number in P14 hearts. 5-AZA alone promoted cardiomyocyte proliferation at P7 and resulted in a higher number of cardiomyocytes in P14 hearts. Dexamethasone significantly decreased cyclin D2, but not p27 expression in P4 hearts. 5-AZA inhibited global DNA methylation and blocked dexamethasone-mediated down-regulation of cyclin D2 in the heart of P4 pups. The findings suggest that dexamethasone acting on glucocorticoid receptors inhibits proliferation and stimulates premature terminal differentiation of cardiomyocytes in the developing heart via increased DNA methylation in a gene specific manner. PMID:25923220

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

PubMed Central

Kojima, Akiko; Kitagawa, Hirotoshi; Omatsu-Kanbe, Mariko; Matsuura, Hiroshi; Nosaka, Shuichi

2010-01-01

BACKGROUND AND PURPOSE The Ca2+ paradox is an important phenomenon associated with Ca2+ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca2+ paradox. EXPERIMENTAL APPROACH Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope. KEY RESULTS The Ca2+ paradox was readily evoked by restoration of the extracellular Ca2+ following 10–20 min of nominally Ca2+-free superfusion. The Ca2+ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd3+, La3+) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca2+ content, assessed by caffeine application, gradually declined during Ca2+-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca2+ leak by tetracaine prevented Ca2+ paradox. The Na+/Ca2+ exchange (NCX) blocker KB-R7943 significantly inhibited Ca2+ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca2+ restoration. The SR Ca2+ content was better preserved during Ca2+ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes. CONCLUSIONS AND IMPLICATIONS These results provide evidence that (i) the Ca2+ paradox is primarily mediated by Ca2+ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca2+ depletion; and (ii) reverse mode NCX contributes little to the Ca2+ paradox, whereas inhibition of NCX during Ca2+ depletion improves SR Ca2+ loading, and is associated with reduced incidence of Ca2+ paradox in mouse ventricular myocytes. PMID:20718730

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart.

PubMed

Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Tena, Juan J; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F; Roy, Anna R; Galjart, Niels; Delgado-Olguin, Paul; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis; Manzanares, Miguel

2017-08-01

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development.

CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart

PubMed Central

Gomez-Velazquez, Melisa; Badia-Careaga, Claudio; Lechuga-Vieco, Ana Victoria; Nieto-Arellano, Rocio; Rollan, Isabel; Alvarez, Alba; Torroja, Carlos; Caceres, Eva F.; Roy, Anna R.; Galjart, Niels; Sanchez-Cabo, Fatima; Enriquez, Jose Antonio; Gomez-Skarmeta, Jose Luis

2017-01-01

Cardiac progenitors are specified early in development and progressively differentiate and mature into fully functional cardiomyocytes. This process is controlled by an extensively studied transcriptional program. However, the regulatory events coordinating the progression of such program from development to maturation are largely unknown. Here, we show that the genome organizer CTCF is essential for cardiogenesis and that it mediates genomic interactions to coordinate cardiomyocyte differentiation and maturation in the developing heart. Inactivation of Ctcf in cardiac progenitor cells and their derivatives in vivo during development caused severe cardiac defects and death at embryonic day 12.5. Genome wide expression analysis in Ctcf mutant hearts revealed that genes controlling mitochondrial function and protein production, required for cardiomyocyte maturation, were upregulated. However, mitochondria from mutant cardiomyocytes do not mature properly. In contrast, multiple development regulatory genes near predicted heart enhancers, including genes in the IrxA cluster, were downregulated in Ctcf mutants, suggesting that CTCF promotes cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Accordingly, loss of CTCF disrupts gene expression and chromatin interactions as shown by chromatin conformation capture followed by deep sequencing. Furthermore, CRISPR-mediated deletion of an intergenic CTCF site within the IrxA cluster alters gene expression in the developing heart. Thus, CTCF mediates local regulatory interactions to coordinate transcriptional programs controlling transitions in morphology and function during heart development. PMID:28846746

Gangliosides and Ceramides Change in a Mouse Model of Blast Induced Traumatic Brain Injury

PubMed Central

2013-01-01

Explosive detonations generate atmospheric pressure changes that produce nonpenetrating blast induced “mild” traumatic brain injury (bTBI). The structural basis for mild bTBI has been extremely controversial. The present study applies matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to track the distribution of gangliosides in mouse brain tissue that were exposed to very low level of explosive detonations (2.5–5.5 psi peak overpressure). We observed major increases of the ganglioside GM2 in the hippocampus, thalamus, and hypothalamus after a single blast exposure. Moreover, these changes were accompanied by depletion of ceramides. No neurological or brain structural signs of injury could be inferred using standard light microscopic techniques. The first source of variability is generated by the Latency between blast and tissue sampling (peak intensity of the blast wave). These findings suggest that subtle molecular changes in intracellular membranes and plasmalemma compartments may be biomarkers for biological responses to mild bTBI. This is also the first report of a GM2 increase in the brains of mature mice from a nongenetic etiology. PMID:23590251

Polycystin-2-dependent control of cardiomyocyte autophagy.

PubMed

Criollo, Alfredo; Altamirano, Francisco; Pedrozo, Zully; Schiattarella, Gabriele G; Li, Dan L; Rivera-Mejías, Pablo; Sotomayor-Flores, Cristian; Parra, Valentina; Villalobos, Elisa; Battiprolu, Pavan K; Jiang, Nan; May, Herman I; Morselli, Eugenia; Somlo, Stefan; de Smedt, Humbert; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

2018-05-01

Considerable evidence points to critical roles of intracellular Ca 2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca 2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca 2+ homeostasis and autophagy. Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2 F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca 2+ chelation using BAPTA-AM, whereas removal of extracellular Ca 2+ had no effect, pointing to a role of intracellular Ca 2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca 2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca 2+ -channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca 2+ . Furthermore, PC2 ablation was associated with impaired Ca 2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca 2+ stores. Finally, we provide evidence that Ca 2+ -mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. Together, this study unveils PC2 as a novel regulator of autophagy acting

Site-targeted complement inhibition by a complement receptor 2-conjugated inhibitor (mTT30) ameliorates post-injury neuropathology in mouse brains.

PubMed

Rich, Megan C; Keene, Chesleigh N; Neher, Miriam D; Johnson, Krista; Yu, Zhao-Xue; Ganivet, Antoine; Holers, V Michael; Stahel, Philip F

2016-03-23

Intracerebral complement activation after severe traumatic brain injury (TBI) leads to a cascade of neuroinflammatory pathological sequelae that propagate host-mediated secondary brain injury and adverse outcomes. There are currently no specific pharmacological agents on the market to prevent or mitigate the development of secondary cerebral insults after TBI. A novel chimeric CR2-fH compound (mTT30) provides targeted inhibition of the alternative complement pathway at the site of tissue injury. This experimental study was designed to test the neuroprotective effects of mTT30 in a mouse model of closed head injury. The administration of 500 μg mTT30 i.v. at 1 h, 4 h and 24 h after head injury attenuated complement C3 deposition in injured brains, reduced the extent of neuronal cell death, and decreased post-injury microglial activation, compared to vehicle-injected placebo controls. These data imply that site-targeted alternative pathway complement inhibition may represent a new promising therapeutic avenue for the future management of severe TBI. Copyright © 2016. Published by Elsevier Ireland Ltd.

Influence of silencing soluble epoxide hydrolase with RNA interference on cardiomyocytes apoptosis induced by doxorubicin.

PubMed

Du, Guangsheng; Lv, Jiagao; He, Li; Ma, Yexin

2011-06-01

In order to investigate the influence of silencing soluble epoxide hydrolase (sEH) with double-stranded small interfering RNA (siRNA) on cardiomyocytes apoptosis induced by doxorubicin (DOX), two plasmids containing siRNA sequences specific to sEH were constructed and transfected into the primary cultured cardiomyocytes by using FuGENE HD transfection agents. The mRNA and protein expression levels of sEH were detected by semiquantitative RT-PCR and Western blotting respectively, and the plasmids that silenced sEH most significantly were selected, and renamed EH-R. The plasmids carrying a nonspecific siRNA coding sequence (PCN) served as the negative control. Cardiomyocytes were divided into four groups: control group, DOX group, PCN+DOX group, and EH-R+DOX group. Apoptosis of cardiomyocytes was induced by DOX at a concentration of 1 μmol/L. Apoptosis rate of cardiomyocytes was determined by flow cytometery. The protein expression levels of Bcl-2 and Bax were detected by Western blotting. The results showed that the expression of sEH was down-regulated by EH-R plasmid. The expression levels of sEH mRNA and protein in the EH-R+DOX group were significantly decreased as compared with other groups (P<0.01). As compared with the control group, the apoptosis rate of cardiomyocytes in three DOX-treated groups was obviously increased, the expression levels of Bax increased, and those of Bcl-2 decreased (P<0.01). However, the expression levels of Bax were decreased, those of Bcl-2 increased and the apoptosis rate of cardiomyocytes obviously decreased in EH-R+DOX group when compared with those in the DOX group and the PCN+DOX group (P<0.01 for each). It was concluded that the recombinant plasmids could be successfully constructed, and transfected into the primary cultured cardiomyocytes. They could ameliorate the DOX-induced cardiomyocytes apoptosis by selectively inhibiting the expression of sEH with RNAi and increasing the expression of Bcl-2.

Free radicals mediate postshock contractile impairment in cardiomyocytes.

PubMed

Tsai, Min-Shan; Sun, Shijie; Tang, Wanchun; Ristagno, Giuseppe; Chen, Wen-Jone; Weil, Max Harry

2008-12-01

Previous studies demonstrated myocardial dysfunction after electrical shock and indicated it may be related to free radicals. Whether the free radicals are generated after electrical shock has not been documented at the cellular level. This study was to investigate whether electrical shock generates intracellular free radicals inside cardiomyocytes and to evaluate whether reducing intracellular free radicals by pretreatment of ascorbic acid would reduce the contractile dysfunction after electrical shock. Randomized prospective animal study. University affiliated research laboratory. Sprague-Dawley rats. Cardiomyocytes isolated from adult male rats were divided into four groups: (1) electrical shock alone; (2) electrical shock pretreated with ascorbic acid; (3) pretreated with ascorbic acid alone; and (4) control. Ascorbic acid (0.2 mM) was administrated in the perfusate of the ascorbic acid + electrical shock and ascorbic acid groups. A 2-J electrical shock was delivered to the electrical shock and ascorbic acid + electrical shock groups. DCFH-DA-loaded cardiomyocytes showed increased intracellular free radicals after electrical shock. The contractions and Ca2+ transients were recorded optically with fura-2 loading. Within 4 mins after electrical shock in the electrical shock group, the length shortening decreased from 8.4% +/- 2.5% to 5.6% +/- 3.4% (p = 0.000) and the Ca2+ transient decreased from 1.15 +/- 0.13 au to 1.08 +/- 0.1 au (p = 0.038). Compared with control, a significant difference in length shortening (p = 0.001) but not Ca2+ transient (p = 0.052) was noted. In the presence of ascorbic acid, electrical shock did not affect length shortening and Ca2+ transient. Electrical shock generates free radicals inside the cardiomyocyte, and causes contractile impairment and associated decrease of Ca transient. Administering ascorbic acid may improve such damage by eliminating free radicals.

Mitochondrial PKC-ε deficiency promotes I/R-mediated myocardial injury via GSK3β-dependent mitochondrial permeability transition pore opening.

PubMed

Wang, Shijun; Zhang, Feng; Zhao, Gang; Cheng, Yong; Wu, Ting; Wu, Bing; Zhang, You-En

2017-09-01

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)-induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase-2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross-clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2 -/- ) and wild-type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin-related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N-acetylcysteine (NAC) or PKC-δ shRNA treatment on glycogen synthase kinase-3β (GSK-3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2 -/- mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC-ε translocation was lower in ALDH2 -/- mice than in WT mice, and PKC-δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre-treatment under I/R injury. In addition, PKC-ε inhibition caused activation of caspase9, caspase3 and Drp1Ser 616 in response to I/R stress. Importantly, expression of phosphorylated GSK-3β (inactive form) was lower in ALDH2 -/- mice than in WT mice, and both were increased by NAC pre-treatment. I/R-induced mitochondrial translocation of GSK-3β was inhibited by PKC-δ shRNA or NAC pre-treatment. In addition

Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction

PubMed Central

Malliaras, Konstantinos; Ibrahim, Ahmed; Tseliou, Eleni; Liu, Weixin; Sun, Baiming; Middleton, Ryan C; Seinfeld, Jeffrey; Wang, Lai; Sharifi, Behrooz G; Marbán, Eduardo

2014-01-01

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium. PMID:24797668

Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes.

PubMed

Gaustad, Kristine G; Boquest, Andrew C; Anderson, Brent E; Gerdes, A Martin; Collas, Philippe

2004-02-06

We report the differentiation of human adipose tissue stem cells (ATSCs) to take on cardiomyocyte properties following transient exposure to a rat cardiomyocyte extract. Reversibly permeabilized ATSCs were incubated for 1h in a nuclear and cytoplasmic extract of rat cardiomyocytes, resealed with CaCl(2), and cultured. Three weeks after exposure to extract, ATSCs expressed several cardiomyocyte markers including sarcomeric alpha-actinin, desmin, and cardiac troponin I, and displayed targeted expression of the gap junction protein connexin 43. Formation of binucleated and striated cells, and spontaneous beating in culture were also observed. A low proportion of intact ATSCs exposed to the extract also showed signs of alpha-actinin and connexin 43 expression. Additional evidence of differentiation was provided by induction of expression of nuclear lamin A/C, a marker of terminally differentiated cells, and a remarkable increase in cell cycle length. Together with our previous data, this study suggests that alteration of cell fate using cellular extracts may be applied to multiple cell types. Cell extracts may also prove useful for investigating the molecular mechanisms of stem cell differentiation.

Plasma exosomes protect the myocardium from ischemia-reperfusion injury.

PubMed

Vicencio, Jose M; Yellon, Derek M; Sivaraman, Vivek; Das, Debashish; Boi-Doku, Claire; Arjun, Sapna; Zheng, Ying; Riquelme, Jaime A; Kearney, Jessica; Sharma, Vikram; Multhoff, Gabriele; Hall, Andrew R; Davidson, Sean M

2015-04-21

Exosomes are nanometer-sized vesicles released from cells into the blood, where they can transmit signals throughout the body. Shown to act on the heart, exosomes' composition and the signaling pathways they activate have not been explored. We hypothesized that endogenous plasma exosomes can communicate signals to the heart and provide protection against ischemia and reperfusion injury. This study sought to isolate and characterize exosomes from rats and healthy volunteers, evaluate their cardioprotective actions, and identify the molecular mechanisms involved. The exosome-rich fraction was isolated from the blood of adult rats and human volunteers and was analyzed by protein marker expression, transmission electron microscopy, and nanoparticle tracking analysis. This was then used in ex vivo, in vivo, and in vitro settings of ischemia-reperfusion, with the protective signaling pathways activated on cardiomyocytes identified using Western blot analyses and chemical inhibitors. Exosomes exhibited the expected size and expressed marker proteins CD63, CD81, and heat shock protein (HSP) 70. The exosome-rich fraction was powerfully cardioprotective in all tested models of cardiac ischemia-reperfusion injury. We identified a pro-survival signaling pathway activated in cardiomyocytes involving toll-like receptor (TLR) 4 and various kinases, leading to activation of the cardioprotective HSP27. Cardioprotection was prevented by a neutralizing antibody against a conserved HSP70 epitope expressed on the exosome surface and by blocking TLR4 in cardiomyocytes, identifying the HSP70/TLR4 communication axis as a critical component in exosome-mediated cardioprotection. Exosomes deliver endogenous protective signals to the myocardium by a pathway involving TLR4 and classic cardioprotective HSPs. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Mouse Mast Cell Protease 4 and 5 Mediate Epidermal Injury Through Disruption of Tight Junctions

PubMed Central

Bankova, Lora G.; Lezcano, Cecilia; Pejler, Gunnar; Stevens, Richard L.; Murphy, George F.; Austen, K. Frank; Gurish, Michael F.

2014-01-01

We previously established a mast cell (MC)-dependent thermal injury model in mice with ulceration and scar formation that depended on non-redundant functions of mouse MC protease 4 (mMCP4) and mMCP5. We hypothesized that MC activation is an early event and now find by histology that exocytosis of granule contents occurred by 2 min after thermal injury in wild type (WT) C57BL/6 mice and in the mMCP4- or mMCP5-deficient mice. The degranulation was equivalent for MCs in the dermis and hypodermis of all three strains, but only the WT mice showed an appreciable increase in epidermal thickness. There was no loss of total MCs, partially degranulated plus intact, over the 4 h of observation. By electron microscopy, MCs in all strains showed early zonal degranulation at 30 s with marked progression in magnitude by 120 s and no mitochondrial injury or cellular necrosis. Concomitantly there was an increase in intercellular spaces indicative of tight junction (TJ) disruption in WT mice but not in the mMCP4- or mMCP5-deficient strains. The desmosomes were intact in all strains. Immunodetection of the TJ protein claudin 4 in WT and mMCP5-deficient mice indicated a significant reduction after scald injury while mMCP4−/− mice showed no significant changes. Taken together, these findings reveal that a second degree burn injury can initiate an immediate novel zonal degranulation of MCs throughout all skin layers and a disruption of the epidermal TJs dependent on the non-redundant presence of mMCP4 and mMCP5. PMID:24523504

Engineering of Pulsatile Conduits from Human Pluripotent Stem Cell Derived Cardiomyocytes

DTIC Science & Technology

2017-06-01

patients with coronary heart disease . Oncotarget [Internet]. 2016 Nov 7; Available from: http://www.ncbi.nlm.nih.gov/pubmed/27835576. 4. Veronesi G...13. SUPPLEMENTARY NOTES 14. ABSTRACT We have derived cardiomyocytes ( heart cells) with high purity using an optimized approach that can coax human...induced pluripotent stem cells (hiPSCs) into heart cells in combination with a two-day lactate selection method that can remove non- cardiomyocytes from

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway.

PubMed

Fan, Qian; Chen, Mai; Zuo, Lin; Shang, Xiying; Huang, Maggie Z; Ciccarelli, Michele; Raake, Philip; Brinks, Henriette; Chuprun, Kurt J; Dorn, Gerald W; Koch, Walter J; Gao, Erhe

2013-01-01

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Clusterin protects H9c2 cardiomyocytes from oxidative stress-induced apoptosis via Akt/GSK-3β signaling pathway

PubMed Central

Jun, Hyoung-Oh; Kim, Dong-hun; Lee, Sae-Won; Lee, Hye Shin; Seo, Ji Hae; Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk; Min, Bon Hong

2011-01-01

Clusterin is a secretory glycoprotein, which is highly up-regulated in a variety of normal and injury tissues undergoing apoptosis including infarct region of the myocardium. Here, we report that clusterin protects H9c2 cardiomyocytes from H2O2-induced apoptosis by triggering the activation of Akt and GSK-3β. Treatment with H2O2 induces apoptosis of H9c2 cells by promoting caspase cleavage and cytochrome c release from mitochondria. However, co-treatment with clusterin reverses the induction of apoptotic signaling by H2O2, thereby recovers cell viability. The protective effect of clusterin on H2O2-induced apoptosis is impaired by PI3K inhibitor LY294002, which effectively suppresses clusterin-induced activation of Akt and GSK-3β. In addition, the protective effect of clusterin is independednt on its receptor megalin, because inhibition of megalin has no effect on clusturin-mediated Akt/GSK-3β phosphoylation and H9c2 cell viability. Collectively, these results suggest that clusterin has a role protecting cardiomyocytes from oxidative stress and the Akt/GSK-3β signaling mediates anti-apoptotic effect of clusterin. PMID:21270507

Cardiomyocyte mitochondrial oxidative stress and cytoskeletal breakdown in the heart with a primary volume overload.

PubMed

Yancey, Danielle M; Guichard, Jason L; Ahmed, Mustafa I; Zhou, Lufang; Murphy, Michael P; Johnson, Michelle S; Benavides, Gloria A; Collawn, James; Darley-Usmar, Victor; Dell'Italia, Louis J

2015-03-15

Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress and mitochondrial dysfunction. Because mitochondria are both a source and target of ROS, we hypothesized that the mitochondrially targeted antioxidant mitoubiquinone (MitoQ) will improve cardiomyocyte damage and LV dysfunction in VO. Isolated cardiomyocytes from Sprague-Dawley rats were exposed to stretch in vitro and VO of aortocaval fistula (ACF) in vivo. ACF rats were treated with and without MitoQ. Isolated cardiomyocytes were analyzed after 3 h of cyclical stretch or 8 wk of ACF with MitoSox red or 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate to measure ROS and with tetramethylrhodamine to measure mitochondrial membrane potential. Transmission electron microscopy and immunohistochemistry were used for cardiomyocyte structural assessment. In vitro cyclical stretch and 8-wk ACF resulted in increased cardiomyocyte mitochondrial ROS production and decreased mitochondrial membrane potential, which were significantly improved by MitoQ. ACF had extensive loss of desmin and β₂-tubulin that was paralleled by mitochondrial disorganization, loss of cristae, swelling, and clustering identified by mitochondria complex IV staining and transmission electron microscopy. MitoQ improved mitochondrial structural damage and attenuated desmin loss/degradation evidenced by immunohistochemistry and protein expression. However, LV dilatation and fractional shortening were unaffected by MitoQ treatment in 8-wk ACF. In conclusion, although MitoQ did not affect LV dilatation or function in ACF, these experiments suggest a connection of cardiomyocyte mitochondria-derived ROS production with cytoskeletal disruption and mitochondrial damage in the VO of ACF.

Cardiomyocyte mitochondrial oxidative stress and cytoskeletal breakdown in the heart with a primary volume overload

PubMed Central

Yancey, Danielle M.; Guichard, Jason L.; Ahmed, Mustafa I.; Zhou, Lufang; Murphy, Michael P.; Johnson, Michelle S.; Benavides, Gloria A.; Collawn, James; Darley-Usmar, Victor

2015-01-01

Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress and mitochondrial dysfunction. Because mitochondria are both a source and target of ROS, we hypothesized that the mitochondrially targeted antioxidant mitoubiquinone (MitoQ) will improve cardiomyocyte damage and LV dysfunction in VO. Isolated cardiomyocytes from Sprague-Dawley rats were exposed to stretch in vitro and VO of aortocaval fistula (ACF) in vivo. ACF rats were treated with and without MitoQ. Isolated cardiomyocytes were analyzed after 3 h of cyclical stretch or 8 wk of ACF with MitoSox red or 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate to measure ROS and with tetramethylrhodamine to measure mitochondrial membrane potential. Transmission electron microscopy and immunohistochemistry were used for cardiomyocyte structural assessment. In vitro cyclical stretch and 8-wk ACF resulted in increased cardiomyocyte mitochondrial ROS production and decreased mitochondrial membrane potential, which were significantly improved by MitoQ. ACF had extensive loss of desmin and β2-tubulin that was paralleled by mitochondrial disorganization, loss of cristae, swelling, and clustering identified by mitochondria complex IV staining and transmission electron microscopy. MitoQ improved mitochondrial structural damage and attenuated desmin loss/degradation evidenced by immunohistochemistry and protein expression. However, LV dilatation and fractional shortening were unaffected by MitoQ treatment in 8-wk ACF. In conclusion, although MitoQ did not affect LV dilatation or function in ACF, these experiments suggest a connection of cardiomyocyte mitochondria-derived ROS production with cytoskeletal disruption and mitochondrial damage in the VO of ACF. PMID:25599572

Structure of cortical cytoskeleton in fibers of mouse muscle cells after being exposed to a 30-day space flight on board the BION-M1 biosatellite.

PubMed

Ogneva, I V; Maximova, M V; Larina, I M

2014-05-15

The aim of the work was to analyze changes in the organization of the cortical cytoskeleton in fibers of the mouse soleus muscle, tibialis anterior muscle and left ventricular cardiomyocytes after completion of a 30-day space flight on board the BION-M1 biosatellite (Russia, 2013). The transversal stiffness of the cortical cytoskeleton of the cardiomyocytes and fibers of the skeletal muscles did not differ significantly within the study groups compared with the vivarium control group. The content of beta- and gamma-actin in the membranous fraction of proteins in the left ventricular cardiomyocytes did not differ significantly within all study groups and correlated with the transversal stiffness. A similar situation was revealed in fibers of the soleus muscle and tibialis anterior muscle. At the same time, the content of beta-actin in the cytoplasmic fraction of proteins was found to be decreased in all types of studied tissues compared with the control levels in the postflight group, with lowered beta-actin gene expression rates in the postflight group. After completion of the space flight, the content of alpha-actinin-4 was found to be reduced in the membranous fraction of proteins from the mouse cardiomyocytes, while its content in the cytoplasmic fraction of proteins did not change significantly. Furthermore, gene expression rates of this protein were decreased at the time of dissection (it was started after 13 h after landing). At the same time, the content of alpha-actinin-1 decreased in the membranous fraction and increased in the cytoplasmic fraction of proteins from the soleus muscle fibers. Copyright © 2014 the American Physiological Society.

Exercise-induced circulating extracellular vesicles protect against cardiac ischemia-reperfusion injury.

PubMed

Bei, Yihua; Xu, Tianzhao; Lv, Dongchao; Yu, Pujiao; Xu, Jiahong; Che, Lin; Das, Avash; Tigges, John; Toxavidis, Vassilios; Ghiran, Ionita; Shah, Ravi; Li, Yongqin; Zhang, Yuhui; Das, Saumya; Xiao, Junjie

2017-07-01

Extracellular vesicles (EVs) serve an important function as mediators of intercellular communication. Exercise is protective for the heart, although the signaling mechanisms that mediate this cardioprotection have not been fully elucidated. Here using nano-flow cytometry, we found a rapid increase in plasma EVs in human subjects undergoing exercise stress testing. We subsequently identified that serum EVs were increased by ~1.85-fold in mice after 3-week swimming. Intramyocardial injection of equivalent quantities of EVs from exercised mice and non-exercised controls provided similar protective effects against acute ischemia/reperfusion (I/R) injury in mice. However, injection of exercise-induced EVs in a quantity equivalent to the increase seen with exercise (1.85 swim group) significantly enhanced the protective effect. Similarly, treatment with exercise-induced increased EVs provided additional anti-apoptotic effect in H 2 O 2 -treated H9C2 cardiomyocytes mediated by the activation of ERK1/2 and HSP27 signaling. Finally, by treating H9C2 cells with insulin-like growth factor-1 to mimic exercise stimulus in vitro, we found an increased release of EVs from cardiomyocytes associated with ALIX and RAB35 activation. Collectively, our results show that exercise-induced increase in circulating EVs enhances the protective effects of endogenous EVs against cardiac I/R injury. Exercise-derived EVs might serve as a potent therapy for myocardial injury in the future.

Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest

PubMed Central

O’Neill, Thomas J.; Mack, Christopher P.; Taylor, Joan M.

2012-01-01

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state impacts normal heart development and pathologic myocardial remodeling, yet the signaling mechanisms that regulate this vital process are incompletely understood. Studies from our lab and others indicate that focal adhesion kinase (FAK) is a critical regulator of cardiac growth and remodeling and we found that expression of the endogenous FAK inhibitor, FAK-related non kinase (FRNK) coincided with postnatal cardiomyocyte arrest. Mis-expression of FRNK in the embryonic heart led to pre-term lethality associated with reduced cardiomyocyte proliferation and led us to speculate that the postnatal FRNK surge might be required to promote quiescence in this growth promoting environment. Herein, we provide strong evidence that endogenous FRNK contributes to post-mitotic arrest. Depletion of FRNK promoted DNA synthesis in post-natal day (P) 10 hearts accompanied by a transient increase in DNA content and multi-nucleation by P14, indicative of DNA replication without cell division. Interestingly, a reduction in tri- and tetra-nucleated cardiomyocytes, concomitant with an increase in bi-nucleated cells by P21, indicated the possibility that FRNK-depleted cardiomyocytes underwent eventual cytokinesis. In support of this conclusion, Aurora B-labeled central spindles (a hallmark of cytokinesis) were observed in tetra-nucleated P20 FRNK−/− but not wt cardiomyocytes, while no evidence of apoptosis was observed. Moreover, hearts from FRNK null mice developed ventricular enlargement that persisted until young adulthood which resulted from myocyte expansion rather than myocyte hypertrophy or interstitial growth. These data indicate that endogenous FRNK serves an important role in limiting DNA synthesis and regulating the un-coupling between DNA synthesis and cytokinesis in the post-natal myocardium. PMID:22555221

Acute ethanol exposure-induced autophagy-mediated cardiac injury via activation of the ROS-JNK-Bcl-2 pathway.

PubMed

Zhu, Zhongxin; Huang, Yewei; Lv, Lingchun; Tao, Youli; Shao, Minglong; Zhao, Congcong; Xue, Mei; Sun, Jia; Niu, Chao; Wang, Yang; Kim, Sunam; Cong, Weitao; Mao, Wei; Jin, Litai

2018-02-01

Binge drinking is associated with increased cardiac autophagy, and often triggers heart injury. Given the essential role of autophagy in various cardiac diseases, this study was designed to investigate the role of autophagy in ethanol-induced cardiac injury and the underlying mechanism. Our study showed that ethanol exposure enhanced the levels of LC3-II and LC3-II positive puncta and promoted cardiomyocyte apoptosis in vivo and in vitro. In addition, we found that ethanol induced autophagy and cardiac injury largely via the sequential triggering of reactive oxygen species (ROS) accumulation, activation of c-Jun NH2-terminal kinase (JNK), phosphorylation of Bcl-2, and dissociation of the Beclin 1/Bcl-2 complex. By contrast, inhibition of ethanol-induced autophagic flux with pharmacologic agents in the hearts of mice and cultured cells significantly alleviated ethanol-induced cardiomyocyte apoptosis and heart injury. Elimination of ROS with the antioxidant N-acetyl cysteine (NAC) or inhibition of JNK with the JNK inhibitor SP600125 reduced ethanol-induced autophagy and subsequent autophagy-mediated apoptosis. Moreover, metallothionein (MT), which can scavenge reactive oxygen and nitrogen species, also attenuated ethanol-induced autophagy and cell apoptosis in MT-TG mice. In conclusion, our findings suggest that acute ethanol exposure induced autophagy-mediated heart toxicity and injury mainly through the ROS-JNK-Bcl-2 signaling pathway. © 2017 Wiley Periodicals, Inc.

Quantifying Electrical Interactions between Cardiomyocytes and Other Cells in Micropatterned Cell Pairs

PubMed Central

Nguyen, Hung; Badie, Nima; McSpadden, Luke; Pedrotty, Dawn; Bursac, Nenad

2014-01-01

Micropatterning is a powerful technique to control cell shape and position on a culture substrate. In this chapter, we describe the method to reproducibly create large numbers of micropatterned heterotypic cell pairs with defined size, shape, and length of cell–cell contact. These cell pairs can be utilized in patch clamp recordings to quantify electrical interactions between cardiomyocytes and non-cardiomyocytes. PMID:25070342

Icariin promotes expression of junctophilin 2 and Ca2+ related function during cardiomyocyte differentiation of murine embryonic stem cells.

PubMed

Liang, Xingguang; Hong, Dongsheng; Huang, Yujie; Rao, Yuefeng; Ma, Kuifen; Huang, Mingzhu; Zhang, Xingguo; Lou, Yijia; Zhao, Qingwei

2015-12-01

Junctophilin2 (JP2) is a critical protein associated with cardiogenesis. Icariin (ICA) facilitated the directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes. However, little is known about the effects of ICA on JP2 during cardiac differentiation. Here, we explored whether ICA has effects on the expression and Ca2+ related function of JP2 during cardiomyocyte differentiation of ES cells in vitro. Embryonid bodies (EBs) formed by hanging drop were treated with 10(-7) mol/L ICA from day 5 to promote the cardiac differentiation. Percentage of beating EBs and number of beating area within EBs were monitored. Cardiomyocytes were purified by discontinuous percoll gradient centrifugation from EBs. The expression of JP2, α-actinin and troponin-T within EBs or isolated cardiomyocytes were analyzed by immunocytochemistry, western blot and flow cytometry. The transient Ca2+ release was characterized in cardiomyocytes treated with/without 10 mmol/L caffeine and 8 mmol/L Ca2+. Our results showed that ES cell-derived cardiomyocytes were well characterized with JP2 proteins. ICA promoted cardiomyocyte differentiation as indicated by an increased percentage of beating EBs and number of beating area within EBs. The expression of JP2, α-actinin and troponin-T were up-regulated both in EBs and isolated cardiomyocytes from EBs. Furthermore, ICA-induced JP2 expression was accompanied by a remarkable increase of the amplitude of Ca2+ transients in cardiomyocytes before/after caffeine and Ca2+ stimulating. In conclusion, ICA promotes in cardiac differentiation partly through regulating JP2 and improved the Ca2+ modulatory function of cardiomyocytes.

Ionizing Radiation Impacts on Cardiac Differentiation of Mouse Embryonic Stem Cells

PubMed Central

Helm, Alexander; Arrizabalaga, Onetsine; Pignalosa, Diana; Schroeder, Insa S.; Durante, Marco

2016-01-01

Little is known about the effects of ionizing radiation on the earliest stages of embryonic development although it is well recognized that ionizing radiation is a natural part of our environment and further exposure may occur due to medical applications. The current study addresses this issue using D3 mouse embryonic stem cells as a model system. Cells were irradiated with either X-rays or carbon ions representing sparsely and densely ionizing radiation and their effect on the differentiation of D3 cells into spontaneously contracting cardiomyocytes through embryoid body (EB) formation was measured. This study is the first to demonstrate that ionizing radiation impairs the formation of beating cardiomyocytes with carbon ions being more detrimental than X-rays. However, after prolonged culture time, the number of beating EBs derived from carbon ion irradiated cells almost reached control levels indicating that the surviving cells are still capable of developing along the cardiac lineage although with considerable delay. Reduced EB size, failure to downregulate pluripotency markers, and impaired expression of cardiac markers were identified as the cause of compromised cardiomyocyte formation. Dysregulation of cardiac differentiation was accompanied by alterations in the expression of endodermal and ectodermal markers that were more severe after carbon ion irradiation than after exposure to X-rays. In conclusion, our data show that carbon ion irradiation profoundly affects differentiation and thus may pose a higher risk to the early embryo than X-rays. PMID:26506910

Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways

PubMed Central

Twu, Cheryl; Liu, Nancy Q.; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P.; MacLellan, W. Robb; Fiala, Milan

2002-01-01

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-α. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-α and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways. PMID:12379743

Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways.

PubMed

Twu, Cheryl; Liu, Nancy Q; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P; MacLellan, W Robb; Fiala, Milan

2002-10-29

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-alpha. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-alpha and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.

SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition.

PubMed

Jia, Zhuqing; Wang, Jiaji; Shi, Qiong; Liu, Siyu; Wang, Weiping; Tian, Yuyao; Lu, Qin; Chen, Ping; Ma, Kangtao; Zhou, Chunyan

2016-02-01

Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.

Human adipose tissue-derived stem cells exhibit proliferation potential and spontaneous rhythmic contraction after fusion with neonatal rat cardiomyocytes.

PubMed

Metzele, Roxana; Alt, Christopher; Bai, Xiaowen; Yan, Yasheng; Zhang, Zhi; Pan, Zhizhong; Coleman, Michael; Vykoukal, Jody; Song, Yao-Hua; Alt, Eckhard

2011-03-01

Various types of stem cells have been shown to have beneficial effects on cardiac function. It is still debated whether fusion of injected stem cells with local resident cardiomyocytes is one of the mechanisms. To better understand the role of fusion in stem cell-based myocardial regeneration, the present study was designed to investigate the fate of human adipose tissue-derived stem cells (hASCs) fused with neonatal rat cardiomyocytes in vitro. hASCs labeled with the green fluorescent probe Vybrant DiO were cocultured with neonatal rat cardiomyocytes labeled with the red fluorescent probe Vybrant DiI and then treated with fusion-inducing hemagglutinating virus of Japan (HVJ). Cells that incorporated both red and green fluorescent signals were considered to be hASCs that had fused with rat cardiomyocytes. Fusion efficiency was 19.86 ± 4.84% at 5 d after treatment with HVJ. Most fused cells displayed cardiomyocyte-like morphology and exhibited spontaneous rhythmic contraction. Both immunofluorescence staining and lentiviral vector labeling showed that fused cells contained separate rat cardiomyocyte and hASC nuclei. Immunofluorescence staining assays demonstrated that human nuclei in fused cells still expressed the proliferation marker Ki67. In addition, hASCs fused with rat cardiomyocytes were positive for troponin I. Whole-cell voltage-clamp analysis demonstrated action potentials in beating fused cells. RT-PCR analysis using rat- or human-specific myosin heavy chain primers revealed that the myosin heavy-chain expression in fused cells was derived from rat cardiomyocytes. Real-time PCR identified expression of human troponin T in fused cells and the presence of rat cardiomyocytes induced a cardiomyogenic protein expression of troponin T in human ASCs. This study illustrates that hASCs exhibit both stem cell (proliferation) and cardiomyocyte properties (action potential and spontaneous rhythmic beating) after fusion with rat cardiomyocytes, supporting the theory

Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

PubMed

Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D

2018-04-17

Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and

Pleiotropic Effects of Chronic Phorbol Ester Treatment to Improve Glucose Transport in Insulin-Resistant Cardiomyocytes.

PubMed

Viglino, Christelle; Khoramdin, Bahareh; Praplan, Guillaume; Montessuit, Christophe

2017-12-01

Stimulation of glucose transport is an important determinant of myocardial susceptibility to ischemia and reperfusion. Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA). Deactivation of the Focal Adhesion Kinase (FAK) by FFA contributes to glucose transport impairment, and could be corrected by chronic treatment with the phorbol ester TPA. However, TPA must have effects in addition to FAK reactivation to restore stimulated glucose transport. Chronic treatment with TPA improved basal and stimulated glucose transport in FFA-exposed, but not in control cardiomyocytes. Chronic FFA exposure induced the activation of PKCδ and PKCϵ. TPA markedly downregulated the expression of PKCα, PKCδ, and PKCϵ, suggesting that PKCδ or PKCϵ activation could contribute to inhibition of glucose transport by FFA. Rottlerin, a specific PKCδ inhibitor, improved glucose transport in FFA-exposed cardiomyocytes; and PKCδ was reduced in the particulate fraction of FFA + TPA-exposed cardiomyocytes. TPA also activated Protein Kinase D 1(PKD1) in FFA-exposed cardiomyocytes, as assessed by autophosphorylation of PKD1 on Y916. Pharmaceutical inhibition of PKD1 only partially prevented the improvement of glucose transport by TPA. Chronic TPA treatment also increased basal and stimulated glycolysis and favored accumulation of lipid droplets in FFA-exposed cardiomyocytes. In conclusion, basal and stimulated glucose transport in cardiomyocytes is reduced by chronic FFA exposure, but restored by concomitant treatment with a phorbol ester. The mechanism of action of phorbol esters may involve downregulation of PKCδ, activation of PKD1 and a general switch from fatty acid to glucose metabolism. J. Cell. Biochem. 9999: 4716-4727, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

C-type natriuretic peptide functions as an innate neuroprotectant in neonatal hypoxic-ischemic brain injury in mouse via natriuretic peptide receptor 2.

PubMed

Ma, Qingyi; Zhang, Lubo

2018-06-01

Neonatal hypoxia-ischemia (HI) is the most common cause of brain injury in neonates, which leads to high neonatal mortality and severe neurological morbidity in later life (Vannucci, 2000; Volpe, 2001). Yet the molecular mechanisms of neuronal death and brain damage induced by neonatal HI remain largely elusive. Herein, using both in vivo and in vitro models, we determine an endogenous neuroprotectant role of c-type natriuretic peptide (CNP) in preserving neuronal survival after HI brain injury in mouse pups. Postnatal day 7 (P7) mouse pups with CNP deficiency (Nppc lbab/lbab ) exhibit increased brain infarct size and worsened long-term locomotor function after neonatal HI compared with wildtype control (Nppc +/+ ). In isolated primary cortical neurons, recombinant CNP dose-dependently protects primary neurons from oxygen-glucose deprivation (OGD) insult. This neuroprotective effect appears to be mediated through its cognate natriuretic peptide receptor 2 (NPR2), in that antagonization of NPR2, but not NPR3, exacerbates neuronal death and counteracts the protective effect of CNP on primary neurons exposed to OGD insult. Immunoblot and confocal microscopy demonstrate the abundant expression of NPR2 in neurons of the neonatal brain and in isolated primary cortical neurons as well. Moreover, similar to CNP deficiency, administration of NPR2 antagonist P19 via intracerebroventricular injection prior to HI results in exacerbated neuronal death and brain injury after HI. Altogether, the present study indicates that CNP and its cognate receptor NPR2 mainly expressed in neurons represent an innate neuroprotective mechanism in neonatal HI brain injury. Copyright © 2018 Elsevier Inc. All rights reserved.

Urotensin II induction of neonatal cardiomyocyte hypertrophy involves the CaMKII/PLN/SERCA 2a signaling pathway.

PubMed

Shi, Hongtao; Han, Qinghua; Xu, Jianrong; Liu, Wenyuan; Chu, Tingting; Zhao, Li

2016-05-25

Although studies have shown that Urotensin II (UII) can induce cardiomyocyte hypertrophy and UII-induced cardiomyocyte hypertrophy model has been widely used for hypertrophy research, but its precise mechanism remains unknown. Recent researches have demonstrated that UII-induced cardiomyocyte hypertrophy has a relationship with the changes of intracellular Ca(2+) concentration. Therefore, the aim of this study was to investigate the mechanisms of cardiomyocyte hypertrophy induced by UII and to explore whether the calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated up-regulating of phospholamban (PLN) Thr17-phosphorylation signaling pathway contributed to UII-induced cardiomyocyte hypertrophy. Primary cultures of neonatal rat cardiomyocytes were stimulated for 48h with UII. Cell size, protein/DNA contents and intracellular Ca(2+) were determined. Phosphorylated and total forms of CaMKII, PLN and the total amount of serco/endo-plasmic reticulum ATPases (SERCA 2a) were quantified by western blot. The responses of cardiomyocytes to UII were also evaluated after pretreatment with the CaMKII inhibitor, KN-93. These results showed that UII increased cell size, protein/DNA ratio and intracellular Ca(2+), consistent with a hypertrophic response. Furthermore, the phosphorylation of CaMKII and its downstream target PLN (Thr17), SERCA 2a levels were up-regulated by UII treatment. Conversely, treatment with KN-93 reversed all those effects of UII. Taken together, the results suggest that UII can induce cardiomyocyte hypertrophy through CaMKII-mediated up-regulating of PLN Thr17-phosphorylation signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.

PubMed

Dubois, Nicole C; Craft, April M; Sharma, Parveen; Elliott, David A; Stanley, Edouard G; Elefanty, Andrew G; Gramolini, Anthony; Keller, Gordon

2011-10-23

To identify cell-surface markers specific to human cardiomyocytes, we screened cardiovascular cell populations derived from human embryonic stem cells (hESCs) against a panel of 370 known CD antibodies. This screen identified the signal-regulatory protein alpha (SIRPA) as a marker expressed specifically on cardiomyocytes derived from hESCs and human induced pluripotent stem cells (hiPSCs), and PECAM, THY1, PDGFRB and ITGA1 as markers of the nonmyocyte population. Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiac precursors and cardiomyocytes from hESC/hiPSC differentiation cultures, yielding populations of up to 98% cardiac troponin T-positive cells. When plated in culture, SIRPA-positive cells were contracting and could be maintained over extended periods of time. These findings provide a simple method for isolating populations of cardiomyocytes from human pluripotent stem cell cultures, and thereby establish a readily adaptable technology for generating large numbers of enriched cardiomyocytes for therapeutic applications.

Acoustical sensing of cardiomyocyte cluster beating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tymchenko, Nina; Kunze, Angelika; Dahlenborg, Kerstin

2013-06-14

Highlights: •An example of the application of QCM-D to live cell studies. •Detection of human pluripotent stem cell-derived cardiomyocyte cluster beating. •Clusters were studied in a thin liquid film and in a large liquid volume. •The QCM-D beating profile provides an individual fingerprint of the hPS-CMCs. -- Abstract: Spontaneously beating human pluripotent stem cell-derived cardiomyocytes clusters (CMCs) represent an excellent in vitro tool for studies of human cardiomyocyte function and for pharmacological cardiac safety assessment. Such testing typically requires highly trained operators, precision plating, or large cell quantities, and there is a demand for real-time, label-free monitoring of small cellmore » quantities, especially rare cells and tissue-like structures. Array formats based on sensing of electrical or optical properties of cells are being developed and in use by the pharmaceutical industry. A potential alternative to these techniques is represented by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique, which is an acoustic surface sensitive technique that measures changes in mass and viscoelastic properties close to the sensor surface (from nm to μm). There is an increasing number of studies where QCM-D has successfully been applied to monitor properties of cells and cellular processes. In the present study, we show that spontaneous beating of CMCs on QCM-D sensors can be clearly detected, both in the frequency and the dissipation signals. Beating rates in the range of 66–168 bpm for CMCs were detected and confirmed by simultaneous light microscopy. The QCM-D beating profile was found to provide individual fingerprints of the hPS-CMCs. The presented results point towards acoustical assays for evaluation cardiotoxicity.« less

The Cardioprotective Effects of Citric Acid and L-Malic Acid on Myocardial Ischemia/Reperfusion Injury

PubMed Central

Tang, Xilan; Liu, Jianxun; Dong, Wei; Li, Peng; Li, Lei; Lin, Chengren; Zheng, Yongqiu; Hou, Jincai; Li, Dan

2013-01-01

Organic acids in Chinese herbs, the long-neglected components, have been reported to possess antioxidant, anti-inflammatory, and antiplatelet aggregation activities; thus they may have potentially protective effect on ischemic heart disease. Therefore, this study aims to investigate the protective effects of two organic acids, that is, citric acid and L-malic acid, which are the main components of Fructus Choerospondiatis, on myocardial ischemia/reperfusion injury and the underlying mechanisms. In in vivo rat model of myocardial ischemia/reperfusion injury, we found that treatments with citric acid and L-malic acid significantly reduced myocardial infarct size, serum levels of TNF-α, and platelet aggregation. In vitro experiments revealed that both citric acid and L-malic acid significantly reduced LDH release, decreased apoptotic rate, downregulated the expression of cleaved caspase-3, and upregulated the expression of phosphorylated Akt in primary neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation injury. These results suggest that both citric acid and L-malic acid have protective effects on myocardial ischemia/reperfusion injury; the underlying mechanism may be related to their anti-inflammatory, antiplatelet aggregation and direct cardiomyocyte protective effects. These results also demonstrate that organic acids, besides flavonoids, may also be the major active ingredient of Fructus Choerospondiatis responsible for its cardioprotective effects and should be attached great importance in the therapy of ischemic heart disease. PMID:23737849

Osthole confers neuroprotection against cortical stab wound injury and attenuates secondary brain injury.

PubMed

Xia, Yang; Kong, Liang; Yao, Yingjia; Jiao, Yanan; Song, Jie; Tao, Zhenyu; You, Zhong; Yang, Jingxian

2015-09-04

Neuroendoscopy is an innovative technique for neurosurgery that can nonetheless result in traumatic brain injury. The accompanying neuroinflammation may lead to secondary tissue damage, which is the major cause of delayed neuronal death after surgery. The present study investigated the capacity of osthole to prevent secondary brain injury and the underlying mechanism of action in a mouse model of stab wound injury. A mouse model of cortical stab wound injury was established by inserting a needle into the cerebral cortex for 20 min to mimic neuroendoscopy. Mice received an intraperitoneal injection of osthole 30 min after surgery and continued for 14 days. Neurological severity was evaluated 12 h and up to 21 days after the trauma. Brains were collected 3-21 days post-injury for histological analysis, immunocytochemistry, quantitative real-time PCR, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and enzyme-linked immunosorbent assays. Neurological function improved in mice treated with osthole and was accompanied by reduced brain water content and accelerated wound closure relative to untreated mice. Osthole treatment reduced the number of macrophages/microglia and peripheral infiltrating of neutrophils and lowered the level of the proinflammatory cytokines interleukin-6 and tumor necrosis factor α in the lesioned cortex. Osthole-treated mice had fewer TUNEL+ apoptotic neurons surrounding the lesion than controls, indicating increased neuronal survival. Osthole reduced secondary brain damage by suppressing inflammation and apoptosis in a mouse model of stab wound injury. These results suggest a new strategy for promoting neuronal survival and function after neurosurgery to improve long-term patient outcome.

Obesity-metabolic derangement exacerbates cardiomyocyte loss distal to moderate coronary artery stenosis in pigs without affecting global cardiac function.

PubMed

Li, Zi-Lun; Ebrahimi, Behzad; Zhang, Xin; Eirin, Alfonso; Woollard, John R; Tang, Hui; Lerman, Amir; Wang, Shen-Ming; Lerman, Lilach O

2014-04-01

Obesity associated with metabolic derangements (ObM) worsens the prognosis of patients with coronary artery stenosis (CAS), but the underlying cardiac pathophysiologic mechanisms remain elusive. We tested the hypothesis that ObM exacerbates cardiomyocyte loss distal to moderate CAS. Obesity-prone pigs were randomized to four groups (n = 6 each): lean-sham, ObM-sham, lean-CAS, and ObM-CAS. Lean and ObM pigs were maintained on a 12-wk standard or atherogenic diet, respectively, and left circumflex CAS was then induced by placing local-irritant coils. Cardiac structure, function, and myocardial oxygenation were assessed 4 wk later by computed-tomography and blood oxygenation level dependent (BOLD) MRI, the microcirculation with micro-computed-tomography, and injury mechanisms by immunoblotting and histology. ObM pigs showed obesity, dyslipidemia, and insulin resistance. The degree of CAS (range, 50-70%) was similar in lean and ObM pigs, and resting myocardial perfusion and global cardiac function remained unchanged. Increased angiogenesis distal to the moderate CAS observed in lean was attenuated in ObM pigs, which also showed microvascular dysfunction and increased inflammation (M1-macrophages, TNF-α expression), oxidative stress (gp91), hypoxia (BOLD-MRI), and fibrosis (Sirius-red and trichrome). Furthermore, lean-CAS showed increased myocardial autophagy, which was blunted in ObM pigs (downregulated expression of unc-51-like kinase-1 and autophagy-related gene-12; P < 0.05 vs. lean CAS) and associated with marked apoptosis. The interaction diet xstenosis synergistically inhibited angiogenic, autophagic, and fibrogenic activities. ObM exacerbates structural and functional myocardial injury distal to moderate CAS with preserved myocardial perfusion, possibly due to impaired cardiomyocyte turnover.

Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

PubMed Central

Kita-Matsuo, Hiroko; Barcova, Maria; Prigozhina, Natalie; Salomonis, Nathan; Wei, Karen; Jacot, Jeffrey G.; Nelson, Brandon; Spiering, Sean; Haverslag, René; Kim, Changsung; Talantova, Maria; Bajpai, Ruchi; Calzolari, Diego; Terskikh, Alexey; McCulloch, Andrew D.; Price, Jeffrey H.; Conklin, Bruce R.; Chen, H. S. Vincent; Mercola, Mark

2009-01-01

Background Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes. Methodology/Principal Findings Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and α-myosin heavy chain (αMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function. Conclusion/Significance The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications. PMID:19352491

Intra-myocardial injection of both growth factors and heart derived Sca-1+/CD31- cells attenuates post-MI LV remodeling more than does cell transplantation alone: neither intervention enhances functionally significant cardiomyocyte regeneration.

PubMed

Wang, Xiaohong; Li, Qinglu; Hu, Qingsong; Suntharalingam, Piradeep; From, Arthur H L; Zhang, Jianyi

2014-01-01

Insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) are two potent cell survival and regenerative factors in response to myocardial injury (MI). We hypothesized that simultaneous delivery of IGF+HGF combined with Sca-1+/CD31- cells would improve the outcome of transplantation therapy in response to the altered hostile microenvironment post MI. One million adenovirus nuclear LacZ-labeled Sca-1+/CD31- cells were injected into the peri-infarction area after left anterior descending coronary artery (LAD) ligation in mice. Recombinant mouse IGF-1+HGF was added to the cell suspension prior to the injection. The left ventricular (LV) function was assessed by echocardiography 4 weeks after the transplantation. The cell engraftment, differentiation and cardiomyocyte regeneration were evaluated by histological analysis. Sca-1+/CD31- cells formed viable grafts and improved LV ejection fraction (EF) (Control, 54.5+/-2.4; MI, 17.6+/-3.1; Cell, 28.2+/-4.2, n = 9, P<0.01). IGF+HGF significantly enhanced the benefits of cell transplantation as evidenced by increased EF (38.8+/-2.2; n = 9, P<0.01) and attenuated adverse structural remodeling. Furthermore, IGF+HGF supplementation increased the cell engraftment rate, promoted the transplanted cell survival, enhanced angiogenesis, and minimally stimulated endogenous cardiomyocyte regeneration in vivo. The in vitro experiments showed that IGF+HGF treatment stimulated Sca-1+/CD31- cell proliferation and inhibited serum free medium induced apoptosis. Supperarray profiling of Sca-1+/CD31- cells revealed that Sca-1+/CD31- cells highly expressed various trophic factor mRNAs and IGF+HGF treatment altered the mRNAs expression patterns of these cells. These data indicate that IGF-1+HGF could serve as an adjuvant to cell transplantation for myocardial repair by stimulating donor cell and endogenous cardiac stem cell survival, regeneration and promoting angiogenesis.

Aleglitazar, a dual peroxisome proliferator-activated receptor-α and -γ agonist, protects cardiomyocytes against the adverse effects of hyperglycaemia.

PubMed

Chen, Yan; Chen, Hongmei; Birnbaum, Yochai; Nanhwan, Manjyot K; Bajaj, Mandeep; Ye, Yumei; Qian, Jinqiao

2017-03-01

To assess the effects of Aleglitazar on hyperglycaemia-induced apoptosis. We incubated human cardiomyocytes, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout or wild-type mice in normoglycaemic or hyperglycaemic conditions (glucose 25 mM). Cells were treated with different concentrations of Aleglitazar for 48 h. We measured viability, apoptosis, caspase-3 activity, cytochrome-C release, total antioxidant capacity and reactive oxygen species formation in the treated cardiomyocytes. Human cardiomyocytes were transfected with short interfering RNA against peroxisome proliferator-activated receptor-α or peroxisome proliferator-activated receptor-γ. Aleglitazar attenuated hyperglycaemia-induced apoptosis, caspase-3 activity and cytochrome-C release and increased viability in human cardiomyocyte, cardiomyocytes from cardiac-specific peroxisome proliferator-activated receptor-γ knockout and wild-type mice. Hyperglycaemia reduced the antioxidant capacity and Aleglitazar significantly blunted this effect. Hyperglycaemia-induced reactive oxygen species production was attenuated by Aleglitazar in both human cardiomyocyte and wild-type mice cardiomyocytes. Aleglitazar improved cell viability in cells exposed to hyperglycaemia. The protective effect was partially blocked by short interfering RNA against peroxisome proliferator-activated receptor-α alone and short interfering RNA against peroxisome proliferator-activated receptor-γ alone and completely blocked by short interfering RNA to both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ. Aleglitazar protects cardiomyocytes against hyperglycaemia-induced apoptosis by combined activation of both peroxisome proliferator-activated receptor-α and peroxisome proliferator-activated receptor-γ in a short-term vitro model.

Insulin/NFκB protects against ischemia-induced necrotic cardiomyocyte death.

PubMed

Díaz, Ariel; Humeres, Claudio; González, Verónica; Gómez, María Teresa; Montt, Natalia; Sanchez, Gina; Chiong, Mario; García, Lorena

2015-11-13

In the heart, insulin controls key functions such as metabolism, muscle contraction and cell death. However, all studies have been focused on insulin action during reperfusion. Here we explore the cardioprotective action of this hormone during ischemia. Rat hearts were perfused ex vivo with an ischemia/reperfusion Langendorff model in absence or presence of insulin. Additionally, cultured rat cardiomyocytes were exposed to simulated ischemia in the absence or presence of insulin. Cytoprotective effects were measured by myocardial infarct size, trypan blue exclusion, released LDH and DNA fragmentation by flow cytometry. We found that insulin protected against cardiac ischemia ex vivo and in vitro. Moreover, insulin protected cardiomyocytes from simulated ischemia by reducing necrotic cell death. Protective effects of insulin were dependent of Akt and NFκB. These novel results show that insulin reduces ischemia-induced cardiomyocyte necrosis through an Akt/NF-κB dependent mechanism. These novel findings clarify the role of insulin during ischemia and further support its use in early GIK perfusion to treat myocardial infarction. Copyright © 2015 Elsevier Inc. All rights reserved.

Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy.

PubMed

Zhang, Ji; Qiao, Congzhen; Chang, Lin; Guo, Yanhong; Fan, Yanbo; Villacorta, Luis; Chen, Y Eugene; Zhang, Jifeng

2016-01-01

Fatty acid binding protein 4 (FABP4) is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG) mice using α myosin-heavy chain (α-MHC) promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC) procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway.

Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy

PubMed Central

Zhang, Ji; Qiao, Congzhen; Chang, Lin; Guo, Yanhong; Fan, Yanbo; Villacorta, Luis; Chen, Y. Eugene; Zhang, Jifeng

2016-01-01

Fatty acid binding protein 4 (FABP4) is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG) mice using α myosin-heavy chain (α-MHC) promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC) procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway. PMID:27294862

Hyperforin inhibits cell proliferation and differentiation in mouse embryonic stem cells.

PubMed

Nakamura, K; Aizawa, K; Yamauchi, J; Tanoue, A

2013-10-01

Hyperforin, a phloroglucinol derivative of St. John's Wort, has been identified as the major molecule responsible for this plant's products anti-depressant effects. It can be expected that exposure to St. John's Wort during pregnancy occurs with some frequency although embryotoxic or teratogenic effects of St. John's Wort and hyperforin have not yet been experimentally examined in detail. In this study, to determine any embryotoxic effects of hyperforin, we have attempted to determine whether hyperforin affects growth and survival processes of employing mouse embryonic stem (mES) cells (representing embryonic tissue) and fibroblasts (representing adult tissues). We used a modified embryonic stem cell test, which has been validated as an in vitro developmental toxicity protocol, mES cells, to assess embryotoxic potential of chemicals under investigation. We have identified that high concentrations of hyperforin inhibited mouse ES cell population growth and induced apoptosis in fibroblasts. Under our cell culture conditions, ES cells mainly differentiated into cardiomyocytes, although various other cell types were also produced. In this condition, hyperforin affected ES cell differentiation into cardiomyocytes in a dose-dependent manner. Analysis of tissue-specific marker expression also revealed that hyperforin at high concentrations partially inhibited ES cell differentiation into mesodermal and endodermal lineages. Hyperforin is currently used in the clinic as a safe and effective antidepressant. Our data indicate that at typical dosages it has only a low risk of embryotoxicity; ingestion of large amounts of hyperforin by pregnant women, however, may pose embryotoxic and teratogenic risks. © 2013 John Wiley & Sons Ltd.

Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells.

PubMed

Lu, Qin; Liu, Yinan; Wang, Yang; Wang, Weiping; Yang, Zhe; Li, Tao; Tian, Yuyao; Chen, Ping; Ma, Kangtao; Jia, Zhuqing; Zhou, Chunyan

2017-06-30

To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10 -4 M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata 4, α- Mhc , β- Mhc , and Tnnt 2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp 1, Brachyury T, Eomes, Isl 1 , Gata 4 , Nkx 2.5 , Tbx 5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor ( Fgf 8), Fgf 10, and Nodal at early stage, and bone morphogenetic protein 2 ( Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin. © 2017 The Author(s).

Stimulation of endogenous cardioblasts by exogenous cell therapy after myocardial infarction.

PubMed

Malliaras, Konstantinos; Ibrahim, Ahmed; Tseliou, Eleni; Liu, Weixin; Sun, Baiming; Middleton, Ryan C; Seinfeld, Jeffrey; Wang, Lai; Sharifi, Behrooz G; Marbán, Eduardo

2014-06-01

Controversy surrounds the identity, origin, and physiologic role of endogenous cardiomyocyte progenitors in adult mammals. Using an inducible genetic labeling approach to identify small non-myocyte cells expressing cardiac markers, we find that activated endogenous cardioblasts are rarely evident in the normal adult mouse heart. However, myocardial infarction results in significant cardioblast activation at the site of injury. Genetically labeled isolated cardioblasts express cardiac transcription factors and sarcomeric proteins, exhibit spontaneous contractions, and form mature cardiomyocytes in vivo after injection into unlabeled recipient hearts. The activated cardioblasts do not arise from hematogenous seeding, cardiomyocyte dedifferentiation, or mere expansion of a preformed progenitor pool. Cell therapy with cardiosphere-derived cells amplifies innate cardioblast-mediated tissue regeneration, in part through the secretion of stromal cell-derived factor 1 by transplanted cells. Thus, stimulation of endogenous cardioblasts by exogenous cells mediates therapeutic regeneration of injured myocardium. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.