GDF15 is a heart-derived hormone that regulates body growth.

PubMed

Wang, Ting; Liu, Jian; McDonald, Caitlin; Lupino, Katherine; Zhai, Xiandun; Wilkins, Benjamin J; Hakonarson, Hakon; Pei, Liming

2017-08-01

The endocrine system is crucial for maintaining whole-body homeostasis. Little is known regarding endocrine hormones secreted by the heart other than atrial/brain natriuretic peptides discovered over 30 years ago. Here, we identify growth differentiation factor 15 (GDF15) as a heart-derived hormone that regulates body growth. We show that pediatric heart disease induces GDF15 synthesis and secretion by cardiomyocytes. Circulating GDF15 in turn acts on the liver to inhibit growth hormone (GH) signaling and body growth. We demonstrate that blocking cardiomyocyte production of GDF15 normalizes circulating GDF15 level and restores liver GH signaling, establishing GDF15 as a bona fide heart-derived hormone that regulates pediatric body growth. Importantly, plasma GDF15 is further increased in children with concomitant heart disease and failure to thrive (FTT). Together these studies reveal a new endocrine mechanism by which the heart coordinates cardiac function and body growth. Our results also provide a potential mechanism for the well-established clinical observation that children with heart diseases often develop FTT. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

NF-κB (p65) negatively regulates myocardin-induced cardiomyocyte hypertrophy through multiple mechanisms.

PubMed

Liao, Xing-Hua; Wang, Nan; Zhao, Dong-Wei; Zheng, De-Liang; Zheng, Li; Xing, Wen-Jing; Zhou, Hao; Cao, Dong-Sun; Zhang, Tong-Cun

2014-12-01

Myocardin is well known to play a key role in the development of cardiomyocyte hypertrophy. But the exact molecular mechanism regulating myocardin stability and transactivity to affect cardiomyocyte hypertrophy has not been studied clearly. We now report that NF-κB (p65) can inhibit myocardin-induced cardiomyocyte hypertrophy. Then we explore the molecular mechanism of this response. First, we show that p65 can functionally repress myocardin transcriptional activity and also reduce the protein expression of myocardin. Second, the function of myocardin can be regulated by epigenetic modifications. Myocardin sumoylation is known to transactivate cardiac genes, but whether p65 can inhibit SUMO modification of myocardin is still not clear. Our data show that p65 weakens myocardin transcriptional activity through attenuating SUMO modification of myocardin by SUMO1/PIAS1, thereby impairing myocardin-mediated cardiomyocyte hypertrophy. Furthermore, the expression of myocardin can be regulated by several microRNAs, which play important roles in the development and function of the heart and muscle. We next investigated potential role of miR-1 in cardiac hypotrophy. Our results show that p65 can upregulate the level of miR-1 and miR-1 can decrease protein expression of myocardin in cardiac myocytes. Notably, miR-1 expression is also controlled by myocardin, leading to a feedback loop. These data thus provide important and novel insights into the function that p65 inhibits myocardin-mediated cardiomyocyte hypertrophy by downregulating the expression and SUMO modification of myocardin and enhancing the expression of miR-1. Copyright © 2014 Elsevier Inc. All rights reserved.

The intrinsic circadian clock within the cardiomyocyte directly regulates myocardial gene expression, metabolism, and contractile function

USDA-ARS?s Scientific Manuscript database

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology remains unknown. We hypothesized that circadian clock within the cardiomyocyte plays a role in regulating myocardia...

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.

c-Abl tyrosine kinase regulates cardiac growth and development.

PubMed

Qiu, Zhaozhu; Cang, Yong; Goff, Stephen P

2010-01-19

The c-Abl protein is a ubiquitously expressed nonreceptor tyrosine kinase involved in the development and function of many mammalian organ systems, including the immune system and bone. Here we show that homozygous Abl mutant embryos and newborns on the C57BL/6J background, but not on other backgrounds, display dramatically enlarged hearts and die perinatally. The heart defects can be largely rescued by cardiomyocyte-specific restoration of the full-length c-Abl protein. The cardiac hyperplasia phenotype is not caused by decreased apoptosis, but rather by abnormally increased cardiomyocyte proliferation during later stages of embryogenesis. Genes involved in cardiac stress and remodeling and cell cycle regulation are also up-regulated in the mutant hearts. These findings reveal an essential role for c-Abl in mammalian heart growth and development.

c-Abl tyrosine kinase regulates cardiac growth and development

PubMed Central

Qiu, Zhaozhu; Cang, Yong; Goff, Stephen P.

2009-01-01

The c-Abl protein is a ubiquitously expressed nonreceptor tyrosine kinase involved in the development and function of many mammalian organ systems, including the immune system and bone. Here we show that homozygous Abl mutant embryos and newborns on the C57BL/6J background, but not on other backgrounds, display dramatically enlarged hearts and die perinatally. The heart defects can be largely rescued by cardiomyocyte-specific restoration of the full-length c-Abl protein. The cardiac hyperplasia phenotype is not caused by decreased apoptosis, but rather by abnormally increased cardiomyocyte proliferation during later stages of embryogenesis. Genes involved in cardiac stress and remodeling and cell cycle regulation are also up-regulated in the mutant hearts. These findings reveal an essential role for c-Abl in mammalian heart growth and development. PMID:20080568

The intrinsic circadian clock within the cardiomyocyte directly regulates myocardial gene expression, metabolism, and contractile function

USDA-ARS?s Scientific Manuscript database

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology remains unknown. We hypothesized that the circadian clock within the cardiomyocyte plays a role in regulating myo...

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.

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

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes.

PubMed

Alam, Shafiul; Abdullah, Chowdhury S; Aishwarya, Richa; Orr, A Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B; Bhuiyan, Md Shenuarin

2017-08-31

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. © 2017 The Author(s).

Sigmar1 regulates endoplasmic reticulum stress-induced C/EBP-homologous protein expression in cardiomyocytes

PubMed Central

Alam, Shafiul; Abdullah, Chowdhury S.; Aishwarya, Richa; Orr, A. Wayne; Traylor, James; Miriyala, Sumitra; Panchatcharam, Manikandan; Pattillo, Christopher B.

2017-01-01

C/EBP-homologous protein (CHOP) is a ubiquitously expressed stress-inducible transcription factor robustly induced by maladaptive endoplasmic reticulum (ER) stresses in a wide variety of cells. Here, we examined a novel function of Sigma 1 receptor (Sigmar1) in regulating CHOP expression under ER stress in cardiomyocytes. We also defined Sigmar1-dependent activation of the adaptive ER-stress pathway in regulating CHOP expression. We used adenovirus-mediated Sigmar1 overexpression as well as Sigmar1 knockdown by siRNA in neonatal rat ventricular cardiomyocytes (NRCs); to induce ER stress, cardiomyocytes were treated with tunicamycin. Sigmar1-siRNA knockdown significantly increased the expression of CHOP and significantly induced cellular toxicity by sustained activation of ER stress in cardiomyocytes. Sigmar1 overexpression decreased the expression of CHOP and significantly decreased cellular toxicity in cells. Using biochemical and immunocytochemical experiments, we also defined the specific ER-stress pathway associated with Sigmar1-dependent regulation of CHOP expression and cellular toxicity. We found that Sigmar1 overexpression significantly increased inositol requiring kinase 1α (IRE1α) phosphorylation and increased spliced X-box-binding proteins (XBP1s) expression as well as nuclear localization. In contrast, Sigmar1 knockdown significantly decreased IRE1α phosphorylation and decreased XBP1s expression as well as nuclear transport. Taken together, these results indicate that Sigmar1-dependent activation of IRE1α-XBP1s ER-stress response pathways are associated with inhibition of CHOP expression and suppression of cellular toxicity. Hence, Sigmar1 is an essential component of the adaptive ER-stress response pathways eliciting cellular protection in cardiomyocytes. PMID:28667101

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

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

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

Adenosine monophosphate-activated protein kinase attenuates cardiomyocyte hypertrophy through regulation of FOXO3a/MAFbx signaling pathway.

PubMed

Chen, Baolin; Wu, Qiang; Xiong, Zhaojun; Ma, Yuedong; Yu, Sha; Chen, Dandan; Huang, Shengwen; Dong, Yugang

2016-09-01

Control of cardiac muscle mass is thought to be determined by a dynamic balance of protein synthesis and degradation. Recent studies have demonstrated that atrophy-related forkhead box O 3a (FOXO3a)/muscle atrophy F-box (MAFbx) signaling pathway plays a central role in the modulation of proteolysis and exert inhibitory effect on cardiomyocyte hypertrophy. In this study, we tested the hypothesis that adenosine monophosphate-activated protein kinase (AMPK) activation attenuates cardiomyocyte hypertrophy by regulating FOXO3a/MAFbx signaling pathway and its downstream protein degradation. The results showed that activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) attenuated cardiomyocyte hypertrophy induced by angiotensin II (Ang II). The antihypertrophic effects of AICAR were blunted by AMPK inhibitor Compound C. In addition, AMPK dramatically increased the activity of transcription factor FOXO3a, up-regulated the expression of its downstream ubiquitin ligase MAFbx, and enhanced cardiomyocyte proteolysis. Meanwhile, the effects of AMPK on protein degradation and cardiomyocyte hypertrophy were blocked after MAFbx was silenced by transfection of cardiomyocytes with MAFbx-siRNA. These results indicate that AMPK plays an important role in the inhibition of cardiomyocyte hypertrophy by activating protein degradation via FOXO3a/MAFbx signaling pathway. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

Sildenafil prevents the up-regulation of transient receptor potential canonical channels in the development of cardiomyocyte hypertrophy

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

Kiso, Hironori; Ohba, Takayoshi; Iino, Kenji

2013-07-05

Highlights: •Transient receptor potential canonical (TRPC1, 3 and 6) are up-regulated by ET-1. •Sildenafil inhibited hypertrophic responses (BNP, Ca entry, NFAT activation). •Sildenafil suppressed TRPC1, 3 and 6 expression. -- Abstract: Background: Transient receptor potential canonical (TRPCs) channels are up-regulated in the development of cardiac hypertrophy. Sildenafil inhibits TRPC6 activation and expression, leading to the prevention of cardiac hypertrophy. However, the effects of sildenafil on the expression of other TRPCs remain unknown. We hypothesized that in addition to its effects of TRPC6, sildenafil blocks the up-regulation of other TRPC channels to suppress cardiomyocyte hypertrophy. Methods and results: In cultured neonatalmore » rat cardiomyocytes, a 48 h treatment with 10 nM endothelin (ET)-1 induced hypertrophic responses characterized by nuclear factor of activated T cells activation and enhancement of brain natriuretic peptide expression and cell surface area. Co-treatment with sildenafil (1 μM, 48 h) inhibited these ET-1-induced hypertrophic responses. Although ET-1 enhanced the gene expression of TRPCs, sildenafil inhibited the enhanced gene expression of TRPC1, C3 and C6. Moreover, co-treatment with sildenafil abolished the augmentation of SOCE in the hypertrophied cardiomyocytes. Conclusions: These results suggest that sildenafil inhibits cardiomyocyte hypertrophy by suppressing the up-regulation of TRPC expression.« less

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.

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

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

TRIM24 protein promotes and TRIM32 protein inhibits cardiomyocyte hypertrophy via regulation of dysbindin protein levels

PubMed Central

Borlepawar, Ankush; Bernt, Alexander; Christen, Lynn; Sossalla, Samuel; Frank, Derk; Frey, Norbert

2017-01-01

We have previously shown that dysbindin is a potent inducer of cardiomyocyte hypertrophy via activation of Rho-dependent serum-response factor (SRF) signaling. We have now performed a yeast two-hybrid screen using dysbindin as bait against a cardiac cDNA library to identify the cardiac dysbindin interactome. Among several putative binding proteins, we identified tripartite motif-containing protein 24 (TRIM24) and confirmed this interaction by co-immunoprecipitation and co-immunostaining. Another tripartite motif (TRIM) family protein, TRIM32, has been reported earlier as an E3 ubiquitin ligase for dysbindin in skeletal muscle. Consistently, we found that TRIM32 also degraded dysbindin in neonatal rat ventricular cardiomyocytes as well. Surprisingly, however, TRIM24 did not promote dysbindin decay but rather protected dysbindin against degradation by TRIM32. Correspondingly, TRIM32 attenuated the activation of SRF signaling and hypertrophy due to dysbindin, whereas TRIM24 promoted these effects in neonatal rat ventricular cardiomyocytes. This study also implies that TRIM32 is a key regulator of cell viability and apoptosis in cardiomyocytes via simultaneous activation of p53 and caspase-3/-7 and inhibition of X-linked inhibitor of apoptosis. In conclusion, we provide here a novel mechanism of post-translational regulation of dysbindin and hypertrophy via TRIM24 and TRIM32 and show the importance of TRIM32 in cardiomyocyte apoptosis in vitro. PMID:28465353

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

GDF11 Modulates Ca2+-Dependent Smad2/3 Signaling to Prevent Cardiomyocyte Hypertrophy.

PubMed

Duran, Javier; Troncoso, Mayarling Francisca; Lagos, Daniel; Ramos, Sebastian; Marin, Gabriel; Estrada, Manuel

2018-05-18

Growth differentiation factor 11 (GDF11), a member of the transforming growth factor-β family, has been shown to act as a negative regulator in cardiac hypertrophy. Ca 2+ signaling modulates cardiomyocyte growth; however, the role of Ca 2+ -dependent mechanisms in mediating the effects of GDF11 remains elusive. Here, we found that GDF11 induced intracellular Ca 2+ increases in neonatal rat cardiomyocytes and that this response was blocked by chelating the intracellular Ca 2+ with BAPTA-AM or by pretreatment with inhibitors of the inositol 1,4,5-trisphosphate (IP₃) pathway. Moreover, GDF11 increased the phosphorylation levels and luciferase activity of Smad2/3 in a concentration-dependent manner, and the inhibition of IP₃-dependent Ca 2+ release abolished GDF11-induced Smad2/3 activity. To assess whether GDF11 exerted antihypertrophic effects by modulating Ca 2+ signaling, cardiomyocytes were exposed to hypertrophic agents (100 nM testosterone or 50 μM phenylephrine) for 24 h. Both treatments increased cardiomyocyte size and [³H]-leucine incorporation, and these responses were significantly blunted by pretreatment with GDF11 over 24 h. Moreover, downregulation of Smad2 and Smad3 with siRNA was accompanied by inhibition of the antihypertrophic effects of GDF11. These results suggest that GDF11 modulates Ca 2+ signaling and the Smad2/3 pathway to prevent cardiomyocyte hypertrophy.

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

Mild hypoxia-induced cardiomyocyte hypertrophy via up-regulation of HIF-1α-mediated TRPC signalling

PubMed Central

Chu, Wenfeng; Wan, Lin; Zhao, Dan; Qu, Xuefeng; Cai, Fulai; Huo, Rong; Wang, Ning; Zhu, Jiuxin; Zhang, Chun; Zheng, Fangfang; Cai, Ruijun; Dong, Deli; Lu, Yanjie; Yang, Baofeng

2012-01-01

Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study was designed to investigate the role of HIF-1α in mild hypoxia-induced cardiomyocytes hypertrophy and its underlying mechanism. Mild hypoxia (MH, 10% O2) caused hypertrophy in cultured neonatal rat cardiac myocytes, which was accompanied with increase of HIF-1α mRNA and accumulation of HIF-1α protein in nuclei. Transient receptor potential canonical (TRPC) channels including TRPC3 and TRPC6, except for TRPC1, were increased, and Ca2+-calcineurin signals were also enhanced in a time-dependent manner under MH condition. MH-induced cardiomyocytes hypertrophy, TRPC up-regulation and enhanced Ca2+-calcineurin signals were inhibited by an HIF-1α specific blocker, SC205346 (30 μM), whereas promoted by HIF-1α overexpression. Electrophysiological voltage-clamp demonstrated that DAG analogue, OAG (30 μM), induced TRPC current by as much as 170% in neonatal rat cardiomyocytes overexpressing HIF-1α compared to negative control. These results implicate that HIF-1α plays a key role in development of cardiac hypertrophy in responses to hypoxic stress. Its mechanism is associated with up-regulating TRPC3, TRPC6 expression, activating TRPC current and subsequently leading to enhanced Ca2+-calcineurin signals. PMID:22129453

Interplay of cell-cell contacts and RhoA/MRTF-A signaling regulates cardiomyocyte identity.

PubMed

Dorn, Tatjana; Kornherr, Jessica; Parrotta, Elvira I; Zawada, Dorota; Ayetey, Harold; Santamaria, Gianluca; Iop, Laura; Mastantuono, Elisa; Sinnecker, Daniel; Goedel, Alexander; Dirschinger, Ralf J; My, Ilaria; Laue, Svenja; Bozoglu, Tarik; Baarlink, Christian; Ziegler, Tilman; Graf, Elisabeth; Hinkel, Rabea; Cuda, Giovanni; Kääb, Stefan; Grace, Andrew A; Grosse, Robert; Kupatt, Christian; Meitinger, Thomas; Smith, Austin G; Laugwitz, Karl-Ludwig; Moretti, Alessandra

2018-06-15

Cell-cell and cell-matrix interactions guide organ development and homeostasis by controlling lineage specification and maintenance, but the underlying molecular principles are largely unknown. Here, we show that in human developing cardiomyocytes cell-cell contacts at the intercalated disk connect to remodeling of the actin cytoskeleton by regulating the RhoA-ROCK signaling to maintain an active MRTF/SRF transcriptional program essential for cardiomyocyte identity. Genetic perturbation of this mechanosensory pathway activates an ectopic fat gene program during cardiomyocyte differentiation, which ultimately primes the cells to switch to the brown/beige adipocyte lineage in response to adipogenesis-inducing signals. We also demonstrate by in vivo fate mapping and clonal analysis of cardiac progenitors that cardiac fat and a subset of cardiac muscle arise from a common precursor expressing Isl1 and Wt1 during heart development, suggesting related mechanisms of determination between the two lineages. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Cardiomyocyte hypertrophy induced by Endonuclease G deficiency requires reactive oxygen radicals accumulation and is inhibitable by the micropeptide humanin.

PubMed

Blasco, Natividad; Cámara, Yolanda; Núñez, Estefanía; Beà, Aida; Barés, Gisel; Forné, Carles; Ruíz-Meana, Marisol; Girón, Cristina; Barba, Ignasi; García-Arumí, Elena; García-Dorado, David; Vázquez, Jesús; Martí, Ramon; Llovera, Marta; Sanchis, Daniel

2018-06-01

The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3β and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Ag+ alters cell growth, neurite extension, cardiomyocyte beating, and fertilized egg constriction.

PubMed

Conrad, A H; Tramp, C R; Long, C J; Wells, D C; Paulsen, A Q; Conrad, G W

1999-11-01

The Russian Space Agency uses electrochemically generated silver ions (Ag+) to purify drinking water for their space station, Mir, and their portion of the International Space Station. U.S. EPA guidelines allow 10.6 micromol x L(-1) Ag+ in human drinking water for up to 10 d. Studies correlate Ag+ exposure with tissue dysfunction in humans, rats, and mice, and with altered ion transport, skeletal muscle contraction, and embryonic cell constriction in other animal cells. Ag+ effects on cell shape change-related functions have not been assessed. Immortalized embryonic human intestinal epithelial cells, freshly explanted embryonic avian nerve cells and cardiomyocytes, and marine fertilized eggs were grown in vitro in medium containing AgNO3. Intestinal cells detach from the substratum and viable cell number decreases by 5-6 d at 5 micromol x L(-1) AgNO3, and faster at higher concentrations. Microtubules appear unaltered in adherent cells. Detached cells are nonviable. Neurite outgrowth and glial cell migration from dorsal root ganglia are inhibited by 3 d at 15 micromol x L(-1) AgNO3 or greater. Contractions stop temporarily in most cardiomyocytes by 5 min at 5 micromol x L(-1) AgNO3 or more, but some cardiomyocytes beat 3 times faster than normal at 7.5-20 micromol x L(-1) AgNO3. Picomolar Ag+ increases marine egg polar lobe constriction within an hour, even in the absence of microtubules. Ag+ alters animal cell growth and shape changes by a MT-independent mechanism. This is the first report of Ag+ effects on vertebrate neurite outgrowth, glial cell migration, or cardiomyocyte beat rate.

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

Temporal regulation of expression of immediate early and second phase transcripts by endothelin-1 in cardiomyocytes

PubMed Central

Cullingford, Timothy E; Markou, Thomais; Fuller, Stephen J; Giraldo, Alejandro; Pikkarainen, Sampsa; Zoumpoulidou, Georgia; Alsafi, Ali; Ekere, Collins; Kemp, Timothy J; Dennis, Jayne L; Game, Laurence; Sugden, Peter H; Clerk, Angela

2008-01-01

Background Endothelin-1 stimulates Gq protein-coupled receptors to promote proliferation in dividing cells or hypertrophy in terminally differentiated cardiomyocytes. In cardiomyocytes, endothelin-1 rapidly (within minutes) stimulates protein kinase signaling, including extracellular-signal regulated kinases 1/2 (ERK1/2; though not ERK5), with phenotypic/physiological changes developing from approximately 12 h. Hypertrophy is associated with changes in mRNA/protein expression, presumably consequent to protein kinase signaling, but the connections between early, transient signaling events and developed hypertrophy are unknown. Results Using microarrays, we defined the early transcriptional responses of neonatal rat cardiomyocytes to endothelin-1 over 4 h, differentiating between immediate early gene (IEG) and second phase RNAs with cycloheximide. IEGs exhibited differential temporal and transient regulation, with expression of second phase RNAs within 1 h. Of transcripts upregulated at 30 minutes encoding established proteins, 28 were inhibited >50% by U0126 (which inhibits ERK1/2/5 signaling), with 9 inhibited 25-50%. Expression of only four transcripts was not inhibited. At 1 h, most RNAs (approximately 67%) were equally changed in total and polysomal RNA with approximately 17% of transcripts increased to a greater extent in polysomes. Thus, changes in expression of most protein-coding RNAs should be reflected in protein synthesis. However, approximately 16% of transcripts were essentially excluded from the polysomes, including some protein-coding mRNAs, presumably inefficiently translated. Conclusion The phasic, temporal regulation of early transcriptional responses induced by endothelin-1 in cardiomyocytes indicates that, even in terminally differentiated cells, signals are propagated beyond the primary signaling pathways through transcriptional networks leading to phenotypic changes (that is, hypertrophy). Furthermore, ERK1/2 signaling plays a major role in

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.

Microtubule Actin Cross-linking Factor 1 regulates cardiomyocyte microtubule distribution and adaptation to hemodynamic overload.

PubMed

Fassett, John T; Xu, Xin; Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J; Chen, Yingjie

2013-01-01

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r(2) = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.

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

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

Renal hypertension prevents run training modification of cardiomyocyte diastolic Ca2+ regulation in male rats.

PubMed

Palmer, B M; Lynch, J M; Snyder, S M; Moore, R L

2001-06-01

The combined effects of endurance run training and renal hypertension on cytosolic Ca2+ concentration ([Ca2+]c) dynamics and Na+-dependent Ca2+ regulation in rat left ventricular cardiomyocytes were examined. Male Fischer 344 rats underwent stenosis of the left renal artery [hypertensive (Ht), n = 18] or a sham operation [normotensive (Nt), n = 20]. One-half of the rats from each group were treadmill trained for >16 wk. Cardiomyocyte fura 2 fluorescence ratio transients were recorded for 7 min during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degrees C. The rate of [Ca2+]c decline was not changed by run training in the Nt group but was reduced in the Ht group. At 7 min, cardiomyocytes were exposed to 10 mM caffeine in the absence of Na+ and Ca2+, which triggered sarcoplasmic reticular Ca2+ release and suppressed Ca2+ efflux via Na+/Ca2+ exchanger. External Na+ was then added, and Na+-dependent Ca2+ efflux rate was recorded. Treadmill training significantly enhanced Na+-dependent Ca2+ efflux rate under these conditions in the Nt group but not in the Ht group. These data provide evidence that renal hypertension prevents the normal run training-induced modifications in diastolic [Ca2+]c regulation mechanisms, including Na+/Ca2+ exchanger.

Aspirin inhibits growth and enhances cardiomyocyte differentiation of bone marrow mesenchymal stem cells.

PubMed

Hao, Wen; Shi, Shutian; Zhou, Shenghui; Wang, Xiao; Nie, Shaoping

2018-05-15

This study aimed to examine the effects of aspirin on the growth and cardiomyocyte differentiation grade of bone marrow mesenchymal stem cells (BMMSCs). BMMSCs were divided into five differentiation groups with different concentrations of aspirin (0 mM, 0.5 mM, 1 mM, 2 mM, or 5 mM), and a undifferentiated control group. Cell growth was measured by cell proliferation, apoptosis assays and DNA cycle analysis. The differentiation grade of BMMSC-derived cardiomyocyte-like cells was examined by measuring the levels of cardiac-specific proteins with cyto-immunofluorescence staining, flow cytometry, and Western blotting. Electrophysiological analyses were performed by patch-clamp experiments and calcium transients were measured by a laser scanning confocal microscope. Cell proliferation decreased as the concentration of aspirin increased. Cell apoptosis increased with increasing aspirin concentration. DNA replication was inhibited in the high dose-aspirin group compared to the low dose- or non-aspirin groups. The number of α-myosin heavy chain (α-MHC) and cardiac troponin I (cTnI) positive cells, cardiac troponin T (cTnT) and connexin 43 (Cx43) positive rates, expression levels of Cx43, Nkx2.5, GATA4 and β1 adrenoceptor increased with increasing aspirin concentration. No sarcomeric cross-striations, spontaneous or induced beating activity or action potentials was observed in each group. Calcium transients were measured in small number cells in 2 mM aspirin group, but the features are atypical. Consequently, aspirin inhibits proliferation and survival of BMMSCs and enhances cardiomyocyte differentiation of BMMSCs. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

Microtubule Actin Cross-Linking Factor 1 Regulates Cardiomyocyte Microtubule Distribution and Adaptation to Hemodynamic Overload

PubMed Central

Kwak, Dongmin; Wang, Huan; Liu, Xiaoyu; Hu, Xinli; Bache, Robert J.; Chen, Yingjie

2013-01-01

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r2 = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload. PMID:24086300

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.

Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

PubMed

Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

2015-02-01

Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy

PubMed Central

Zaglia, Tania; Ceriotti, Paola; Campo, Antonio; Borile, Giulia; Armani, Andrea; Carullo, Pierluigi; Prando, Valentina; Coppini, Raffaele; Vida, Vladimiro; Stølen, Tomas O.; Ulrik, Wisløff; Cerbai, Elisabetta; Stellin, Giovanni; Faggian, Giuseppe; De Stefani, Diego; Sandri, Marco; Rizzuto, Rosario; Di Lisa, Fabio; Pozzan, Tullio; Catalucci, Daniele; Mongillo, Marco

2017-01-01

The mitochondrial Ca2+ uniporter complex (MCUC) is a multimeric ion channel which, by tuning Ca2+ influx into the mitochondrial matrix, finely regulates metabolic energy production. In the heart, this dynamic control of mitochondrial Ca2+ uptake is fundamental for cardiomyocytes to adapt to either physiologic or pathologic stresses. Mitochondrial calcium uniporter (MCU), which is the core channel subunit of MCUC, has been shown to play a critical role in the response to β-adrenoreceptor stimulation occurring during acute exercise. The molecular mechanisms underlying the regulation of MCU, in conditions requiring chronic increase in energy production, such as physiologic or pathologic cardiac growth, remain elusive. Here, we show that microRNA-1 (miR-1), a member of the muscle-specific microRNA (myomiR) family, is responsible for direct and selective targeting of MCU and inhibition of its translation, thereby affecting the capacity of the mitochondrial Ca2+ uptake machinery. Consistent with the role of miR-1 in heart development and cardiomyocyte hypertrophic remodeling, we additionally found that MCU levels are inversely related with the myomiR content, in murine and, remarkably, human hearts from both physiologic (i.e., postnatal development and exercise) and pathologic (i.e., pressure overload) myocardial hypertrophy. Interestingly, the persistent activation of β-adrenoreceptors is likely one of the upstream repressors of miR-1 as treatment with β-blockers in pressure-overloaded mouse hearts prevented its down-regulation and the consequent increase in MCU content. Altogether, these findings identify the miR-1/MCU axis as a factor in the dynamic adaptation of cardiac cells to hypertrophy. PMID:29073097

Mitochondrial fission is required for cardiomyocyte hypertrophy mediated by a Ca2+-calcineurin signaling pathway.

PubMed

Pennanen, Christian; Parra, Valentina; López-Crisosto, Camila; Morales, Pablo E; Del Campo, Andrea; Gutierrez, Tomás; Rivera-Mejías, Pablo; Kuzmicic, Jovan; Chiong, Mario; Zorzano, Antonio; Rothermel, Beverly A; Lavandero, Sergio

2014-06-15

Cardiomyocyte hypertrophy has been associated with diminished mitochondrial metabolism. Mitochondria are crucial organelles for the production of ATP, and their morphology and function are regulated by the dynamic processes of fusion and fission. The relationship between mitochondrial dynamics and cardiomyocyte hypertrophy is still poorly understood. Here, we show that treatment of cultured neonatal rat cardiomyocytes with the hypertrophic agonist norepinephrine promotes mitochondrial fission (characterized by a decrease in mitochondrial mean volume and an increase in the relative number of mitochondria per cell) and a decrease in mitochondrial function. We demonstrate that norepinephrine acts through α1-adrenergic receptors to increase cytoplasmic Ca(2+), activating calcineurin and promoting migration of the fission protein Drp1 (encoded by Dnml1) to mitochondria. Dominant-negative Drp1 (K38A) not only prevented mitochondrial fission, it also blocked hypertrophic growth of cardiomyocytes in response to norepinephrine. Remarkably, an antisense adenovirus against the fusion protein Mfn2 (AsMfn2) was sufficient to increase mitochondrial fission and stimulate a hypertrophic response without agonist treatment. Collectively, these results demonstrate the importance of mitochondrial dynamics in the development of cardiomyocyte hypertrophy and metabolic remodeling. © 2014. Published by The Company of Biologists Ltd.

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

Epigenomic Reprogramming of Adult Cardiomyocyte-Derived Cardiac Progenitor Cells

PubMed Central

Zhang, Yiqiang; Zhong, Jiang F; Qiu, Hongyu; Robb MacLellan, W.; Marbán, Eduardo; Wang, Charles

2015-01-01

It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration. PMID:26657817

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

β-Blocker carvedilol protects cardiomyocytes against oxidative stress-induced apoptosis by up-regulating miR-133 expression.

PubMed

Xu, Chaoqian; Hu, Yingying; Hou, Liangyu; Ju, Jin; Li, Xiaoguang; Du, Ning; Guan, Xiaoxiang; Liu, Zhenhong; Zhang, Tianze; Qin, Wei; Shen, Nannan; Bilal, Muhammad U; Lu, Yanjie; Zhang, Yong; Shan, Hongli

2014-10-01

Oxidative stress is a causal factor and key promoter of a variety of cardiovascular diseases associated with apoptotic cell death by causing deregulation of related genes. Though carvedilol, a β-adrenergic blocker, has been shown to produce cytoprotective effects against cardiomyocyte apoptosis, the mechanisms are not fully understood. The present study was designed to investigate whether the beneficial effects of carvedilol are related to microRNAs which have emerged as critical players in cardiovascular pathophysiology via post-transcriptional regulation of protein-coding genes. In vivo, we demonstrated that carvedilol ameliorated impaired cardiac function of infarct rats and restored miR-133 expression. In vitro, carvedilol protected cardiomyocytes from H2O2 induced apoptosis detected by TUNEL staining and MTT assays, and increased miR-133 expression in cardiomyocytes. Overexpression of miR-133, a recognized anti-apoptotic miRNA, produced similar effects to carvedilol: reduction of reactive oxygen species (ROS) and malondialdehyde (MDA) content and increment of superoxide dismutase (SOD) activity and glutathione peroxidase (GPx) level, so as to protect cardiomyocytes from apoptosis by downregulating caspase-9 and caspase-3 expression in the presence of H2O2. Transfection with AMO-133 (antisense inhibitor oligodeoxyribonucleotides) alone abolished the beneficial effects of carvedilol. Caspase-9-specific inhibitor z-LEHD-fmk, caspase-3-specific inhibitor z-DEVD-fmk, caspase-9 siRNA and caspase-3 siRNA were used to establish caspase-3 as a downstream target of miR-133. In conclusion, our data indicated that carvedilol protected cardiomyocytes by increasing miR-133 expression and suppressing caspase-9 and subsequent apoptotic pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

An integrated mechanism of cardiomyocyte nuclear Ca(2+) signaling.

PubMed

Ibarra, Cristián; Vicencio, Jose Miguel; Varas-Godoy, Manuel; Jaimovich, Enrique; Rothermel, Beverly A; Uhlén, Per; Hill, Joseph A; Lavandero, Sergio

2014-10-01

In cardiomyocytes, Ca(2+) plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca(2+) within subcellular microdomains: transcription is regulated by Ca(2+) release within nuclear microdomains, and excitation-contraction coupling is regulated by cytosolic Ca(2+). Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca(2+) signals. However, signaling pathways coupling surface receptor activation to nuclear Ca(2+) release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca(2+) signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca(2+) release from perinuclear Ca(2+) stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca(2+) release. In this review, we discuss mechanisms for the selective control of nuclear Ca(2+) signals with special focus on emerging models of agonist receptor activation. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

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

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

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

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.

Rapamycin and CHIR99021 Coordinate Robust Cardiomyocyte Differentiation From Human Pluripotent Stem Cells Via Reducing p53-Dependent Apoptosis.

PubMed

Qiu, Xiao-Xu; Liu, Yang; Zhang, Yi-Fan; Guan, Ya-Na; Jia, Qian-Qian; Wang, Chen; Liang, He; Li, Yong-Qin; Yang, Huang-Tian; Qin, Yong-Wen; Huang, Shuang; Zhao, Xian-Xian; Jing, Qing

2017-10-02

Cardiomyocytes differentiated from human pluripotent stem cells can serve as an unexhausted source for a cellular cardiac disease model. Although small molecule-mediated cardiomyocyte differentiation methods have been established, the differentiation efficiency is relatively unsatisfactory in multiple lines due to line-to-line variation. Additionally, hurdles including line-specific low expression of endogenous growth factors and the high apoptotic tendency of human pluripotent stem cells also need to be overcome to establish robust and efficient cardiomyocyte differentiation. We used the H9-human cardiac troponin T-eGFP reporter cell line to screen for small molecules that promote cardiac differentiation in a monolayer-based and growth factor-free differentiation model. We found that collaterally treating human pluripotent stem cells with rapamycin and CHIR99021 during the initial stage was essential for efficient and reliable cardiomyocyte differentiation. Moreover, this method maintained consistency in efficiency across different human embryonic stem cell and human induced pluripotent stem cell lines without specifically optimizing multiple parameters (the efficiency in H7, H9, and UQ1 human induced pluripotent stem cells is 98.3%, 93.3%, and 90.6%, respectively). This combination also increased the yield of cardiomyocytes (1:24) and at the same time reduced medium consumption by about 50% when compared with the previous protocols. Further analysis indicated that inhibition of the mammalian target of rapamycin allows efficient cardiomyocyte differentiation through overcoming p53-dependent apoptosis of human pluripotent stem cells during high-density monolayer culture via blunting p53 translation and mitochondrial reactive oxygen species production. We have demonstrated that mammalian target of rapamycin exerts a stage-specific and multifaceted regulation over cardiac differentiation and provides an optimized approach for generating large numbers of functional

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

Palmitoylation regulates intracellular trafficking of β2 adrenergic receptor/arrestin/phosphodiesterase 4D complexes in cardiomyocytes.

PubMed

Liu, Ruijie; Wang, Dayong; Shi, Qian; Fu, Qin; Hizon, Steven; Xiang, Yang K

2012-01-01

β(2) adrenergic receptor (β(2)AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β(2)AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β(2)AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β(2)AR association with arrestin/PDE4D. Replacing cysteine 341 on β(2)AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β(2)AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β(2)AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β(2)AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β(2)AR.

Palmitoylation Regulates Intracellular Trafficking of β2 Adrenergic Receptor/Arrestin/Phosphodiesterase 4D Complexes in Cardiomyocytes

PubMed Central

Liu, Ruijie; Wang, Dayong; Shi, Qian; Fu, Qin; Hizon, Steven; Xiang, Yang K.

2012-01-01

β2 adrenergic receptor (β2AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β2AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β2AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β2AR association with arrestin/PDE4D. Replacing cysteine 341 on β2AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β2AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β2AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β2AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β2AR. PMID:22912718

Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation

PubMed Central

Aix, Esther; Gutiérrez-Gutiérrez, Óscar; Sánchez-Ferrer, Carlota; Aguado, Tania

2016-01-01

The molecular mechanisms that drive mammalian cardiomyocytes out of the cell cycle soon after birth remain largely unknown. Here, we identify telomere dysfunction as a critical physiological signal for cardiomyocyte cell-cycle arrest. We show that telomerase activity and cardiomyocyte telomere length decrease sharply in wild-type mouse hearts after birth, resulting in cardiomyocytes with dysfunctional telomeres and anaphase bridges and positive for the cell-cycle arrest protein p21. We further show that premature telomere dysfunction pushes cardiomyocytes out of the cell cycle. Cardiomyocytes from telomerase-deficient mice with dysfunctional telomeres (G3 Terc−/−) show precocious development of anaphase-bridge formation, p21 up-regulation, and binucleation. In line with these findings, the cardiomyocyte proliferative response after cardiac injury was lost in G3 Terc−/− newborns but rescued in G3 Terc−/−/p21−/− mice. These results reveal telomere dysfunction as a crucial signal for cardiomyocyte cell-cycle arrest after birth and suggest interventions to augment the regeneration capacity of mammalian hearts. PMID:27241915

Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes

PubMed Central

Ram, Rashmi; Wescott, Andrew P.; Varandas, Katherine; Dirksen, Robert T.

2013-01-01

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1. PMID:24186093

Mena associates with Rac1 and modulates connexin 43 remodeling in cardiomyocytes.

PubMed

Ram, Rashmi; Wescott, Andrew P; Varandas, Katherine; Dirksen, Robert T; Blaxall, Burns C

2014-01-01

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1.

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.

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout.

PubMed

Sokolova, Niina; Vendelin, Marko; Birkedal, Rikke

2009-12-17

coupled to respiration. This argues against diffusion restriction by the outer mitochondrial membrane. These results from rainbow trout cardiomyocytes resemble those from other low-performance hearts such as neonatal rat and rabbit hearts. Thus, it seems that metabolic regulation is related to cardiac performance, and it is likely that rainbow trout can be used as a model animal for further studies of the localization and role of diffusion restrictions in low-performance hearts.

Effect of Substrate Mechanics on Cardiomyocyte Maturation and Growth

PubMed Central

Tallawi, Marwa; Rai, Ranjana; Boccaccini, Aldo. R.

2015-01-01

Cardiac tissue engineering constructs are a promising therapeutic treatment for myocardial infarction, which is one of the leading causes of death. In order to further advance the development and regeneration of engineered cardiac tissues using biomaterial platforms, it is important to have a complete overview of the effects that substrates have on cardiomyocyte (CM) morphology and function. This article summarizes recent studies that investigate the effect of mechanical cues on the CM differentiation, maturation, and growth. In these studies, CMs derived from embryos, neonates, and mesenchymal stem cells were seeded on different substrates of various elastic modulus. Measuring the contractile function by force production, work output, and calcium handling, it was seen that cell behavior on substrates was optimized when the substrate stiffness mimicked that of the native tissue. The contractile function reflected changes in the sarcomeric protein confirmation and organization that promoted the contractile ability. The analysis of the literature also revealed that, in addition to matrix stiffness, mechanical stimulation, such as stretching the substrate during cell seeding, also played an important role during cell maturation and tissue development. PMID:25148904

Evidence Suggesting that the Cardiomyocyte Circadian Clock Modulates Responsiveness of the Heart to Hypertrophic Stimuli in Mice

PubMed Central

Durgan, David J.; Tsai, Ju-Yun; Grenett, Maximiliano H.; Pat, Betty M.; Ratcliffe, William F.; Villegas-Montoya, Carolina; Garvey, Merissa E.; Nagendran, Jeevan; Dyck, Jason R.B.; Bray, Molly S.; Gamble, Karen L.; Gimble, Jeffrey M.; Young, Martin E.

2011-01-01

Circadian dyssynchrony of an organism (at the whole body level) with its environment, either through light/dark cycle or genetic manipulation of clock genes, augments various cardiometabolic diseases. The cardiomyocyte circadian clock has recently been shown to influence multiple myocardial processes, ranging from transcriptional regulation and energy metabolism, to contractile function. We therefore reasoned that chronic dyssychrony of the cardiomyocyte circadian clock with its environment would precipitate myocardial maladaptation to a circadian challenge (simulated shift work; SSW). To test this hypothesis, 2 and 20 month old wild-type and CCM (Cardiomyocyte Clock Mutant; a model with genetic temporal suspension of the cardiomyocyte circadian clock at the active-to-sleep phase transition) mice were subjected to chronic (16-wks) bi-weekly 12-hr phase shifts in the light/dark cycle (i.e., SSW). Assessment of adaptation/maladaptation at whole body homeostatic, gravimetric, humoral, histological, transcriptional, and cardiac contractile function levels revealed essentially identical responses between wild-type and CCM littermates. However, CCM hearts exhibit increased bi-ventricular weight, cardiomyocyte size, and molecular markers of hypertrophy (anf, mcip1) independent of aging and/or SSW. Similarly, a second genetic model of selective temporal suspension of the cardiomyocyte circadian clock (Cardiomyocyte-specific BMAL1 Knockout [CBK] mice) exhibits increased bi-ventricular weight and mcip1 expression. Wild-type mice exhibit 5-fold greater cardiac hypertrophic growth (and 6-fold greater anf mRNA induction) when challenged with the hypertrophic agonist isoproterenol at the active-to-sleep phase transition, relative to isoproterenol administration at the sleep-to-active phase transition. This diurnal variation was absent in CCM mice. Collectively, these data suggest that the cardiomyocyte circadian clock likely influences responsiveness of the heart to hypertrophic

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.

Vasodilator therapy with hydralazine induces angiotensin AT2 receptor-mediated cardiomyocyte growth in mice lacking guanylyl cyclase-A

PubMed Central

Li, Y; Saito, Y; Kuwahara, K; Rong, X; Kishimoto, I; Harada, M; Horiuchi, M; Murray, M; Nakao, K

2010-01-01

Background and purpose: Recent clinical guidelines advocate the use of the isosorbide dinitrate/hydralazine combination in treatment for heart failure. However, clinical and laboratory evidence suggest that some vasodilators may induce cardiac hypertrophy under uncertain conditions. This study investigated the effects and underlying mechanism of action of the vasodilator hydralazine on cardiac growth. Experimental approach: Wild-type mice and animals deficient in guanylyl cyclase-A (GCA) and/or angiotensin receptors (AT1 and AT2 subtypes) were treated with hydralazine (≈24 mg·kg−1·day−1 in drinking water) for 5 weeks. Cardiac mass and/or cardiomyocyte cross-sectional area, fibrosis (van Giessen-staining) and cardiac gene expression (real-time RT-PCR) were measured. Key results: Hydralazine lowered blood pressure in mice of all genotypes. However, this treatment increased the heart and left ventricular to body weight ratios, as well as cardiomyocyte cross-sectional area, and cardiac expression of atrial natriuretic peptide mRNA in mice lacking GCA. Hydralazine did not affect cardiac hypertrophy in wild-type mice and mice lacking either AT1 or AT2 receptors alone. However, the pro-hypertrophic effect of hydralazine was prevented in mice lacking both GCA and AT2, but not GCA and AT1 receptors. However, hydralazine did decrease cardiac collagen deposition and collagen I mRNA (signs of cardiac fibrosis) in mice that were deficient in GCA, or both GCA and AT2 receptors. Conclusions and implications: The vasodilator hydralazine induced AT2 receptor-mediated cardiomyocyte growth under conditions of GCA deficiency. However, attenuation of cardiac fibrosis by hydralazine could be beneficial in the management of cardiac diseases. PMID:20136844

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.

Autoantibodies in dilated cardiomyopathy induce vascular endothelial growth factor expression in cardiomyocytes

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

Saygili, Erol, E-mail: erol.saygili@med.uni-duesseldorf.de; Noor-Ebad, Fawad; Schröder, Jörg W.

2015-09-11

Background: Autoantibodies have been identified as major predisposing factors for dilated cardiomyopathy (DCM). Patients with DCM show elevated serum levels of vascular endothelial growth factor (VEGF) whose source is unknown. Besides its well-investigated effects on angiogenesis, evidence is present that VEGF signaling is additionally involved in fibroblast proliferation and cardiomyocyte hypertrophy, hence in cardiac remodeling. Whether autoimmune effects in DCM impact cardiac VEGF signaling needs to be elucidated. Methods: Five DCM patients were treated by the immunoadsorption (IA) therapy on five consecutive days. The eluents from the IA columns were collected and prepared for cell culture. Cardiomyocytes from neonatal ratsmore » (NRCM) were incubated with increasing DCM-immunoglobulin-G (IgG) concentrations for 48 h. Polyclonal IgG (Venimmun N), which was used to restore IgG plasma levels in DCM patients after the IA therapy was additionally used for control cell culture purposes. Results: Elevated serum levels of VEGF decreased significantly after IA (Serum VEGF (ng/ml); DCM pre-IA: 45 ± 9.1 vs. DCM post–IA: 29 ± 6.7; P < 0.05). In cell culture, pretreatment of NRCM by DCM-IgG induced VEGF expression in a time and dose dependent manner. Biologically active VEGF that was secreted by NRCM significantly increased BNP mRNA levels in control cardiomyocytes and induced cell-proliferation of cultured cardiac fibroblast (Fibroblast proliferation; NRCM medium/HC-IgG: 1 ± 0.0 vs. NRCM medium/DCM-IgG 100 ng/ml: 5.6 ± 0.9; P < 0.05). Conclusion: The present study extends the knowledge about the possible link between autoimmune signaling in DCM and VEGF induction. Whether this observation plays a considerable role in cardiac remodeling during DCM development needs to be further elucidated. - Highlights: • Mechanisms of remodeling in dilated cardiomyopathy (DCM) are not fully understood. • Autoantibodies have been identified as major predisposing

Cyclosporine A regulate oxidative stress-induced apoptosis in cardiomyocytes: mechanisms via ROS generation, iNOS and Hsp70

PubMed Central

Chen, Huei-Wen; Chien, Chiang-Ting; Yu, Sung-Liang; Lee, Yuan-Teh; Chen, Wen-Jone

2002-01-01

Previous study suggested that cyclosporine A (CsA) could partially reduce ischaemia/reperfusion-induced injury in isolated heart, but the mechanism was still unclear. In this study, the possible mechanisms of cyclosporine A in regulating oxidative stress-induced cardiomyocyte apoptosis were examined. Morphological (cell shrinkage, apoptotic body formation, and DNA fragmentation) and biochemical (annexin-V staining for exposed phosphatidylserine residues) evidences showed that both hydrogen peroxide (H2O2) and hypoxia/reoxygenation could induce apoptotic change in the embryonal rat heart myoblast-derived cells (H9c2). These effects were inhibited by pre-treatment with CsA at concentration of 0.01–1.0 μM for 24 h, but were increased with 10.0 μM CsA. While examining the mechanisms of CsA in protecting cardiomyocyte apoptosis, we found that the collapse of mitochondria membrane potential (ΔΨm) induced by oxidative stress was partially reversed by CsA (0.01–1.0 μM). Compared to the control, CSA at the concentration of 0.1 and 10.0 μM significantly increased the level of intracellular reactive oxygen species (ROS) to 117.2±12.4% and 234.4±9.3%, respectively. Co-incubating with the antioxidant, ascorbic acid (10.0 μM), could partially reduce the protective effect of CsA (0.01–1.0 μM) and the toxic effect of 10.0 μM CsA. Pre-treatment with CsA at concentration of 0.01–1.0 μM for 24 h produced up-regulation of heat shock protein 70 (Hsp 70), inducible nitric oxide synthase (iNOS) and also induced NO production, indicating that these factors might be associated with the cell protective effects of CsA. These results suggest that CsA could protect the oxidative stress-induced cardiomyocyte apoptosis not only by preventing the loss of ΔΨm in mitochondria, but also through ROS generation, Hsp70, and iNOS up-regulation. PMID:12411407

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.

Role of α-crystallin B in regulation of stress induced cardiomyocyte apoptosis.

PubMed

Ganguly, Subhalakshmi; Mitra, Arkadeep; Sarkar, Sagartirtha

2014-01-01

Cardiovascular disease is the leading cause of death worldwide. Recently emerging evidence suggests that cardiomyocyte apoptosis is one of the major pathogenic factors in heart diseases leading to heart failure. Cardiomyocytes undergo apoptosis in response to a wide variety of cellular stresses including protein folding stress at Endoplasmic reticulum (ER). Stressed myocytes elicit an adaptive response referred as Unfolded Protein Response (UPR) by inducing accumulation of heat shock proteins (HSPs) to mitigate the ER stress. HSPs act as molecular chaperons by assisting correct folding of the aggregated misfolded proteins in ER lumen. α-Crystallin B (CRYAB) is an abundant small HSP that confers protection to cardiomyocytes against various stress stimuli. Recent evidence indicates that CRYAB directly interacts with several components of ER stress and also mitochondrial apoptotic pathway. Based on currently available literature this mini review will focus on how CRYAB confers protection to stressed myocardium thereby emphasizing its function as antiapoptotic molecule. Understanding the interplay between CRYAB and the key components in the apoptotic signaling cascade mediated by ER and mitochondria will help in development of novel therapies for cardiac diseases.

Chinonin, a novel drug against cardiomyocyte apoptosis induced by hypoxia and reoxygenation.

PubMed

Shen, J G; Quo, X S; Jiang, B; Li, M; Xin, W; Zhao, B L

2000-02-21

The inhibitory effects of Chinonin, a natural antioxidant extracted from a Chinese medicine, on apoptotic and necrotic cell death of cardiomyocytes in hypoxia-reoxygenation process were observed in this study. The possible mechanisms of Chinonin on scavenging reactive oxygen species and regulating apoptotic related genes bcl-2 and p53 were also investigated. Neonatal rat cardiomyocytes were subjected to 24-h hypoxia and 4-h reoxygenation. Cell death was evaluated by DNA electrophoresis on agarose gel, cell death ELISA and annexin-V-FLUOS/propidium iodide (PI) double staining cytometry. Hypoxia caused the increase of apoptotic rates and the release of lactate dehydrogenase (LDH), while reoxygenation not only further increased the apoptotic rates and leakage of LDH, but also induced necrosis of cardiomyocytes. In addition, hypoxia increased the levels of NO(2)(-)/NO(3)(-) and thiobarbituric acid reacted substances (TBARS), while reoxygenation decreased NO(2)(-)/NO(3)(-), but further increased TBARS in the cultured media. Moreover, hypoxia up-regulated the expression levels of bcl-2 and p53 proteins, while reoxygenation down-regulated bcl-2 and further up-regulated p53. Chinonin significantly decreased the rates of apoptotic and necrotic cardiomyocytes, and inhibited the leakage of LDH. It also diminished NO(2)(-)/NO(3)(-) and TBARS, down-regulated the expression level of p53 protein, and up-regulated bcl-2 protein, respectively. The results suggest that Chinonin has preventive effects against apoptotic and necrotic cell death and its protective mechanisms are related to the antioxidant properties of scavenging nitric oxide and oxygen free radicals, and the modulating effects on the expression levels of bcl-2 and p53 proteins.

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

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.

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

Aldosterone down-regulates the slowly activated delayed rectifier potassium current in adult guinea pig cardiomyocytes.

PubMed

Lv, Yankun; Bai, Song; Zhang, Hua; Zhang, Hongxue; Meng, Jing; Li, Li; Xu, Yanfang

2015-12-01

There is emerging evidence that the mineralocorticoid hormone aldosterone is associated with arrhythmias in cardiovascular disease. However, the effect of aldosterone on the slowly activated delayed rectifier potassium current (IK s ) remains poorly understood. The present study was designed to investigate the modulation of IK s by aldosterone. Adult guinea pigs were treated with aldosterone for 28 days via osmotic pumps. Standard glass microelectrode recordings and whole-cell patch-clamp techniques were used to record action potentials in papillary muscles and IK s in ventricular cardiomyocytes. The aldosterone-treated animals exhibited a prolongation of the QT interval and action potential duration with a higher incidence of early afterdepolarizations. Patch-clamp recordings showed a significant down-regulation of IK s density in the ventricular myocytes of these treated animals. These aldosterone-induced electrophysiological changes were fully prevented by a combined treatment with spironolactone, a mineralocorticoid receptor (MR) antagonist. In addition, in in vitro cultured ventricular cardiomyocytes, treatment with aldosterone (sustained exposure for 24 h) decreased the IK s density in a concentration-dependent manner. Furthermore, a significant corresponding reduction in the mRNA/protein expression of IKs channel pore and auxiliary subunits, KCNQ1 and KCNE1 was detected in ventricular tissue from the aldosterone-treated animals. Aldosterone down-regulates IK s by inhibiting the expression of KCNQ1 and KCNE1, thus delaying the ventricular repolarization. These results provide new insights into the mechanism underlying K(+) channel remodelling in heart disease and may explain the highly beneficial effects of MR antagonists in HF. © 2015 The British Pharmacological Society.

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

Cardiomyocyte marker expression in a human lymphocyte cell line using mouse cardiomyocyte extract.

PubMed

Vojdani, Zahra; Tavakolinejad, Sima; Talaei-Khozani, Tahereh; Esmaeilpour, Tahereh; Rasooli, Manuchehr

2011-03-01

Cell transplantation shows potential for the treatment of cardiac diseases. Embryonic stem cells, cord blood and mesenchymal stem cells have been suggested as sources for transplantation therapy. Because of some technical limitations with the use of stem cells, transdifferentiation of fully differentiated cells is a potentially useful alternative. We investigated whether human peripheral blood cells could transdifferentiate into cardiomyocyte. Transdifferentiation was induced in a human B lymphocyte cell line (Raji). Cardiomyocyte extract was prepared from adult mouse cardiomyocytes. The cells were treated with 5-aza-2-deoxycytidine and trichostatin A, permeabilized with streptolysin O, and exposed to the mouse cardiomyocyte extract. They were cultured for 10 days, 3 weeks and 4 weeks. Cardiomyocyte markers were detected with immunohistochemistry and flow cytometry. Immunocytochemistry revealed that some cells expressed myosin heavy chain, α-actinin and cardiac troponin T after 3 and 4 weeks. Flow cytometry confirmed these data. In cells exposed to trichostatin A and 5-aza-2-deoxycytidine and permeabilized in the presence of the cardiomyocyte extract, troponin T expression was seen in 3.53% of the cells and 3.11% of them expressed α-actinin. After exposure to the cardiomyocyte extract, some permeabilized cells adhered to the plate loosely; however, the morphology did not change significantly, and they continued to show a rounded shape after 4 weeks. Our treated lymphocytes expressed cardiomyocyte markers. Our results suggest that lymphocytes may be useful in future research as a source of cells for reprogramming procedures.

p38α Mitogen-Activated Protein Kinase Plays a Critical Role in Cardiomyocyte Survival but Not in Cardiac Hypertrophic Growth in Response to Pressure Overload

PubMed Central

Nishida, Kazuhiko; Yamaguchi, Osamu; Hirotani, Shinichi; Hikoso, Shungo; Higuchi, Yoshiharu; Watanabe, Tetsuya; Takeda, Toshihiro; Osuka, Soh; Morita, Takashi; Kondoh, Gen; Uno, Yoshihiro; Kashiwase, Kazunori; Taniike, Masayuki; Nakai, Atsuko; Matsumura, Yasushi; Miyazaki, Jun-ichi; Sudo, Tatsuhiko; Hongo, Kenichi; Kusakari, Yoichiro; Kurihara, Satoshi; Chien, Kenneth R.; Takeda, Junji; Hori, Masatsugu; Otsu, Kinya

2004-01-01

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38α is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38α in hearts. First, we generated mice with floxed p38α alleles and crossbred them with mice expressing the Cre recombinase under the control of the α-myosin heavy-chain promoter to obtain cardiac-specific p38α knockout mice. These cardiac-specific p38α knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38α plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation. PMID:15572667

S100A8/MYD88/NF-қB: a novel pathway involved in cardiomyocyte hypertrophy driven by thyroid hormone.

PubMed

Takano, Ana Paula Cremasco; Munhoz, Carolina Demarchi; Moriscot, Anselmo Sigari; Gupta, Sudhiranjan; Barreto-Chaves, Maria Luiza Morais

2017-06-01

Recent studies have evidenced the involvement of inflammation-related pathways to the development of cardiac hypertrophy and other consequences on the cardiovascular system, including the calcium-binding protein S100A8. However, this has never been investigated in the thyroid hormone (TH)-prompted cardiac hypertrophy. Thus, we aimed to test whether S100A8 and related signaling molecules, myeloid differentiation factor-88 (MyD88) and nuclear factor kappa B (NF-қB), could be associated with the cardiomyocyte hypertrophy induced by TH. Our results demonstrate that the S100A8/MyD88/NF-қB signaling pathway is activated in cardiomyocytes following TH stimulation. The knockdown of S100A8 and MyD88 indicates the contribution of those molecules to cardiomyocyte hypertrophy in response to TH, as evaluated by cell surface area, leucine incorporation assay, and gene expression. Furthermore, S100A8 and MyD88 are crucial mediators of NF-қB activation, which is also involved in the hypertrophic growth of TH-treated cardiomyocytes. Supporting the in vitro data, the contribution of NF-қB for TH-induced cardiac hypertrophy is confirmed in vivo, by using transgenic mice with cardiomyocyte-specific suppression of NF-қB. These data identify a novel pathway regulated by TH that mediates cardiomyocyte hypertrophy. However, the potential role of this new pathway in short and long-term cardiac effects of TH remains to be further investigated. Inflammation-related signaling is activated by T3 in cardiomyocytes. S100A8 and MyD88 have a crucial role in cardiomyocyte hypertrophy by T3. S100A8 and MyD88 mediate NF-қB activation by T3. NF-қB contributes to T3-induced cardiac hypertrophy in vitro and in vivo.

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.

[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.

Pi3kcb links Hippo-YAP and PI3K-AKT signaling pathways to promote cardiomyocyte proliferation and survival.

PubMed

Lin, Zhiqiang; Zhou, Pingzhu; von Gise, Alexander; Gu, Fei; Ma, Qing; Chen, Jinghai; Guo, Haidong; van Gorp, Pim R R; Wang, Da-Zhi; Pu, William T

2015-01-02

Yes-associated protein (YAP), the nuclear effector of Hippo signaling, regulates cellular growth and survival in multiple organs, including the heart, by interacting with TEA (transcriptional enhancer activator)-domain sequence-specific DNA-binding proteins. Recent studies showed that YAP stimulates cardiomyocyte proliferation and survival. However, the direct transcriptional targets through which YAP exerts its effects are poorly defined. To identify direct YAP targets that mediate its mitogenic and antiapoptotic effects in the heart. We identified direct YAP targets by combining differential gene expression analysis in YAP gain- and loss-of-function with genome-wide identification of YAP-bound loci using chromatin immunoprecipitation and high throughput sequencing. This screen identified Pik3cb, encoding p110β, a catalytic subunit of phosphoinositol-3-kinase, as a candidate YAP effector that promotes cardiomyocyte proliferation and survival. YAP and TEA-domain occupied a conserved enhancer within the first intron of Pik3cb, and this enhancer drove YAP-dependent reporter gene expression. Yap gain- and loss-of-function studies indicated that YAP is necessary and sufficient to activate the phosphoinositol-3-kinase-Akt pathway. Like Yap, Pik3cb gain-of-function stimulated cardiomyocyte proliferation, and Pik3cb knockdown dampened YAP mitogenic activity. Reciprocally, impaired heart function in Yap loss-of-function was significantly rescued by adeno-associated virus-mediated Pik3cb expression. Pik3cb is a crucial direct target of YAP, through which the YAP activates phosphoinositol-3-kinase-AKT pathway and regulates cardiomyocyte proliferation and survival. © 2014 American Heart Association, Inc.

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.

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

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

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

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

Deficiency of insulin-like growth factor 1 reduces vulnerability to chronic alcohol intake-induced cardiomyocyte mechanical dysfunction: role of AMPK.

PubMed

Ge, Wei; Li, Qun; Turdi, Subat; Wang, Xiao-Ming; Ren, Jun

2011-08-01

Circulating insulin-like growth factor I (IGF-1) levels are closely associated with cardiac performance although the role of IGF-1 in alcoholic cardiac dysfunction is unknown. This study was designed to evaluate the impact of severe liver IGF-1 deficiency (LID) on chronic alcohol-induced cardiomyocyte contractile and intracellular Ca(2+) dysfunction. Adult male C57 and LID mice were placed on a 4% alcohol diet for 15 weeks. Cardiomyocyte contractile and intracellular Ca(2+) properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-relengthening (TR(90) ), change in fura-fluorescence intensity (ΔFFI) and intracellular Ca(2+) decay. Levels of apoptotic regulators caspase-3, Bcl-2 and c-Jun NH2-terminal kinase (JNK), the ethanol metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), as well as the cellular fuel gauge AMP-activated protein kinase (AMPK) were evaluated. Chronic alcohol intake enlarged myocyte cross-sectional area, reduced PS, ± dL/dt and ΔFFI as well as prolonged TR(90) and intracellular Ca(2+) decay, the effect of which was greatly attenuated by IGF-1 deficiency. The beneficial effect of LID against alcoholic cardiac mechanical defect was ablated by IGF-1 replenishment. Alcohol intake increased caspase-3 activity/expression although it down-regulated Bcl-2, ALDH2 and pAMPK without affecting JNK and AMPK. IGF-1 deficiency attenuated alcoholism-induced responses in all these proteins with the exception of Bcl-2. In addition, the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside abrogated short-term ethanol incubation-elicited cardiac mechanical dysfunction. Taken together, these data suggested that IGF-1 deficiency may reduce the sensitivity to ethanol-induced myocardial mechanical dysfunction. Our data further depicted a likely role of Caspase-3, ALDH2 and AMPK activation in IGF-1 deficiency induced 'desensitization' of alcoholic cardiomyopathy. © 2011 The

Increased expression of 78 kD glucose-regulated protein promotes cardiomyocyte apoptosis in a rat model of liver cirrhosis

PubMed Central

Zhang, Lili; Zhang, Huiying; Lv, Minli; Jia, Jiantao; Fan, Yimin; Tian, Xiaoxia; Li, Xujiong; Li, Baohong; Ji, Jingquan; Wang, Limin; Zhao, Zhongfu; Han, Dewu; Ji, Cheng

2015-01-01

Aims: This study was to investigate the role and underlying mechanism of 78 kD glucose-regulated protein (GRP78) in cardiomyocyte apoptosis in a rat model of liver cirrhosis. Methods: A rat model of liver cirrhosis was established with multiple pathogenic factors. A total of 42 male SD rats were randomly divided into the liver cirrhosis group and control group. Cardiac structure analysis was performed to assess alterations in cardiac structure. Cardiomyocytes apoptosis was detected by TdT-mediated dUTP nick end labeling method. Expression of GRP78, CCAAT/enhancer-binding protein homologous protein (CHOP), caspase-12, nuclear factor kappa-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65) and B cell lymphoma-2 (Bcl-2) was detected by immunohistochemical staining. Results: The ratios of left ventricular wall thickness to heart weight and heart weight to body weight were significantly increased with the progression of liver cirrhosis (P < 0.05). Apoptosis index of cardiomyocytes was significantly increased with the progression of liver cirrhosis (P < 0.05). The expression levels of GRP78, CHOP and caspase-12 were significantly increased in the progression of liver cirrhosis (P < 0.05). The expression levels of NF-κB p65 and Bcl-2 were highest in the 4-wk liver cirrhosis, and they were decreased in the 6-wk and 8-wk in the progression of liver cirrhosis. GRP78 expression levels were positively correlated with apoptosis index, CHOP and caspase-12 expression levels (P < 0.05). CHOP expression levels were negatively correlated with NF-κB p65 and Bcl-2 expression levels (P < 0.05). Conclusion: Increased expression of GRP78 promotes cardiomyocyte apoptosis in rats with cirrhotic cardiomyopathy. PMID:26464674

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.

Stimulus-dependent regulation of nuclear Ca2+ signaling in cardiomyocytes: a role of neuronal calcium sensor-1.

PubMed

Nakao, Shu; Wakabayashi, Shigeo; Nakamura, Tomoe Y

2015-01-01

In cardiomyocytes, intracellular calcium (Ca2+) transients are elicited by electrical and receptor stimulations, leading to muscle contraction and gene expression, respectively. Although such elevations of Ca2+levels ([Ca2+]) also occur in the nucleus, the precise mechanism of nuclear [Ca2+] regulation during different kinds of stimuli, and its relationship with cytoplasmic [Ca2+] regulation are not fully understood. To address these issues, we used a new region-specific fluorescent protein-based Ca2+ indicator, GECO, together with the conventional probe Fluo-4 AM. We confirmed that nuclear Ca2+ transients were elicited by both electrical and receptor stimulations in neonatal mouse ventricular myocytes. Kinetic analysis revealed that electrical stimulation-elicited nuclear Ca2+ transients are slower than cytoplasmic Ca2+ transients, and chelating cytoplasmic Ca2+ abolished nuclear Ca2+ transients, suggesting that nuclear Ca2+ are mainly derived from the cytoplasm during electrical stimulation. On the other hand, receptor stimulation such as with insulin-like growth factor-1 (IGF-1) preferentially increased nuclear [Ca2+] compared to cytoplasmic [Ca2+]. Experiments using inhibitors revealed that electrical and receptor stimulation-elicited Ca2+ transients were mainly mediated by ryanodine receptors and inositol 1,4,5-trisphosphate receptors (IP3Rs), respectively, suggesting different mechanisms for the two signals. Furthermore, IGF-1-elicited nuclear Ca2+ transient amplitude was significantly lower in myocytes lacking neuronal Ca2+ sensor-1 (NCS-1), a Ca2+ binding protein implicated in IP3R-mediated pathway in the heart. Moreover, IGF-1 strengthened the interaction between NCS-1 and IP3R. These results suggest a novel mechanism for receptor stimulation-induced nuclear [Ca2+] regulation mediated by IP3R and NCS-1 that may further fine-tune cardiac Ca2+ signal regulation.

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

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

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

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.

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.

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

PubMed

Wang, Wei Eric; Li, Liangpeng; Xia, Xuewei; Fu, Wenbin; Liao, Qiao; Lan, Cong; Yang, Dezhong; Chen, Hongmei; Yue, Rongchuan; Zeng, Cindy; Zhou, Lin; Zhou, Bin; Duan, Dayue Darrel; Chen, Xiongwen; Houser, Steven R; Zeng, Chunyu

2017-08-29

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca 2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca 2+ -dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca 2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca 2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia

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

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

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

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

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.

Insulin stimulates mitochondrial fusion and function in cardiomyocytes via the Akt-mTOR-NFκB-Opa-1 signaling pathway.

PubMed

Parra, Valentina; Verdejo, Hugo E; Iglewski, Myriam; Del Campo, Andrea; Troncoso, Rodrigo; Jones, Deborah; Zhu, Yi; Kuzmicic, Jovan; Pennanen, Christian; Lopez-Crisosto, Camila; Jaña, Fabián; Ferreira, Jorge; Noguera, Eduard; Chiong, Mario; Bernlohr, David A; Klip, Amira; Hill, Joseph A; Rothermel, Beverly A; Abel, Evan Dale; Zorzano, Antonio; Lavandero, Sergio

2014-01-01

Insulin regulates heart metabolism through the regulation of insulin-stimulated glucose uptake. Studies have indicated that insulin can also regulate mitochondrial function. Relevant to this idea, mitochondrial function is impaired in diabetic individuals. Furthermore, the expression of Opa-1 and mitofusins, proteins of the mitochondrial fusion machinery, is dramatically altered in obese and insulin-resistant patients. Given the role of insulin in the control of cardiac energetics, the goal of this study was to investigate whether insulin affects mitochondrial dynamics in cardiomyocytes. Confocal microscopy and the mitochondrial dye MitoTracker Green were used to obtain three-dimensional images of the mitochondrial network in cardiomyocytes and L6 skeletal muscle cells in culture. Three hours of insulin treatment increased Opa-1 protein levels, promoted mitochondrial fusion, increased mitochondrial membrane potential, and elevated both intracellular ATP levels and oxygen consumption in cardiomyocytes in vitro and in vivo. Consequently, the silencing of Opa-1 or Mfn2 prevented all the metabolic effects triggered by insulin. We also provide evidence indicating that insulin increases mitochondrial function in cardiomyocytes through the Akt-mTOR-NFκB signaling pathway. These data demonstrate for the first time in our knowledge that insulin acutely regulates mitochondrial metabolism in cardiomyocytes through a mechanism that depends on increased mitochondrial fusion, Opa-1, and the Akt-mTOR-NFκB pathway.

Insulin Stimulates Mitochondrial Fusion and Function in Cardiomyocytes via the Akt-mTOR-NFκB-Opa-1 Signaling Pathway

PubMed Central

Parra, Valentina; Verdejo, Hugo E.; Iglewski, Myriam; del Campo, Andrea; Troncoso, Rodrigo; Jones, Deborah; Zhu, Yi; Kuzmicic, Jovan; Pennanen, Christian; Lopez‑Crisosto, Camila; Jaña, Fabián; Ferreira, Jorge; Noguera, Eduard; Chiong, Mario; Bernlohr, David A.; Klip, Amira; Hill, Joseph A.; Rothermel, Beverly A.; Abel, Evan Dale; Zorzano, Antonio; Lavandero, Sergio

2014-01-01

Insulin regulates heart metabolism through the regulation of insulin-stimulated glucose uptake. Studies have indicated that insulin can also regulate mitochondrial function. Relevant to this idea, mitochondrial function is impaired in diabetic individuals. Furthermore, the expression of Opa-1 and mitofusins, proteins of the mitochondrial fusion machinery, is dramatically altered in obese and insulin-resistant patients. Given the role of insulin in the control of cardiac energetics, the goal of this study was to investigate whether insulin affects mitochondrial dynamics in cardiomyocytes. Confocal microscopy and the mitochondrial dye MitoTracker Green were used to obtain three-dimensional images of the mitochondrial network in cardiomyocytes and L6 skeletal muscle cells in culture. Three hours of insulin treatment increased Opa-1 protein levels, promoted mitochondrial fusion, increased mitochondrial membrane potential, and elevated both intracellular ATP levels and oxygen consumption in cardiomyocytes in vitro and in vivo. Consequently, the silencing of Opa-1 or Mfn2 prevented all the metabolic effects triggered by insulin. We also provide evidence indicating that insulin increases mitochondrial function in cardiomyocytes through the Akt-mTOR-NFκB signaling pathway. These data demonstrate for the first time in our knowledge that insulin acutely regulates mitochondrial metabolism in cardiomyocytes through a mechanism that depends on increased mitochondrial fusion, Opa-1, and the Akt-mTOR-NFκB pathway. PMID:24009260

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.

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.

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

Calcineurin B homologous protein 3 negatively regulates cardiomyocyte hypertrophy via inhibition of glycogen synthase kinase 3 phosphorylation.

PubMed

Kobayashi, Soushi; Nakamura, Tomoe Y; Wakabayashi, Shigeo

2015-07-01

Cardiac hypertrophy is a leading cause of serious heart diseases. Although many signaling molecules are involved in hypertrophy, the functions of some proteins in this process are still unknown. Calcineurin B homologous protein 3 (CHP3)/tescalcin is an EF-hand Ca(2+)-binding protein that is abundantly expressed in the heart; however, the function of CHP3 is unclear. Here, we aimed to identify the cardiac functions of CHP3. CHP3 was expressed in hearts at a wide range of developmental stages and was specifically detected in neonatal rat ventricular myocytes (NRVMs) but not in cardiac fibroblasts in culture. Moreover, knockdown of CHP3 expression using adenoviral-based RNA interference in NRVMs resulted in enlargement of cardiomyocyte size, concomitant with increased expression of a pathological hypertrophy marker ANP. This same treatment elevated glycogen synthase kinase (GSK3α/β) phosphorylation, which is known to inhibit GSK3 function. In contrast, CHP3 overexpression blocked the insulin-induced phosphorylation of GSK3α/β without affecting the phosphorylation of Akt, which is an upstream kinase of GSK3α/β, in HEK293 cells, and it inhibited both IGF-1-induced phosphorylation of GSK3β and cardiomyocyte hypertrophy in NRVMs. Co-immunoprecipitation experiments revealed that GSK3β interacted with CHP3. However, a Ca(2+)-binding-defective mutation of CHP3 (CHP3-D123A) also interacted with GSK3β and had the same inhibitory effect on GSK3α/β phosphorylation, suggesting that the action of CHP3 was independent of Ca(2+). These findings suggest that CHP3 functions as a novel negative regulator of cardiomyocyte hypertrophy via inhibition of GSK3α/β phosphorylation and subsequent enzymatic activation of GSK3α/β. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cardiac-specific overexpression of insulin-like growth factor I (IGF-1) rescues lipopolysaccharide-induced cardiac dysfunction and activation of stress signaling in murine cardiomyocytes.

PubMed

Zhao, Peng; Turdi, Subat; Dong, Feng; Xiao, Xiaoyan; Su, Guohai; Zhu, Xinglei; Scott, Glenda I; Ren, Jun

2009-07-01

Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, plays a key role in cardiac dysfunction in sepsis. Low circulating levels of insulin-like growth factor 1 (IGF-1) are found in sepsis, although the influence of IGF-1 on septic cardiac defect is unknown. This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca2+ dysfunction, activation of stress signal and endoplasmic reticulum (ER) stress. Mechanical and intracellular Ca2+ properties were examined in cardiomyocytes from Fast Violet B and cardiac-specific IGF-1 overexpression mice treated with or without LPS (4 mg kg(-1), 6 h). Reactive oxygen species (ROS), protein carbonyl formation and apoptosis were measured. Activation of mitogen-activated protein kinase pathways (p38, c-jun N-terminal kinase [JNK] and extracellular signal-related kinase [ERK]), ER stress and apoptotic markers were evaluated using Western blot analysis. Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening and prolonged duration of relengthening in LPS-treated Fast Violet B cardiomyocytes associated with reduced intracellular Ca2+ decay. Accumulation of ROS protein carbonyl and apoptosis were elevated after LPS treatment. Western blot analysis revealed activated p38 and JNK, up-regulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2. Total protein expression of p38, JNK, and ERK was unaffected by either LPS or IGF-1. Interestingly, these LPS-induced changes in mechanical and intracellular Ca2+ properties, ROS, protein carbonyl, apoptosis, stress signal activation, and ER stress markers were effectively ablated by IGF-1. In vitro LPS exposure (1 microg mL(-1)) produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting, which was alleviated by exogenous IGF-1 (50 nM). These data collectively suggested a beneficial of IGF-1 in

Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction.

PubMed

Ngkelo, Anta; Richart, Adèle; Kirk, Jonathan A; Bonnin, Philippe; Vilar, Jose; Lemitre, Mathilde; Marck, Pauline; Branchereau, Maxime; Le Gall, Sylvain; Renault, Nisa; Guerin, Coralie; Ranek, Mark J; Kervadec, Anaïs; Danelli, Luca; Gautier, Gregory; Blank, Ulrich; Launay, Pierre; Camerer, Eric; Bruneval, Patrick; Menasche, Philippe; Heymes, Christophe; Luche, Elodie; Casteilla, Louis; Cousin, Béatrice; Rodewald, Hans-Reimer; Kass, David A; Silvestre, Jean-Sébastien

2016-06-27

Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit-independent MC-deficient (Cpa3(Cre/+)) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca(2+) desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force-Ca(2+) interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators. ©2016 Ngkelo et al.

Mast cells regulate myofilament calcium sensitization and heart function after myocardial infarction

PubMed Central

Richart, Adèle; Vilar, Jose; Lemitre, Mathilde; Marck, Pauline; Branchereau, Maxime; Guerin, Coralie; Gautier, Gregory; Blank, Ulrich; Heymes, Christophe; Luche, Elodie; Cousin, Béatrice; Rodewald, Hans-Reimer

2016-01-01

Acute myocardial infarction (MI) is a severe ischemic disease responsible for heart failure and sudden death. Inflammatory cells orchestrate postischemic cardiac remodeling after MI. Studies using mice with defective mast/stem cell growth factor receptor c-Kit have suggested key roles for mast cells (MCs) in postischemic cardiac remodeling. Because c-Kit mutations affect multiple cell types of both immune and nonimmune origin, we addressed the impact of MCs on cardiac function after MI, using the c-Kit–independent MC-deficient (Cpa3Cre/+) mice. In response to MI, MC progenitors originated primarily from white adipose tissue, infiltrated the heart, and differentiated into mature MCs. MC deficiency led to reduced postischemic cardiac function and depressed cardiomyocyte contractility caused by myofilament Ca2+ desensitization. This effect correlated with increased protein kinase A (PKA) activity and hyperphosphorylation of its targets, troponin I and myosin-binding protein C. MC-specific tryptase was identified to regulate PKA activity in cardiomyocytes via protease-activated receptor 2 proteolysis. This work reveals a novel function for cardiac MCs modulating cardiomyocyte contractility via alteration of PKA-regulated force–Ca2+ interactions in response to MI. Identification of this MC-cardiomyocyte cross-talk provides new insights on the cellular and molecular mechanisms regulating the cardiac contractile machinery and a novel platform for therapeutically addressable regulators. PMID:27353089

Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis.

PubMed

Angeloni, Cristina; Turroni, Silvia; Bianchi, Laura; Fabbri, Daniele; Motori, Elisa; Malaguti, Marco; Leoncini, Emanuela; Maraldi, Tullia; Bini, Luca; Brigidi, Patrizia; Hrelia, Silvana

2013-01-01

Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.

[Octanol preconditioning alleviates mouse cardiomyocyte swelling induced by simulated ischemia/reperfusion challenge in vitro].

PubMed

Luo, Yukun; Fang, Jun; Fan, Lin; Lin, Chaogui; Chen, Zhaoyang; Chen, Lianglong

2012-10-01

To investigate the role of connexin 43-formed hemichannels in cell volume regulation induced by simulated ischemia/reperfusion (SI/R). Mouse cardiomyocytes isolated on a Langendorff apparatus with enzyme solution were aliquoted into control, SI/R and SI/R +octanol groups. Calcein-AM was used to stain the cells and the cell volume was measured with confocal microscope by stack scanning. Trypan blue was used to measure the cell viability after the treatments. Calcein-AM staining and cofocal microscopy yielded stable and reproducible results for cell volume measurement. Mouse cardiomyocytes subjected to simulated SI/R showed obvious cell swelling as compared with the control cells [(126∓6)% vs 100%, P<0.05], and octanol preconditioning significantly attenuated the cell swelling [(113∓6)%, P<0.05]. SI/R caused a significant reduction of the cell viability compared to the control cells [(19∓2)% vs (45∓3)%, P<0.01], and octanol preconditioning obviously reduced the viability of the cells with SI/R challenge [(31∓2)%, P<0.01]. Connexin 43-formed hemichannels are involved in the regulation of cardiomyocyte volumes induced by SI/R challenge, and octanol can alleviate the cell swelling to enhance the viability of the cardiomyocytes following SI/R.

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

Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes

PubMed Central

Yasui, Taku; Masaki, Takeshi; Arita, Yoh; Ishibashi, Tomohiko; Inagaki, Tadakatsu; Okazawa, Makoto; Oka, Toru; Shioyama, Wataru; Yamauchi-Takihara, Keiko; Komuro, Issei; Sakata, Yasushi; Nakaoka, Yoshikazu

2016-01-01

Grb2-associated binder (Gab) docking proteins regulate signals downstream of a variety of growth factors and receptor tyrosine kinases. Neuregulin-1 (NRG-1), a member of epidermal growth factor family, plays a critical role for cardiomyocyte proliferation and prevention of heart failure via ErbB receptors. We previously reported that Gab1 and Gab2 in the myocardium are essential for maintenance of myocardial function in the postnatal heart via transmission of NRG-1/ErbB-signaling through analysis of Gab1/Gab2 cardiomyocyte-specific double knockout mice. In that study, we also found that there is an unknown high-molecular weight (high-MW) Gab1 isoform (120 kDa) expressed exclusively in the heart, in addition to the ubiquitously expressed low-MW (100 kDa) Gab1. However, the high-MW Gab1 has been molecularly ill-defined to date. Here, we identified the high-MW Gab1 as a striated muscle-specific isoform. The high-MW Gab1 has an extra exon encoding 27 amino acid residues between the already-known 3rd and 4th exons of the ubiquitously expressed low-MW Gab1. Expression analysis by RT-PCR and immunostaining with the antibody specific for the high-MW Gab1 demonstrate that the high-MW Gab1 isoform is exclusively expressed in striated muscle including heart and skeletal muscle. The ratio of high-MW Gab1/ total Gab1 mRNAs increased along with heart development. The high-MW Gab1 isoform in heart underwent tyrosine-phosphorylation exclusively after intravenous administration of NRG-1, among several growth factors. Adenovirus-mediated overexpression of the high-MW Gab1 induces more sustained activation of AKT after stimulation with NRG-1 in cardiomyocytes compared with that of β-galactosidase. On the contrary, siRNA-mediated knockdown of the high-MW Gab1 significantly attenuated AKT activation after stimulation with NRG-1 in cardiomyocytes. Taken together, these findings suggest that the striated muscle-specific high-MW isoform of Gab1 has a crucial role for NRG-1/ErbB signaling

Molecular Characterization of Striated Muscle-Specific Gab1 Isoform as a Critical Signal Transducer for Neuregulin-1/ErbB Signaling in Cardiomyocytes.

PubMed

Yasui, Taku; Masaki, Takeshi; Arita, Yoh; Ishibashi, Tomohiko; Inagaki, Tadakatsu; Okazawa, Makoto; Oka, Toru; Shioyama, Wataru; Yamauchi-Takihara, Keiko; Komuro, Issei; Sakata, Yasushi; Nakaoka, Yoshikazu

2016-01-01

Grb2-associated binder (Gab) docking proteins regulate signals downstream of a variety of growth factors and receptor tyrosine kinases. Neuregulin-1 (NRG-1), a member of epidermal growth factor family, plays a critical role for cardiomyocyte proliferation and prevention of heart failure via ErbB receptors. We previously reported that Gab1 and Gab2 in the myocardium are essential for maintenance of myocardial function in the postnatal heart via transmission of NRG-1/ErbB-signaling through analysis of Gab1/Gab2 cardiomyocyte-specific double knockout mice. In that study, we also found that there is an unknown high-molecular weight (high-MW) Gab1 isoform (120 kDa) expressed exclusively in the heart, in addition to the ubiquitously expressed low-MW (100 kDa) Gab1. However, the high-MW Gab1 has been molecularly ill-defined to date. Here, we identified the high-MW Gab1 as a striated muscle-specific isoform. The high-MW Gab1 has an extra exon encoding 27 amino acid residues between the already-known 3rd and 4th exons of the ubiquitously expressed low-MW Gab1. Expression analysis by RT-PCR and immunostaining with the antibody specific for the high-MW Gab1 demonstrate that the high-MW Gab1 isoform is exclusively expressed in striated muscle including heart and skeletal muscle. The ratio of high-MW Gab1/ total Gab1 mRNAs increased along with heart development. The high-MW Gab1 isoform in heart underwent tyrosine-phosphorylation exclusively after intravenous administration of NRG-1, among several growth factors. Adenovirus-mediated overexpression of the high-MW Gab1 induces more sustained activation of AKT after stimulation with NRG-1 in cardiomyocytes compared with that of β-galactosidase. On the contrary, siRNA-mediated knockdown of the high-MW Gab1 significantly attenuated AKT activation after stimulation with NRG-1 in cardiomyocytes. Taken together, these findings suggest that the striated muscle-specific high-MW isoform of Gab1 has a crucial role for NRG-1/ErbB signaling

Comparative analysis of cardiomyocyte differentiation from human embryonic stem cells under 3-D and 2-D culture conditions.

PubMed

Pal, Rajarshi; Mamidi, Murali Krishna; Das, Anjan Kumar; Bhonde, Ramesh

2013-02-01

Post-myocardial infarction cardiomyocytes are the most important target cell types for cardiac repair. Many of the applications envisaged for human embryonic stem cells (hESC)-derived cardiomyocytes demand that the differentiation procedure be robust, cost effective and high yielding. Various lines of evidence including our earlier study suggest that hESCs have distinct preferences to become heart cells. However, a direct comparison between different protocols has not yet been reported to date. Here, we performed a logical and systematic comparison of cardiomyocytes obtained from hESCs via embryoid bodies (EBs) in suspension versus adherent static cultures of feeder-free hES colonies representing three-dimensional (3-D) and two-dimensional (2-D) culture systems, respectively. An in-depth characterization of the beating cells revealed appropriate cardiac marker expression both at gene and protein levels. Despite using similar media, 3-D and 2-D cultures showed significant variation in growth and ability to form beating areas. While the expression of pre-cardiac mesoderm markers like GATA-4, HAND1, Myf5, Msx1, and BMP-IIR remained unaltered; levels of functional heart-specific markers such as MLC-2A/2V, cTnT, ANP, Phospholamban, α-MHC and KV4.3 were substantially up-regulated in 3-D compared to 2-D cultures. Concurrently we observed a sharp decline in the expression of ESC, ectoderm and endoderm markers including Oct-4, Sox-2, NFH, Sox-1, Sox-17 and AFP. Further immunocytochemistry and flow cytometry demonstrated a higher percentage of cells positive for Brachyury, desmin and cardiac troponin in 3-D cultures. Our results underscore the higher efficiency of cardiomyocytes derived via 3-D cultures. This finding enriches our basic understanding of the differentiation pattern in hESC-derived cardiomyocytes. Copyright © 2012 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Basal and β-adrenergic cardiomyocytes contractility dysfunction induced by dietary protein restriction is associated with downregulation of SERCA2a expression and disturbance of endoplasmic reticulum Ca2+ regulation in rats.

PubMed

Penitente, Arlete R; Novaes, Rômulo D; Silva, Marcelo E; Silva, Márcia F; Quintão-Júnior, Judson F; Guatimosim, Silvia; Cruz, Jader S; Chianca, Deoclécio A; Natali, Antônio J; Neves, Clóvis A

2014-01-01

The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR) are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20) and a protein-restricted group (PRG, n = 20), receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV) were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca(2+)sparks analysis. PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca(2+)sparks were observed in PRG cardiomyocytes. The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca(2+) intracellular kinetics. © 2014 S. Karger AG, Basel.

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

CYP epoxygenase 2J2 prevents cardiac fibrosis by suppression of transmission of pro-inflammation from cardiomyocytes to macrophages

PubMed Central

Yang, Lei; Ni, Li; Duan, Quanlu; Wang, Xingxu; Chen, Chen; Chen, Song; Chaugai, Sandip; Zeldin, D.C.; Tang, Jia Rong; Wang, Dao Wen

2017-01-01

Cytochrome P450 epoxygenase (CYP450)-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; but the underlying mechanism remains unclear. The present study aimed to elucidate how EETs regulated cardiac fibrosis in response to isoprenaline (Iso) or angiotensin (Ang) II. Cardiac-specific human CYP2J2 transgenic mice (Tr) and wild-type (WT) C57BL/6 littermates were infused with Iso- or Ang II. Two weeks after infusion, Tr mice showed more alleviative cardiac fibrosis and inflammation compared with WT mice. In vitro, we found Iso or Ang II induced nuclear transfer of NF-κB p65 and inflammatory cytokines expression in cardiomyocytes. Furthermore, inflammation response emerged in macrophages cultured in cardiomyocytes-conditioned medium. When pretreatment with 14,15-EET in cardiomyocytes, the inflammatory response was markedly suppressed and the transmission of inflammation from cardiomyocytes to macrophages was reduced. In conclusion, CYP2J2 and EETs prevent cardiac fibrosis and cardiac dysfunction by suppressing transmission of pro-inflammation from cardiomyocytes to macrophages in heart, suggesting that elevation of EETs level could be a potential strategy to prevent cardiac fibrosis. PMID:25686540

Identification of Fat Mass and Obesity Associated (FTO) Protein Expression in Cardiomyocytes: Regulation by Leptin and Its Contribution to Leptin-Induced Hypertrophy

PubMed Central

Gan, Xiaohong Tracey; Zhao, Ganjian; Huang, Cathy X.; Rowe, Adrianna C.; Purdham, Daniel M.; Karmazyn, Morris

2013-01-01

The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the

Identification of fat mass and obesity associated (FTO) protein expression in cardiomyocytes: regulation by leptin and its contribution to leptin-induced hypertrophy.

PubMed

Gan, Xiaohong Tracey; Zhao, Ganjian; Huang, Cathy X; Rowe, Adrianna C; Purdham, Daniel M; Karmazyn, Morris

2013-01-01

The recently-identified fat mass and obesity-associated (FTO) protein is associated with various physiological functions including energy and body weight regulation. Ubiquitously expressed, FTO was identified in heart homogenates although its function is unknown. We studied whether FTO is specifically expressed within the cardiac myocyte and its potential role pertaining to the hypertrophic effect of the adipokine leptin. Most experiments were performed using cultured neonatal rat cardiomyocytes which showed nuclei-specific FTO expression. Leptin significantly increased FTO expression which was associated with myocyte hypertrophy although both events were abrogated by FTO knockdown with siRNA. Administration of a leptin receptor antibody to either normal or obese rats significant reduced myocardial FTO protein expression. Responses in cardiomyocytes were accompanied by JAK2/STAT3 activation whereas JAK2/STAT3 inhibition abolished these effects. Expression of the cut-like homeobox 1(CUX1) transcriptional factor was significantly increased by leptin although this was restricted to the cathepsin L-dependent, proteolytically-derived shorter p110CUX1 isoform whereas the longer p200CUX1 protein was not significantly affected. Cathepsin L expression and activity were both significantly increased by leptin whereas a cathepsin L peptide inhibitor or siRNA specific for CUX1 completely prevented the leptin-induced increase in FTO expression. The cathepsin L peptide inhibitor or siRNA-induced knockdown of either CUX1 or FTO abrogated the hypertrophic response to leptin. Two other pro-hypertrophic factors, endothelin-1 or angiotensin II had no effect on FTO expression and FTO knockdown did not alter the hypertrophic response to either agent. This study demonstrates leptin-induced FTO upregulation in cardiomyocytes via JAK2/STAT3- dependent CUX1 upregulation and suggests an FTO regulatory function of leptin. It also demonstrates for the first time a functional role of FTO in the

Contribution of α-smooth muscle actin and extracellular matrix to the in vitro reorganization of cardiomyocyte contractile system.

PubMed

Bildyug, Natalya; Bozhokina, Ekaterina; Khaitlina, Sofia

2016-04-01

Cardiomyocytes in culture undergo reversible rearrangement of their contractile apparatus with the conversion of typical myofibrils into the structures of non-muscle type and the loss of contractility. Along with these transformations, the cardiomyocytes gain the capacity to synthesize extracellular matrix. Here we show that during cultivation of rat neonatal cardiomyocytes, the inherent α-cardiac actin isoform is transiently replaced by α-smooth-muscle actin, whose expression is accompanied by transformation of myofibrils into stress-fiber-like structures. The following down-regulation of α-smooth muscle actin parallels restoration of myofibrillar system and correlates with the accumulation of extracellular collagen and laminin, initially missing from the cardiomyocytes culture. © 2016 International Federation for Cell Biology.

Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-{beta} family signaling

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

Nomura, Tetsuya; Department of Cardiovascular Medicine, Kyoto Prefectural University of Medicine, Kyoto 602-8566; Ueyama, Tomomi

2008-01-25

The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-{beta} family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activinmore » A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle.« less

Cardiomyocyte exosomes regulate glycolytic flux in endothelium by direct transfer of GLUT transporters and glycolytic enzymes.

PubMed

Garcia, Nahuel A; Moncayo-Arlandi, Javier; Sepulveda, Pilar; Diez-Juan, Antonio

2016-03-01

Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship, which is essential for maintaining the metabolic requirements of the heart. Little is known about the mechanisms that regulate nutrient flow from ECs to associated CMs, especially in situations of acute stress when local active processes are required to regulate endothelial transport. We examined whether CM-derived exosomes can modulate glucose transport and metabolism in ECs. In conditions of glucose deprivation, CMs increase the synthesis and secretion of exosomes. These exosomes are loaded with functional glucose transporters and glycolytic enzymes, which are internalized by ECs, leading to increased glucose uptake, glycolytic activity, and pyruvate production in recipient cells. These findings establish CM-derived exosomes as key components of the cardio-endothelial communication system which, through intercellular protein complementation, would allow a rapid response from ECs to increase glucose transport and a putative uptake of metabolic fuels from blood to CMs. This CM-EC protein complementation process might have implications for metabolic regulation in health and disease. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.

[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

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

Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

PubMed

Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

2014-04-01

In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin. © 2013 Wiley Periodicals, Inc.

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

PubMed

Li, Xiao-Li; Zeng, Di; Chen, Yan; Ding, Lu; Li, Wen-Ju; Wei, Ting; Ou, Dong-Bo; Yan, Song; Wang, Bin; Zheng, Qiang-Sun

2017-02-01

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α 1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α 2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α 1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. These results demonstrate that activation of ARs, particularly of α 1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling. © 2016 John Wiley & Sons Ltd.

Plant Growth Regulators.

ERIC Educational Resources Information Center

Nickell, Louis G.

1978-01-01

Describes the effect of "plant growth regulators" on plants, such as controlling the flowering, fruit development, plant size, and increasing crop yields. Provides a list of plant growth regulators which includes their chemical, common, and trade names, as well as their different use(s). (GA)

miR-138 protects cardiomyocytes from hypoxia-induced apoptosis via MLK3/JNK/c-jun pathway

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

He, Siyi; Liu, Peng; Jian, Zhao

2013-11-29

Highlights: •First time to find miR-138 is up-regulated in hypoxic cardiomyocytes. •First time to find miR-138 targets MLK3 and regulates JNK/c-jun pathway. •Rare myocardial biopsy of patients with CHD were collected. •Both silence and overexpression of miR-138 were implemented. •Various methods were used to detect cell function. -- Abstract: Cardiomyocytes experience a series of complex endogenous regulatory mechanisms against apoptosis induced by chronic hypoxia. MicroRNAs are a class of endogenous small non-coding RNAs that regulate cellular pathophysiological processes. Recently, microRNA-138 (miR-138) has been found related to hypoxia, and beneficial for cell proliferation. Therefore, we intend to study the role ofmore » miR-138 in hypoxic cardiomyocytes and the main mechanism. Myocardial samples of patients with congenital heart disease (CHD) were collected to test miR-138 expression. Agomir or antagomir of miR-138 was transfected into H9C2 cells to investigate its effect on cell apoptosis. Higher miR-138 expression was observed in patients with cyanotic CHD, and its expression gradually increased with prolonged hypoxia time in H9C2 cells. Using MTT and LDH assays, cell growth was significantly greater in the agomir group than in the negative control (NC) group, while antagomir decreased cell survival. Dual luciferase reporter gene and Western-blot results confirmed MLK3 was a direct target of miR-138. It was found that miR-138 attenuated hypoxia-induced apoptosis using TUNEL, Hoechst staining and Annexin V-PE/7-AAD flow cytometry analysis. We further detected expression of apoptosis-related proteins. In the agomir group, the level of pro-apoptotic proteins such as cleaved-caspase-3, cleaved-PARP and Bad significantly reduced, while Bcl-2 and Bcl-2/Bax ratio increased. Opposite changes were observed in the antagomir group. Downstream targets of MLK3, JNK and c-jun, were also suppressed by miR-138. Our study demonstrates that up-regulation of mi

Analysis of proteome changes in doxorubicin-treated adult rat cardiomyocyte

PubMed Central

Kumar, Suresh N.; Konorev, Eugene A.; Aggarwal, Deepika; Kalyanaraman, Balaraman

2011-01-01

Doxorubicin-induced cardiomyopathy in cancer patients is well established. The proposed mechanism of cardiac damage includes generation of reactive oxygen species, mitochondrial dysfunction and cardiomyocyte apoptosis. Exposure of adult rat cardiomyocytes to low levels of DOX for 48 h induced apoptosis. Analysis of protein expression showed a differential regulation of several key proteins including the voltage dependent anion selective channel protein 2 and methylmalonate semialdehyde dehydrogenase. In comparison, proteomic evaluation of DOX-treated rat heart showed a slightly different set of protein changes that suggests nuclear accumulation of DOX. Using a new solubilization technique, changes in low abundant protein profiles were monitored. Altered protein expression, modification and function related to oxidative stress response may play an important role in DOX cardiotoxicity. PMID:21338723

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

Ischemia-reperfusion injury and pregnancy initiate time-dependent and robust signs of up-regulation of cardiac progenitor cells.

PubMed

Genead, Rami; Fischer, Helene; Hussain, Alamdar; Jaksch, Marie; Andersson, Agneta B; Ljung, Karin; Bulatovic, Ivana; Franco-Cereceda, Anders; Elsheikh, Elzafir; Corbascio, Matthias; Smith, C I Edvard; Sylvén, Christer; Grinnemo, Karl-Henrik

2012-01-01

To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease.

Recent advances on the role of long non-coding RNA H19 in regulating mammalian muscle growth and development.

PubMed

Qin, Chen Yu; Cai, He; Qing, Han Rui; Li, Li; Zhang, Hong Ping

2017-12-20

As one of the first identified long non-coding RNAs (lncRNAs), H19 plays a wide range of roles in vivo, including not only as a tumor suppressor and oncogene involved in disease process, but also as a regulator of growth and development of multiple tissues in mammalian embryos. The function of H19 in muscles (both skeletal and cardiac muscle) draws widespread attention due to the following two reasons. On one hand, H19 promotes myogenic differentiation and myogenesis of skeletal muscle satellite cells (SMSCs) via regulating Igf2 in cis. On the other hand, H19 also modulates the target genes in trans, including sponging let-7, miR-106 or miR-29 to mediate myocyte glucose uptake, cardiomyocyte proliferation and tendon repair, as well as promote embryonic development and muscle regeneration through binding to MBD1 as a chromatin modifier. In this review, we summarize the role of H19 in mammalian muscles, which will provide a reference for further research to unveil the molecular mechanism of muscle growth and development.

Role of Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy

PubMed Central

Miller, Clint L.; Oikawa, Masayoshi; Cai, Yujun; Wojtovich, Andrew P.; Nagel, David J.; Xu, Xiangbin; Xu, Haodong; Florio, Vince; Rybalkin, Sergei D.; Beavo, Joseph A.; Chen, Yiu-Fai; Li, Jian-Dong; Blaxall, Burns C.; Abe, Jun-ichi; Yan, Chen

2009-01-01

Rationale Cyclic nucleotide phosphodiesterases (PDE) through the degradation of second messenger cyclic guanosine monophosphate (cGMP) play critical roles in maintaining cardiomyocyte homeostasis. Ca2+/CaM-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca2+/CaM and cGMP signaling, however its function in cardiomyocytes is unknown. Objective Herein we investigate the role of Ca2+/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes (NRVM and ARVM) and in the heart in vivo. Methods and Results Inhibition of PDE1 activity using a PDE1 selective inhibitor IC86340 or downregulation of PDE1A using siRNA prevented phenylephrine (PE) induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal (NRVM) and adult (ARVM) rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic ISO infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts, however PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated NRVM and ARVM treated with neurohumoral stimuli such as angiotensin II (Ang II) and ISO. Further, PDE1A plays a critical role in PE-induced reduction of intracellular cGMP and PKG activity, and thereby cardiomyocyte hypertrophy in vitro. Conclusions These results elucidate a novel role for Ca2+/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca2+ and cGMP signaling cross-talk during cardiac hypertrophy. PMID:19797176

Nuclear inositol 1,4,5-trisphosphate is a necessary and conserved signal for the induction of both pathological and physiological cardiomyocyte hypertrophy.

PubMed

Arantes, Lilian A M; Aguiar, Carla J; Amaya, Maria Jimena; Figueiró, Núbia C G; Andrade, Lídia M; Rocha-Resende, Cibele; Resende, Rodrigo R; Franchini, K G; Guatimosim, Silvia; Leite, M Fatima

2012-10-01

It is well established that inositol 1,4,5-trisphosphate (IP3) dependent Ca(2+) signaling plays a crucial role in cardiomyocyte hypertrophy. However, it is not yet known whether nuclear IP3 represents a Ca(2+) mobilizing pathway involved in this process. The goal of the current work was to investigate the specific role of nuclear IP3 in cardiomyocyte hypertrophic response. In this work, we used an adenovirus construct that selectively buffers IP3 in the nuclear region of neonatal cardiomyocytes. We showed for the first time that nuclear IP3 mediates endothelin-1 (ET-1) induced hypertrophy. We also found that both calcineurin (Cn)/nuclear factor of activated T Cells (NFAT) and histone deacetylase-5 (HDAC5) pathways require nuclear IP3 to mediate pathological cardiomyocyte growth. Additionally, we found that nuclear IP3 buffering inhibited insulin-like growth factor-1 (IGF-1) induced hypertrophy and prevented reexpression of fetal gene program. Together, these results demonstrated that nuclear IP3 is an essential and a conserved signal for both pathological and physiological forms of cardiomyocyte hypertrophy. Copyright © 2012. Published by Elsevier Ltd.

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

Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-β in cardiac myocytes.

PubMed

Shyu, Kou-Gi; Cheng, Wen-Pin; Wang, Bao-Wei; Chang, Hang

2014-03-01

The expression of MURC (muscle-restricted coiled-coil protein), a hypertrophy-regulated gene, increases during pressure overload. Hypoxia can cause myocardial hypertrophy; however, how hypoxia affects the regulation of MURC in cardiomyocytes undergoing hypertrophy is still unknown. The aim of the present study was to test the hypothesis that hypoxia induces MURC expression in cardiomyocytes during hypertrophy. The expression of MURC was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia and in an in vivo model of AMI (acute myocardial infarction) to induce myocardial hypoxia in adult rats. MURC protein and mRNA expression were significantly enhanced by hypoxia. MURC proteins induced by hypoxia were significantly blocked after the addition of PD98059 or ERK (extracellular-signal-regulated kinase) siRNA 30 min before hypoxia. Gel-shift assay showed increased DNA-binding activity of SRF (serum response factor) after hypoxia. PD98059, ERK siRNA and an anti-TGF-β (transforming growth factor-β) antibody abolished the SRF-binding activity enhanced by hypoxia or exogenous administration of TGF-β. A luciferase promoter assay demonstrated increased transcriptional activity of SRF in cardiomyocytes by hypoxia. Increased βMHC (β-myosin heavy chain) and BNP (B-type natriuretic peptide) protein expression and increased protein synthesis was identified after hypoxia with the presence of MURC in hypertrophic cardiomyocytes. MURC siRNA inhibited the hypertrophic marker protein expression and protein synthesis induced by hypoxia. AMI in adult rats also demonstrated increased MURC protein expression in the left ventricular myocardium. In conclusion, hypoxia in cultured rat neonatal cardiomyocytes increased MURC expression via the induction of TGF-β, SRF and the ERK pathway. These findings suggest that MURC plays a role in hypoxia-induced hypertrophy in cardiomyocytes.

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.

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

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.

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

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

PubMed

Fragoso-Medina, Jorge; Rodriguez, Gabriela; Zarain-Herzberg, Angel

2018-05-01

The cardiac sarco/endoplasmic reticulum Ca 2+ -ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPβ transcription factors to the SERCA2 gene proximal promoter.

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

Activator of G Protein Signaling 8 (AGS8) Is Required for Hypoxia-induced Apoptosis of Cardiomyocytes

PubMed Central

Sato, Motohiko; Jiao, Qibin; Honda, Takashi; Kurotani, Reiko; Toyota, Eiji; Okumura, Satoshi; Takeya, Tatsuo; Minamisawa, Susumu; Lanier, Stephen M.; Ishikawa, Yoshihiro

2009-01-01

Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of Gβγ subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a Gβγ-dependent manner by providing a scaffold composed of Gβγ and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the Gβγ inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-Gβγ signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM. PMID:19723622

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

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

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

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

Cardiomyocyte Ca2+ handling and structure is regulated by degree and duration of mechanical load variation.

PubMed

Ibrahim, Michael; Kukadia, Punam; Siedlecka, Urszula; Cartledge, James E; Navaratnarajah, Manoraj; Tokar, Sergiy; Van Doorn, Carin; Tsang, Victor T; Gorelik, Julia; Yacoub, Magdi H; Terracciano, Cesare M

2012-12-01

Cardiac transverse (t)-tubules are altered during disease and may be regulated by stretch-sensitive molecules. The relationship between variations in the degree and duration of load and t-tubule structure remains unknown, as well as its implications for local Ca(2+)-induced Ca(2+) release (CICR). Rat hearts were studied after 4 or 8 weeks of moderate mechanical unloading [using heterotopic abdominal heart-lung transplantation (HAHLT)] and 6 or 10 weeks of pressure overloading using thoracic aortic constriction. CICR, cell and t-tubule structure were assessed using confocal-microscopy, patch-clamping and scanning ion conductance microscopy. Moderate unloading was compared with severe unloading [using heart-only transplantation (HAHT)]. Mechanical unloading reduced cardiomyocyte volume in a time-dependent manner. Ca(2+) release synchronicity was reduced at 8 weeks moderate unloading only. Ca(2+) sparks increased in frequency and duration at 8 weeks of moderate unloading, which also induced t-tubule disorganization. Overloading increased cardiomyocyte volume and disrupted t-tubule morphology at 10 weeks but not 6 weeks. Moderate mechanical unloading for 4 weeks had milder effects compared with severe mechanical unloading (37% reduction in cell volume at 4 weeks compared to 56% reduction after severe mechanical unloading) and did not cause depression and delay of the Ca(2+) transient, increased Ca(2+) spark frequency or impaired t-tubule and cell surface structure. These data suggest that variations in chronic mechanical load influence local CICR and t-tubule structure in a time- and degree-dependent manner, and that physiological states of increased and reduced cell size, without pathological changes are possible. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes

PubMed Central

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M.; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H.; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-01-01

Aims Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein–protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. Methods and results We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. Conclusions This study

Prenatal Mechanistic Target of Rapamycin Complex 1 (m TORC1) Inhibition by Rapamycin Treatment of Pregnant Mice Causes Intrauterine Growth Restriction and Alters Postnatal Cardiac Growth, Morphology, and Function.

PubMed

Hennig, Maria; Fiedler, Saskia; Jux, Christian; Thierfelder, Ludwig; Drenckhahn, Jörg-Detlef

2017-08-04

Fetal growth impacts cardiovascular health throughout postnatal life in humans. Various animal models of intrauterine growth restriction exhibit reduced heart size at birth, which negatively influences cardiac function in adulthood. The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient and growth factor availability with cell growth, thereby regulating organ size. This study aimed at elucidating a possible involvement of mTORC1 in intrauterine growth restriction and prenatal heart growth. We inhibited mTORC1 in fetal mice by rapamycin treatment of pregnant dams in late gestation. Prenatal rapamycin treatment reduces mTORC1 activity in various organs at birth, which is fully restored by postnatal day 3. Rapamycin-treated neonates exhibit a 16% reduction in body weight compared with vehicle-treated controls. Heart weight decreases by 35%, resulting in a significantly reduced heart weight/body weight ratio, smaller left ventricular dimensions, and reduced cardiac output in rapamycin- versus vehicle-treated mice at birth. Although proliferation rates in neonatal rapamycin-treated hearts are unaffected, cardiomyocyte size is reduced, and apoptosis increased compared with vehicle-treated neonates. Rapamycin-treated mice exhibit postnatal catch-up growth, but body weight and left ventricular mass remain reduced in adulthood. Prenatal mTORC1 inhibition causes a reduction in cardiomyocyte number in adult hearts compared with controls, which is partially compensated for by an increased cardiomyocyte volume, resulting in normal cardiac function without maladaptive left ventricular remodeling. Prenatal rapamycin treatment of pregnant dams represents a new mouse model of intrauterine growth restriction and identifies an important role of mTORC1 in perinatal cardiac growth. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

Class I HDACs control a JIP1-dependent pathway for kinesin-microtubule binding in cardiomyocytes

PubMed Central

Blakeslee, Weston W.; Lin, Ying-Hsi; Stratton, Matthew S.; Tatman, Philip D.; Hu, Tianjing; Ferguson, Bradley S.; McKinsey, Timothy A.

2018-01-01

Class I histone deacetylase (HDAC) inhibitors block hypertrophy and fibrosis of the heart by suppressing pathological signaling and gene expression programs in cardiac myocytes and fibroblasts. The impact of HDAC inhibition in unstressed cardiac cells remains poorly understood. Here, we demonstrate that treatment of cultured cardiomyocytes with small molecule HDAC inhibitors leads to dramatic induction of c-Jun amino-terminal kinase (JNK)-interacting protein-1 (JIP1) mRNA and protein expression. In contrast to prior findings, elevated levels of endogenous JIP1 in cardiomyocytes failed to significantly alter JNK signaling or cardiomyocyte hypertrophy. Instead, HDAC inhibitor-mediated induction of JIP1 was required to stimulate expression of the kinesin heavy chain family member, KIF5A. We provide evidence for an HDAC-dependent regulatory circuit that promotes formation of JIP1:KIF5A:microtubule complexes that regulate intracellular transport of cargo such as autophagosomes. These findings define a novel role for class I HDACs in the control of the JIP1/kinesin axis in cardiomyocytes, and suggest that HDAC inhibitors could be used to alter microtubule transport in the heart. PMID:28886967

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.

Retinoic Acid Protects Cardiomyocytes from High Glucose-Induced Apoptosis via Inhibition of Sustained Activation of NF-κB Signaling

PubMed Central

Nizamutdinova, Irina T.; Guleria, Rakeshwar S.; Singh, Amar B.; Kendall, Jonathan A.; Baker, Kenneth M.; Pan, Jing

2012-01-01

We have previously shown that retinoic acid (RA) has protective effects on high glucose (HG)-induced cardiomyocyte apoptosis. To further elucidate the molecular mechanisms of RA effects, we determined the interaction between nuclear factor (NF)-κB and RA signaling. HG induced a sustained phosphorylation of IKK/IκBα and transcriptional activation of NF-κB in cardiomyocytes. Activated NF-κB signaling has an important role in HG-induced cardiomyocyte apoptosis and gene expression of interleukin-6 (IL-6), tumor necrosis factor (TNF)-α and monocyte chemoattractant protein-1 (MCP-1). All-trans RA (ATRA) and LGD1069, through activation of RAR/RXR-mediated signaling, inhibited the HG-mediated effects in cardiomyocytes. The inhibitory effect of RA on NF-κB activation was mediated through inhibition of IKK/IκBα phosphorylation. ATRA and LGD1069 treatment promoted protein phosphatase 2A (PP2A) activity, which was significantly suppressed by HG stimulation. The RA effects on IKK and IκBα were blocked by okadaic acid or silencing the expression of PP2Ac-subunit, indicating that the inhibitory effect of RA on NF-κB is regulated through activation of PP2A and subsequent dephosphorylation of IKK/IκBα. Moreover, ATRA and LGD1069 reversed the decreased PP2A activity and inhibited the activation of IKK/IκBα and gene expression of MCP-1, IL-6 and TNF-α in the hearts of Zucker diabetic fatty rats. In summary, our findings suggest that the suppressed activation of PP2A contributed to sustained activation of NF-κB in HG-stimulated cardiomyocytes; and that the protective effect of RA on hyperglycemia-induced cardiomyocyte apoptosis and inflammatory responses is partially regulated through activation of PP2A and suppression of NF-κB-mediated signaling and downstream targets. PMID:22718360

Quantitative Non-canonical Amino Acid Tagging (QuaNCAT) Proteomics Identifies Distinct Patterns of Protein Synthesis Rapidly Induced by Hypertrophic Agents in Cardiomyocytes, Revealing New Aspects of Metabolic Remodeling*

PubMed Central

Liu, Rui; Kenney, Justin W.; Manousopoulou, Antigoni; Johnston, Harvey E.; Kamei, Makoto; Woelk, Christopher H.; Xie, Jianling; Schwarzer, Michael; Proud, Christopher G.

2016-01-01

Cardiomyocytes undergo growth and remodeling in response to specific pathological or physiological conditions. In the former, myocardial growth is a risk factor for cardiac failure and faster protein synthesis is a major factor driving cardiomyocyte growth. Our goal was to quantify the rapid effects of different pro-hypertrophic stimuli on the synthesis of specific proteins in ARVC and to determine whether such effects are caused by alterations on mRNA abundance or the translation of specific mRNAs. Cardiomyocytes have very low rates of protein synthesis, posing a challenging problem in terms of studying changes in the synthesis of specific proteins, which also applies to other nondividing primary cells. To study the rates of accumulation of specific proteins in these cells, we developed an optimized version of the Quantitative Noncanonical Amino acid Tagging LC/MS proteomic method to label and selectively enrich newly synthesized proteins in these primary cells while eliminating the suppressive effects of pre-existing and highly abundant nonisotope-tagged polypeptides. Our data revealed that a classical pathologic (phenylephrine; PE) and the recently identified insulin stimulus that also contributes to the development of pathological cardiac hypertrophy (insulin), both increased the synthesis of proteins involved in, e.g. glycolysis, the Krebs cycle and beta-oxidation, and sarcomeric components. However, insulin increased synthesis of many metabolic enzymes to a greater extent than PE. Using a novel validation method, we confirmed that synthesis of selected candidates is indeed up-regulated by PE and insulin. Synthesis of all proteins studied was up-regulated by signaling through mammalian target of rapamycin complex 1 without changes in their mRNA levels, showing the key importance of translational control in the rapid effects of hypertrophic stimuli. Expression of PKM2 was up-regulated in rat hearts following TAC. This isoform possesses specific regulatory

Regulation of body growth by microRNAs.

PubMed

Lui, Julian C

2017-11-15

Regulation of body growth remains a fascinating and unresolved biological mystery. One key component of body growth is skeletal and longitudinal bone growth. Children grow taller because their bones grew longer, and the predominant driver of longitudinal bone growth is a cartilaginous structure found near the ends of long bone named the growth plate. Numerous recent studies have started to unveil the importance of microRNAs in regulation of growth plate functions, therefore contributing to regulation of linear growth. In addition to longitudinal growth, other organs in our body need to increase in size and cell number as we grow, and the regulation of organ growth involves both systemic factors like hormones; and other intrinsic mechanisms, which we are just beginning to understand. This review aims to summarize some recent important findings on how microRNAs are involved in both of these processes: the regulation of longitudinal bone growth, and the regulation of organs and overall body growth. Published by Elsevier B.V.

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.

PubMed

Yang, Xiulan; Rodriguez, Marita; Pabon, Lil; Fischer, Karin A; Reinecke, Hans; Regnier, Michael; Sniadecki, Nathan J; Ruohola-Baker, Hannele; Murry, Charles E

2014-07-01

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens. Copyright © 2014 Elsevier Ltd. All rights reserved.

α-Enolase plays a catalytically independent role in doxorubicin-induced cardiomyocyte apoptosis and mitochondrial dysfunction.

PubMed

Gao, Si; Li, Hong; Feng, Xiao-jun; Li, Min; Liu, Zhi-ping; Cai, Yi; Lu, Jing; Huang, Xiao-yang; Wang, Jiao-jiao; Li, Qin; Chen, Shao-rui; Ye, Jian-tao; Liu, Pei-qing

2015-02-01

α-Enolase is a glycolytic enzyme with "second jobs" beyond its catalytic activity. However, its possible contribution to cardiac dysfunction remains to be determined. The present study aimed to investigate the role of α-enolase in doxorubicin (Dox)-induced cardiomyopathy as well as the underlying mechanisms. The expression of α-enolase was detected in rat hearts and primary cultured rat cardiomyocytes with or without Dox administration. An adenovirus carrying short-hairpin interfering RNA targeting α-enolase was constructed and transduced specifically into the heart by intramyocardial injection. Heart function, cell apoptosis and mitochondrial function were measured following Dox administration. In addition, by using gain- and loss-of-function approaches to regulate α-enolase expression in primary cultured rat cardiomyocytes, we investigated the role of endogenous, wide type and catalytically inactive mutant α-enolase in cardiomyocyte apoptosis and ATP generation. Furthermore, the involvement of α-enolase in AMPK phosphorylation was also studied. The mRNA and protein expression of cardiac α-enolase was significantly upregulated by Dox. Genetic silencing of α-enolase in rat hearts and cultured cardiomyocytes attenuated Dox-induced apoptosis and mitochondrial dysfunction. In contrast, overexpression of wide-type or catalytically inactive α-enolase in cardiomyocytes mimicked the detrimental role of Dox in inducing apoptosis and ATP reduction. AMPK dephosphorylation was further demonstrated to be involved in the proapoptotic and ATP-depriving effects of α-enolase. Our findings provided the evidence that α-enolase has a catalytically independent role in inducing cardiomyocyte apoptosis and mitochondrial dysfunction, which could be at least partially contributed to the inhibition of AMPK phosphorylation. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

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

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.

Ursodeoxycholic acid protects cardiomyocytes against cobalt chloride induced hypoxia by regulating transcriptional mediator of cells stress hypoxia inducible factor 1α and p53 protein.

PubMed

Mohamed, Anis Syamimi; Hanafi, Noorul Izzati; Sheikh Abdul Kadir, Siti Hamimah; Md Noor, Julina; Abdul Hamid Hasani, Narimah; Ab Rahim, Sharaniza; Siran, Rosfaiizah

2017-10-01

In hepatocytes, ursodeoxycholic acid (UDCA) activates cell signalling pathways such as p53, intracellular calcium ([Ca 2+ ] i ), and sphingosine-1-phosphate (S1P)-receptor via Gα i -coupled-receptor. Recently, UDCA has been shown to protect the heart against hypoxia-reoxygenation injury. However, it is not clear whether UDCA cardioprotection against hypoxia acts through a transcriptional mediator of cells stress, HIF-1α and p53. Therefore, in here, we aimed to investigate whether UDCA could protect cardiomyocytes (CMs) against hypoxia by regulating expression of HIF-1α, p53, [Ca 2+ ] i , and S1P-Gα i -coupled-receptor. Cardiomyocytes were isolated from newborn rats (0-2 days), and hypoxia was induced by using cobalt chloride (CoCl 2 ). Cardiomyocytes were treated with UDCA and cotreated with either FTY720 (S1P-receptor agonist) or pertussis toxin (PTX; Gα i inhibitor). Cells were subjected for proliferation assay, beating frequency, QuantiGene Plex assay, western blot, immunofluorescence, and calcium imaging. Our findings showed that UDCA counteracted the effects of CoCl 2 on cell viability, beating frequency, HIF-1α, and p53 protein expression. We found that these cardioprotection effects of UDCA were similar to FTY720, S1P agonist. Furthermore, we observed that UDCA protects CMs against CoCl 2 -induced [Ca 2+ ] i dynamic alteration. Pharmacological inhibition of the Gα i -sensitive receptor did not abolish the cardioprotection of UDCA against CoCl 2 detrimental effects, except for cell viability and [Ca 2+ ] i . Pertussis toxin is partially effective in inhibiting UDCA protection against CoCl 2 effects on CM cell viability. Interestingly, PTX fully inhibits UDCA cardioprotection on CoCl 2 -induced [Ca 2+ ] i dynamic changes. We conclude that UDCA cardioprotection against CoCl 2 -induced hypoxia is similar to FTY720, and its actions are not fully mediated by the Gα i -coupled protein sensitive pathways. Ursodeoxycholic acid is the most hydrophilic bile

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.

[Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

PubMed

Anatskaia, O V; Sidorenko, N V; Matveev, I V; Kropotov, A V; Vinogradov, A E

2012-01-01

Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

Process for producing vegetative and tuber growth regulator

NASA Technical Reports Server (NTRS)

Yorio, Neil C. (Inventor); Stutte, Gary W. (Inventor)

1999-01-01

A process of making a vegetative and tuber growth regulator. The vegetative and tuber growth regulator is made by growing potato plants in a recirculating hydroponic system for a sufficient time to produce the growth regulator. Also, the use of the vegetative and growth regulator on solanaceous plants, tuber forming plants and ornamental seedlings by contacting the roots or shoots of the plant with a sufficient amount of the growth regulator to regulate the growth of the plant and one more of canopy size, plant height, stem length, internode number and presence of tubers in fresh mass. Finally, a method for regulating the growth of potato plants using a recirculating hydroponic system is described.

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

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

Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxicmore » microenvironment in vitro, well characterized HCM were treated with 100 µm (48 h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1 µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic

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.

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells

PubMed Central

Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment. PMID:29216265

OCT4 expression mediates partial cardiomyocyte reprogramming of mesenchymal stromal cells.

PubMed

Yannarelli, Gustavo; Pacienza, Natalia; Montanari, Sonia; Santa-Cruz, Diego; Viswanathan, Sowmya; Keating, Armand

2017-01-01

Mesenchymal stem/stromal cells (MSCs) are in numerous cell therapy clinical trials, including for injured myocardium. Acquisition of cardiomyocyte characteristics by MSCs may improve cardiac regeneration but the mechanisms regulating this process are unclear. Here, we investigated whether the pluripotency transcription factor OCT4 is involved in the activation of cardiac lineage genetic programs in MSCs. We employed our established co-culture model of MSCs with rat embryonic cardiomyocytes showing co-expression of cardiac markers on MSCs independent of cell fusion. Bone marrow-derived MSCs were isolated from transgenic mice expressing GFP under the control of the cardiac-specific α-myosin heavy chain promoter. After 5 days of co-culture, MSCs expressed cardiac specific genes, including Nkx2.5, atrial natriuretic factor and α-cardiac actin. The frequency of GFP+ cells was 7.6±1.9%, however, these cells retained the stromal cell phenotype, indicating, as expected, only partial differentiation. Global OCT4 expression increased 2.6±0.7-fold in co-cultured MSCs and of interest, 87±5% vs 79±4% of MSCs expressed OCT4 by flow cytometry in controls and after co-culture, respectively. Consistent with the latter observation, the GFP+ cells did not express nuclear OCT4 and showed a significant increase in OCT4 promoter methylation compared with undifferentiated MSCs (92% vs 45%), inferring that OCT4 is regulated by an epigenetic mechanism. We further showed that siRNA silencing of OCT4 in MSCs resulted in a reduced frequency of GFP+ cells in co-culture to less than 1%. Our data infer that OCT4 expression may have a direct effect on partial cardiomyocyte reprogramming of MSCs and suggest a new mechanism(s) associated with MSC multipotency and a requirement for crosstalk with the cardiac microenvironment.

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

Prolyl hydroxylation regulates protein degradation, synthesis, and splicing in human induced pluripotent stem cell-derived cardiomyocytes.

PubMed

Stoehr, Andrea; Yang, Yanqin; Patel, Sajni; Evangelista, Alicia M; Aponte, Angel; Wang, Guanghui; Liu, Poching; Boylston, Jennifer; Kloner, Philip H; Lin, Yongshun; Gucek, Marjan; Zhu, Jun; Murphy, Elizabeth

2016-06-01

Protein hydroxylases are oxygen- and α-ketoglutarate-dependent enzymes that catalyse hydroxylation of amino acids such as proline, thus linking oxygen and metabolism to enzymatic activity. Prolyl hydroxylation is a dynamic post-translational modification that regulates protein stability and protein-protein interactions; however, the extent of this modification is largely uncharacterized. The goals of this study are to investigate the biological consequences of prolyl hydroxylation and to identify new targets that undergo prolyl hydroxylation in human cardiomyocytes. We used human induced pluripotent stem cell-derived cardiomyocytes in combination with pulse-chase amino acid labelling and proteomics to analyse the effects of prolyl hydroxylation on protein degradation and synthesis. We identified 167 proteins that exhibit differences in degradation with inhibition of prolyl hydroxylation by dimethyloxalylglycine (DMOG); 164 were stabilized. Proteins involved in RNA splicing such as serine/arginine-rich splicing factor 2 (SRSF2) and splicing factor and proline- and glutamine-rich (SFPQ) were stabilized with DMOG. DMOG also decreased protein translation of cytoskeletal and sarcomeric proteins such as α-cardiac actin. We searched the mass spectrometry data for proline hydroxylation and identified 134 high confidence peptides mapping to 78 unique proteins. We identified SRSF2, SFPQ, α-cardiac actin, and cardiac titin as prolyl hydroxylated. We identified 29 prolyl hydroxylated proteins that showed a significant difference in either protein degradation or synthesis. Additionally, we performed next-generation RNA sequencing and showed that the observed decrease in protein synthesis was not due to changes in mRNA levels. Because RNA splicing factors were prolyl hydroxylated, we investigated splicing ± inhibition of prolyl hydroxylation and detected 369 alternative splicing events, with a preponderance of exon skipping. This study provides the first extensive

Polycystin-1 is a Cardiomyocyte Mechanosensor That Governs L-type Ca2+ Channel Protein Stability

PubMed Central

Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K.; Morales, Cyndi R.; Contreras, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J.; Somlo, Stefan; Rothermel, Beverly A.; Gillette, Thomas G.; Lavandero, Sergio; Hill, Joseph A.

2015-01-01

Background L-type calcium channel (LTCC) activity is critical to afterload-induced hypertrophic growth of the heart. However, mechanisms governing mechanical stress-induced activation of LTCC activity are obscure. Polycystin-1 (PC-1) is a G-protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. Methods and Results We subjected neonatal rat ventricular myocytes (NRVMs) to mechanical stretch by exposing them to hypo-osmotic (HS) medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on LTCC activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Over-expression of a C-terminal fragment of PC-1 was sufficient to trigger NRVM hypertrophy. Exposing NRVMs to HS medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 KO) and subjected them to mechanical stress in vivo (transverse aortic constriction, TAC). At baseline, PC-1 KO mice manifested decreased cardiac function relative to littermate controls, and α1C LTCC protein levels were significantly lower in PC-1 KO hearts. Whereas control mice manifested robust TAC-induced increases in cardiac mass, PC-1 KO mice showed no significant growth. Likewise, TAC-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals Conclusions PC-1 is a cardiomyocyte mechanosensor and is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. PMID:25888683

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

Hydrogen sulfide protects cardiomyocytes from hypoxia/reoxygenation-induced apoptosis by preventing GSK-3beta-dependent opening of mPTP.

PubMed

Yao, Ling-Ling; Huang, Xiao-Wei; Wang, Yong-Gang; Cao, Yin-Xiang; Zhang, Cai-Cai; Zhu, Yi-Chun

2010-05-01

Hydrogen sulfide (H(2)S) is an endogenously generated gaseous transmitter, which has recently been suggested to regulate cardiovascular functions. The present study aims to clarify the mechanisms underlying the cardioprotective effects of H(2)S. Signaling elements were examined in cardiomyocytes cultured under hypoxia/reoxygenation conditions and in a rat model of ischemia-reperfusion. In cultured cardiomyocytes, sodium hydrosulfide (NaHS; 10, 30, and 50 mumol/l) showed concentration-dependent inhibitory effects on cardiomyocyte apoptosis induced by hypoxia/reoxygenation. These effects were associated with an increase in phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) (Ser9) and a decrease in Bax translocation, caspase-3 activation, and mitochondrial permeability transition pore (mPTP) opening. Transfection of a phosphorylation-resistant mutant of GSK-3beta at Ser9 attenuated the effects of NaHS in reducing cardiomyocyte apoptosis, Bax translocation, caspase-3 activation, and mPTP opening. In a rat model of ischemia-reperfusion, NaHS administration reduced myocardial infarct size and increased the phosphorylation of GSK-3beta (Ser9) at a dose of 30 mumol/kg. In conclusion, the H(2)S donor prevents cardiomyocyte apoptosis by inducing phosphorylation of GSK-3beta (Ser9) and subsequent inhibition of mPTP opening.

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.

Myofibril growth during cardiac hypertrophy is regulated through dual phosphorylation and acetylation of the actin capping protein CapZ

PubMed Central

Lin, Ying-Hsi; Warren, Chad M.; Li, Jieli; McKinsey, Timothy A.; Russell, Brenda

2016-01-01

The mechanotransduction signaling pathways initiated in heart muscle by increased mechanical loading are known to lead to long-term transcriptional changes and hypertrophy, but the rapid events for adaptation at the sarcomeric level are not fully understood. The goal of this study was to test the hypothesis that actin filament assembly during cardiomyocyte growth is regulated by post-translational modifications (PTMs) of CapZβ1. In rapidly hypertrophying neonatal rat ventricular myocytes (NRVMs) stimulated by phenylephrine (PE), two-dimensional gel electrophoresis (2DGE) of CapZβ1 revealed a shift toward more negative charge. Consistent with this, mass spectrometry identified CapZβ1 phosphorylation on serine-204 and acetylation on lysine-199, two residues which are near the actin binding surface of CapZβ1. Ectopic expression of dominant negative PKCε (dnPKCε) in NRVMs blunted the PE-induced increase in CapZ dynamics, as evidenced by the kinetic constant (Kfrap) of fluorescence recovery after photobleaching (FRAP), and concomitantly reduced phosphorylation and acetylation of CapZβ1. Furthermore, inhibition of class I histone deacetylases (HDACs) increased lysine-199 acetylation on CapZβ1, which increased Kfrap of CapZ and stimulated actin dynamics. Finally, we show that PE treatment of NRVMs results in decreased binding of HDAC3 to myofibrils, suggesting a signal-dependent mechanism for the regulation of sarcomere-associated CapZβ1 acetylation. Taken together, this dual regulation through phosphorylation and acetylation of CapZβ1 provides a novel model for the regulation of myofibril growth during cardiac hypertrophy. PMID:27185186

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes.

PubMed

Shafa, Mehdi; Krawetz, Roman; Zhang, Yuan; Rattner, Jerome B; Godollei, Anna; Duff, Henry J; Rancourt, Derrick E

2011-12-14

Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Murine D3-MHC-neo(r) ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a

Frequency of mononuclear diploid cardiomyocytes underlies natural variation in heart regeneration.

PubMed

Patterson, Michaela; Barske, Lindsey; Van Handel, Ben; Rau, Christoph D; Gan, Peiheng; Sharma, Avneesh; Parikh, Shan; Denholtz, Matt; Huang, Ying; Yamaguchi, Yukiko; Shen, Hua; Allayee, Hooman; Crump, J Gage; Force, Thomas I; Lien, Ching-Ling; Makita, Takako; Lusis, Aldons J; Kumar, S Ram; Sucov, Henry M

2017-09-01

Adult mammalian cardiomyocyte regeneration after injury is thought to be minimal. Mononuclear diploid cardiomyocytes (MNDCMs), a relatively small subpopulation in the adult heart, may account for the observed degree of regeneration, but this has not been tested. We surveyed 120 inbred mouse strains and found that the frequency of adult mononuclear cardiomyocytes was surprisingly variable (>7-fold). Cardiomyocyte proliferation and heart functional recovery after coronary artery ligation both correlated with pre-injury MNDCM content. Using genome-wide association, we identified Tnni3k as one gene that influences variation in this composition and demonstrated that Tnni3k knockout resulted in elevated MNDCM content and increased cardiomyocyte proliferation after injury. Reciprocally, overexpression of Tnni3k in zebrafish promoted cardiomyocyte polyploidization and compromised heart regeneration. Our results corroborate the relevance of MNDCMs in heart regeneration. Moreover, they imply that intrinsic heart regeneration is not limited nor uniform in all individuals, but rather is a variable trait influenced by multiple genes.

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

Stimulation of cardiomyocyte regeneration in neonatal mice and in human myocardium with neuregulin reveals a therapeutic window

PubMed Central

Polizzotti, Brian D.; Ganapathy, Balakrishnan; Walsh, Stuart; Choudhury, Sangita; Ammanamanchi, Niyatie; Bennett, David G.; dos Remedios, Cristobal G.; Haubner, Bernhard J.; Penninger, Josef M.; Kuhn, Bernhard

2015-01-01

Background Pediatric patients with heart failure are treated with medical therapies that were developed for adult patients. These therapies have been shown to be ineffective in pediatric trials, leading to the recognition that new pediatric-specific therapies must be developed. We have previously shown that administration of the recombinant growth factor neuregulin-1 (rNRG1) stimulates heart muscle cell (cardiomyocyte) regeneration in adult mice. We hypothesized that rNRG1 administration may be more effective in the neonatal period, which could provide a new therapeutic paradigm for treating heart failure in pediatric patients. Methods We used a cryoinjury model to induce myocardial dysfunction and scar formation for evaluating the effectiveness of rNRG1-administration in neonatal mice. We evaluated the ability of rNRG1 to stimulate cardiomyocyte proliferation in intact cultured myocardium from pediatric patients. Results After cryoinjury in neonatal mice, early administration of rNRG1 from birth for 34 days improved myocardial function and reduced the prevalence of transmural scars. In contrast, late administration of rNRG1 from 4 to 34 days after cryoinjury transiently improved myocardial function. The mechanisms of early administration involved cardiomyocyte protection (38%) and proliferation (62%). rNRG1 induced cardiomyocyte proliferation in myocardium from infants with heart disease less than 6 months of age. Conclusion Our results identify a more effective time period within which to execute future clinical trials of rNRG1 for stimulating cardiomyocyte regeneration. PMID:25834111

GSK-3β promotes PA-induced apoptosis through changing β-arrestin 2 nucleus location in H9c2 cardiomyocytes.

PubMed

Chang, Fen; Liu, Jing; Fu, Hui; Wang, Jinlan; Li, Fang; Yue, Hongwei; Li, Wenjing; Zhao, Jing; Yin, Deling

2016-09-01

Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.

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...

Neuroendocrine regulation of somatic growth in fishes.

PubMed

Dai, XiangYan; Zhang, Wei; Zhuo, ZiJian; He, JiangYan; Yin, Zhan

2015-02-01

Growth is a polygenic trait that is under the influence of multiple physiological pathways regulating energy metabolism and muscle growth. Among the possible growth-regulating pathways in vertebrates, components of the somatotropic axis are thought to have the greatest influence. There is growing body of literature focusing on the somatotropic axis and its role regulating growth in fish. This includes research into growth hormone, upstream hypothalamic hormones, insulin-like growth factors, and downstream signaling molecules. Many of these signals have both somatic effects stimulating the growth of tissues and metabolic effects that play a role in nutrient metabolism. Signals of other endocrine axes exhibit profound effects on the function of the somatotropic axis in vivo. In this review we highlight recent advances in our understanding of the teleost fish endocrine somatotropic axis, including emerging research using genetic modified models. These studies have revealed new aspects and challenges associated with regulation of the important steps of somatic growth.

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.

Measuring Fast Calcium Fluxes in Cardiomyocytes

PubMed Central

Golebiewska, Urszula; Scarlata, Suzanne

2011-01-01

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase Cβ- Gαq pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated Gαq7. This entrapment has the effect of stabilizing the activated state of Gαq and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad

Measuring fast calcium fluxes in cardiomyocytes.

PubMed

Golebiewska, Urszula; Scarlata, Suzanne

2011-11-29

Cardiomyocytes have multiple Ca(2+) fluxes of varying duration that work together to optimize function (1,2). Changes in Ca(2+) activity in response to extracellular agents is predominantly regulated by the phospholipase Cβ- Gα(q;) pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine (3,4). We have recently found that plasma membrane protein domains called caveolae(5,6) can entrap activated Gα(q;)(7). This entrapment has the effect of stabilizing the activated state of Gα(q;) and resulting in prolonged Ca(2+) signals in cardiomyocytes and other cell types(8). We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca(2+) indicator. In our studies, we used Ca(2+) Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca(2+) responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca(2+) waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca(2+) waves

Inward rectifier potassium channels in the HL-1 cardiomyocyte-derived cell line.

PubMed

Goldoni, Dana; Zhao, YouYou; Green, Brian D; McDermott, Barbara J; Collins, Anthony

2010-11-01

HL-1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K(+) channels. Our aim was to identify and characterize inward rectifier K(+) channels in HL-1 cells. External Ba(2+) (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole-cell patch-clamp recordings. The reversal potential of the Ba(2+)-sensitive current shifted with external [K(+)] as expected for K(+)-selective channels. The slope conductance of the inward Ba(2+)-sensitive current increased with external [K(+)]. The apparent Kd for Ba(2+) was voltage dependent, ranging from 15 µM at -150  mV to 148 µM at -75  mV in 120  mM external K(+). This current was insensitive to 10 µM glybenclamide. A component of whole-cell current was sensitive to 150 µM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), although it did not correspond to the Ba(2+)-sensitive component. The effect of external 1 mM Cs(+) was similar to that of Ba(2+). Polymerase chain reaction using HL-1 cDNA as template and primers specific for the cardiac inward rectifier K(ir)2.1 produced a fragment of the expected size that was confirmed to be K(ir)2.1 by DNA sequencing. In conclusion, HL-1 cells express a current that is characteristic of cardiac inward rectifier K(+) channels, and express K(ir)2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype. © 2010 Wiley-Liss, Inc.

The Regulation of Filamentous Growth in Yeast

PubMed Central

Cullen, Paul J.; Sprague, George F.

2012-01-01

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host–cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways—rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)—also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior. PMID:22219507

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.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels: NEW TARGETS OF ALDOSTERONE IN CARDIOMYOCYTES.

PubMed

Sabourin, Jessica; Bartoli, Fiona; Antigny, Fabrice; Gomez, Ana Maria; Benitah, Jean-Pierre

2016-06-17

Store-operated Ca(2+) entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca(2+) influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca(2+)]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Polycystin-1 Is a Cardiomyocyte Mechanosensor That Governs L-Type Ca2+ Channel Protein Stability.

PubMed

Pedrozo, Zully; Criollo, Alfredo; Battiprolu, Pavan K; Morales, Cyndi R; Contreras-Ferrat, Ariel; Fernández, Carolina; Jiang, Nan; Luo, Xiang; Caplan, Michael J; Somlo, Stefan; Rothermel, Beverly A; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

2015-06-16

L-type calcium channel activity is critical to afterload-induced hypertrophic growth of the heart. However, the mechanisms governing mechanical stress-induced activation of L-type calcium channel activity are obscure. Polycystin-1 (PC-1) is a G protein-coupled receptor-like protein that functions as a mechanosensor in a variety of cell types and is present in cardiomyocytes. We subjected neonatal rat ventricular myocytes to mechanical stretch by exposing them to hypo-osmotic medium or cyclic mechanical stretch, triggering cell growth in a manner dependent on L-type calcium channel activity. RNAi-dependent knockdown of PC-1 blocked this hypertrophy. Overexpression of a C-terminal fragment of PC-1 was sufficient to trigger neonatal rat ventricular myocyte hypertrophy. Exposing neonatal rat ventricular myocytes to hypo-osmotic medium resulted in an increase in α1C protein levels, a response that was prevented by PC-1 knockdown. MG132, a proteasomal inhibitor, rescued PC-1 knockdown-dependent declines in α1C protein. To test this in vivo, we engineered mice harboring conditional silencing of PC-1 selectively in cardiomyocytes (PC-1 knockout) and subjected them to mechanical stress in vivo (transverse aortic constriction). At baseline, PC-1 knockout mice manifested decreased cardiac function relative to littermate controls, and α1C L-type calcium channel protein levels were significantly lower in PC-1 knockout hearts. Whereas control mice manifested robust transverse aortic constriction-induced increases in cardiac mass, PC-1 knockout mice showed no significant growth. Likewise, transverse aortic constriction-elicited increases in hypertrophic markers and interstitial fibrosis were blunted in the knockout animals PC-1 is a cardiomyocyte mechanosensor that is required for cardiac hypertrophy through a mechanism that involves stabilization of α1C protein. © 2015 American Heart Association, Inc.

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

Defects of mtDNA Replication Impaired Mitochondrial Biogenesis During Trypanosoma cruzi Infection in Human Cardiomyocytes and Chagasic Patients: The Role of Nrf1/2 and Antioxidant Response

PubMed Central

Wan, Xianxiu; Gupta, Shivali; Zago, Maria P.; Davidson, Mercy M.; Dousset, Pierre; Amoroso, Alejandro; Garg, Nisha Jain

2012-01-01

Background Mitochondrial dysfunction is a key determinant in chagasic cardiomyopathy development in mice; however, its relevance in human Chagas disease is not known. We determined if defects in mitochondrial biogenesis and dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1 (PGC-1)–regulated transcriptional pathways constitute a mechanism or mechanisms underlying mitochondrial oxidative-phosphorylation (OXPHOS) deficiency in human Chagas disease. Methods and Results We utilized human cardiomyocytes and left-ventricular tissue from chagasic and other cardiomyopathy patients and healthy donors (n>6/group). We noted no change in citrate synthase activity, yet mRNA and/or protein levels of subunits of the respiratory complexes were significantly decreased in Trypanosoma cruzi–infected cardiomyocytes (0 to 24 hours) and chagasic hearts. We observed increased mRNA and decreased nuclear localization of PGC-1-coactivated transcription factors, yet the expression of genes for PPARγ-regulated fatty acid oxidation and nuclear respiratory factor (NRF1/2)–regulated mtDNA replication and transcription machinery was enhanced in infected cardiomyocytes and chagasic hearts. The D-loop formation was normal or higher, but mtDNA replication and mtDNA content were decreased by 83% and 40% to 65%, respectively. Subsequently, we noted that reactive oxygen species (ROS), oxidative stress, and mtDNA oxidation were significantly increased, yet NRF1/2-regulated antioxidant gene expression remained compromised in infected cardiomyocytes and chagasic hearts. Conclusions The replication of mtDNA was severely compromised, resulting in a significant loss of mtDNA and expression of OXPHOS genes in T cruzi–infected cardiomyocytes and chagasic hearts. Our data suggest increased ROS generation and selective functional incapacity of NRF2-mediated antioxidant gene expression played a role in the defects in mtDNA replication and unfitness of mtDNA for

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.

Rho-associated kinases play an essential role in cardiac morphogenesis and cardiomyocyte proliferation.

PubMed

Zhao, Zhiyong; Rivkees, Scott A

2003-01-01

Rho-associated coiled-coil kinases (ROCKs), initially identified as effectors for Rho GTPases, play a role in cardiac cell physiology and are also expressed in the developing heart. However, their role in cardiac development is not known. To investigate the role of these kinases in cardiac development, we examined cardiac development in cultured murine embryos treated with the ROCK inhibitor Y27632. After inhibition of ROCK activity, we found disturbed cardiac chamber formation and trabeculation. To further examine the mechanisms by which ROCK blockade causes cardiac hypoplasia, we assessed programmed cell death and cell proliferation in the hearts. We found decreased cell proliferation in the Y27632-treated hearts, but no changes in programmed cell death. We further observed that ROCK inhibition decreased cardiac myocyte proliferation, suggesting that ROCK kinases regulate cardiomyocyte division. To identify factors involved in ROCK action in regulation of cardiac cell division, we examined expression of cell cycle proteins by using Western blot analysis. We found that ROCK blockade decreased expression of cell cycle proteins, cyclin D3, CDK6, and p27(KIP1) in the hearts and cardiomyocytes, which are required for initiation of cell cycle and G1/S phase transition. These observations show that ROCK kinases play a role in cardiac development and that ROCK kinases regulate cardiac cell proliferation and cell cycle protein expression. Copyright 2002 Wiley-Liss, Inc.

(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.

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.

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.

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

Mechanosensitive Gene Regulation by Myocardin-Related Transcription Factors is Required for Cardiomyocyte Integrity in Load-Induced Ventricular Hypertrophy.

PubMed

Trembley, Michael A; Quijada, Pearl; Agullo-Pascual, Esperanza; Tylock, Kevin M; Colpan, Mert; Dirkx, Ronald A; Myers, Jason R; Mickelsen, Deanne M; de Mesy Bentley, Karen; Rothenberg, Eli; Moravec, Christine S; Alexis, Jeffrey D; Gregorio, Carol C; Dirksen, Robert T; Delmar, Mario; Small, Eric M

2018-05-01

Background -Hypertrophic cardiomyocyte (CM) growth and dysfunction accompanies various forms of heart disease. The mechanisms responsible for transcriptional changes that impact cardiac physiology and the transition to heart failure (HF) are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling CM electrical activity and force transmission, and is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. Methods -Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy (SMLM) were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor-A (MRTF-A) and -B specifically in adult CMs to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. Results -We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure (HF). Although mice lacking MRTFs in adult CMs display normal cardiac physiology at baseline, pressure overload leads to rapid HF characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and CM adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by SMLM may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. Conclusions -Taken together, our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates crosstalk between the actin and microtubule cytoskeleton and maintains ID integrity and CM homeostasis in heart

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.

UCR1C is a novel activator of phosphodiesterase 4 (PDE4) long isoforms and attenuates cardiomyocyte hypertrophy.

PubMed

Wang, Li; Burmeister, Brian T; Johnson, Keven R; Baillie, George S; Karginov, Andrei V; Skidgel, Randal A; O'Bryan, John P; Carnegie, Graeme K

2015-05-01

Hypertrophy increases the risk of heart failure and arrhythmia. Prevention or reversal of the maladaptive hypertrophic phenotype has thus been proposed to treat heart failure. Chronic β-adrenergic receptor (β-AR) stimulation induces cardiomyocyte hypertrophy by elevating 3',5'-cyclic adenosine monophosphate (cAMP) levels and activating downstream effectors such protein kinase A (PKA). Conversely, hydrolysis of cAMP by phosphodiesterases (PDEs) spatiotemporally restricts cAMP signaling. Here, we demonstrate that PDE4, but not PDE3, is critical in regulating cardiomyocyte hypertrophy, and may represent a potential target for preventing maladaptive hypertrophy. We identify a sequence within the upstream conserved region 1 of PDE4D, termed UCR1C, as a novel activator of PDE4 long isoforms. UCR1C activates PDE4 in complex with A-kinase anchoring protein (AKAP)-Lbc resulting in decreased PKA signaling facilitated by AKAP-Lbc. Expression of UCR1C in cardiomyocytes inhibits hypertrophy in response to chronic β-AR stimulation. This effect is partially due to inhibition of nuclear PKA activity, which decreases phosphorylation of the transcription factor cAMP response element-binding protein (CREB). In conclusion, PDE4 activation by UCR1C attenuates cardiomyocyte hypertrophy by specifically inhibiting nuclear PKA activity. Published by Elsevier Inc.

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

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

PubMed Central

2011-01-01

Background Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within

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

β-Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α-PP2A-Mediated Dephosphorylation.

PubMed

Weeks, Kate L; Ranieri, Antonella; Karaś, Agnieszka; Bernardo, Bianca C; Ashcroft, Alexandra S; Molenaar, Chris; McMullen, Julie R; Avkiran, Metin

2017-03-25

Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal-responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor-2. β-Adrenoceptor (β-AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation-independent nuclear export and phosphorylation-dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β-AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. A novel 3-dimensional confocal microscopy method that objectively quantifies the whole-cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β-AR agonist isoproterenol to induce β 1 -AR-mediated and protein kinase A-dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor-2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol-induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol-induced HDAC5 dephosphorylation. Co-immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3-fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol-induced HDAC5 dephosphorylation. β-AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A-dependent but requires B55α-PP2A-mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A

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.

Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I

PubMed Central

Frantz, Stefan; Klaiber, Michael; Baba, Hideo A.; Oberwinkler, Heike; Völker, Katharina; Gaβner, Birgit; Bayer, Barbara; Abeβer, Marco; Schuh, Kai; Feil, Robert; Hofmann, Franz; Kuhn, Michaela

2013-01-01

Aims Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. Methods and results To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to β-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca2+]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca2+i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca2+i-handling, and contractility via cGKI. Conclusion These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca2+i handling and contractility. PMID:22199120

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.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells.

PubMed

Szaraz, Peter; Gratch, Yarden S; Iqbal, Farwah; Librach, Clifford L

2017-08-09

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells. Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein α, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. Our results demonstrate that this

Cavβ2 transcription start site variants modulate calcium handling in newborn rat cardiomyocytes.

PubMed

Moreno, Cristian; Hermosilla, Tamara; Morales, Danna; Encina, Matías; Torres-Díaz, Leandro; Díaz, Pablo; Sarmiento, Daniela; Simon, Felipe; Varela, Diego

2015-12-01

In the heart, the main pathway for calcium influx is mediated by L-type calcium channels, a multi-subunit complex composed of the pore-forming subunit CaV1.2 and the auxiliary subunits CaVα2δ1 and CaVβ2. To date, five distinct CaVβ2 transcriptional start site (TSS) variants (CaVβ2a-e) varying only in the composition and length of the N-terminal domain have been described, each of them granting distinct biophysical properties to the L-type current. However, the physiological role of these variants in Ca(2+) handling in the native tissue has not been explored. Our results show that four of these variants are present in neonatal rat cardiomyocytes. The contribution of those CaVβ2 TSS variants on endogenous L-type current and Ca(2+) handling was explored by adenoviral-mediated overexpression of each CaVβ2 variant in cultured newborn rat cardiomyocytes. As expected, all CaVβ2 TSS variants increased L-type current density and produced distinctive changes on L-type calcium channel (LTCC) current activation and inactivation kinetics. The characteristics of the induced calcium transients were dependent on the TSS variant overexpressed. Moreover, the amplitude of the calcium transients varied depending on the subunit involved, being higher in cardiomyocytes transduced with CaVβ2a and smaller in CaVβ2d. Interestingly, the contribution of Ca(2+) influx and Ca(2+) release on total calcium transients, as well as the sarcoplasmic calcium content, was found to be TSS-variant-dependent. Remarkably, determination of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) messenger RNA (mRNA) abundance and cell size change indicates that CaVβ2 TSS variants modulate the cardiomyocyte hypertrophic state. In summary, we demonstrate that expression of individual CaVβ2 TSS variants regulates calcium handling in cardiomyocytes and, consequently, has significant repercussion in the development of hypertrophy.

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

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.

sRAGE attenuates angiotensin II-induced cardiomyocyte hypertrophy by inhibiting RAGE-NFκB-NLRP3 activation.

PubMed

Lim, Soyeon; Lee, Myung Eun; Jeong, Jisu; Lee, Jiye; Cho, Soyoung; Seo, Miran; Park, Sungha

2018-05-23

The receptor for advanced glycation endproducts (RAGE) is an innate immunity receptor that has been implicated in the pathogenesis of atherosclerotic cardiovascular disease. However, the possibility that RAGE-mediated signaling is involved in angiotensin II (Ang II)-induced cardiac left ventricular hypertrophy has yet to be investigated. We therefore determined whether RAGE has a role in regulating pathological cardiac hypertrophy. Protein abundance was estimated using Western blotting and intracellular ROS level and phospho-p65 were detected using fluorescence microscopy. Enzyme-linked immunosorbent assay was used to detect HMGB1 and IL-1β. All in vitro experiments were performed using H9C2 cells. To induce cardiomyocyte hypertrophy, 300 nM Ang II was treated for 48 h and 2 µg/ml sRAGE was treated 1 h prior to addition of Ang II. sRAGE attenuated Ang II-induced cardiomyocyte hypertrophy by downregulating RAGE and angiotensin II type 1 receptor expression. Secretion levels of high motility group box 1 and interleukin-1β, estimated from a cell culture medium, were significantly reduced by sRAGE. Activated PKCs and ERK1/2, important signals in left ventricular hypertrophy (LVH) development, were downregulated by sRAGE treatment. Furthermore, we found that nuclear factor-κB and NOD-like receptor protein 3 (NLRP3) were associated with RAGE-mediated cardiomyocyte hypertrophy. In the context of these results, we conclude that RAGE induces cardiac hypertrophy through the activation of the PKCs-ERK1/2 and NF-κB-NLRP3-IL1β signaling pathway, and suggest that RAGE-NLRP3 may be an important mediator of Ang II-induced cardiomyocyte hypertrophy. In addition, we determined that inhibition of RAGE activation with soluble RAGE (sRAGE) has a protective effect on Ang II-induced cardiomyocyte hypertrophy.

Identifying panaxynol, a natural activator of nuclear factor erythroid-2 related factor 2 (Nrf2) from American ginseng as a suppressor of inflamed macrophage-induced cardiomyocyte hypertrophy

PubMed Central

Qu, Chen; Li, Bin; Lai, Yimu; Li, Hechu; Windust, Anthony; Hofseth, Lorne J.; Nagarkatti, Mitzi; Nagarkatti, Prakash; Wang, Xing Li; Tang, Dongqi; Janicki, Joseph S.; Tian, Xingsong; Cui, Taixing

2015-01-01

Ethnopharmacological relevance American ginseng is capable of ameliorating cardiac dysfunction and activating Nrf2, a master regulator of antioxidant defense, in the heart. This study was designed to isolate compounds from American ginseng and to determine those responsible for the Nrf2-mediated resolution of inflamed macrophage-induced cardiomyocyte hypertrophy. Materials and methods A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. A bioassay-based fractionization of American ginseng was performed to identify the putative substances which could activate Nrf2-mediated suppression of pro-inflammatory cytokine expression in macrophages and macrophage-mediated pro-hypertrophic growth in cardiomyocytes. Results A hexane fraction of an anti-inflammatory crude extract of American ginseng was found to be most effective in suppressing the inflammatory responses in macrophages. Preparative, reverse-phase HPLC and a comparative analysis by analytical scale LC–UV/MS revealed the hexane fraction contains predominantly C17 polyacetylenes and linolenic acid. Panaxynol, one of the major polyacetylenes, was found to be a potent Nrf2 activator. Panaxynol posttranscriptionally activated Nrf2 by inhibiting Kelch-like ECH-associated protein (Keap) 1-mediated degradation without affecting the binding of Keap1 and Nrf2. Moreover, panaxynol suppressed a selected set of cytokine expression via the activation of Nrf2 while minimally regulating nuclear factor-kappa B (NF-κB)-mediated cytokine expression in macrophages. It also dramatically inhibited the inflamed macrophage-mediated cardiomyocyte death and hypertrophy by activating Nrf2 in macrophages. Conclusions These results demonstrate that American ginseng-derived panaxynol is a specific Nrf2 activator and panaxynol-activated Nrf2 signaling is at least partly responsible for American ginseng-induced health benefit in the heart. PMID

[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.

Modulation of cardiac fibrosis by Krüppel-like factor 6 through transcriptional control of thrombospondin 4 in cardiomyocytes

PubMed Central

Sawaki, Daigo; Hou, Lianguo; Tomida, Shota; Sun, Junqing; Zhan, Hong; Aizawa, Kenichi; Son, Bo-Kyung; Kariya, Taro; Takimoto, Eiki; Otsu, Kinya; Conway, Simon J.; Manabe, Ichiro; Komuro, Issei; Friedman, Scott L.; Nagai, Ryozo; Suzuki, Toru

2015-01-01

Aims Krüppel-like factors (KLFs) are a family of transcription factors which play important roles in the heart under pathological and developmental conditions. We previously identified and cloned Klf6 whose homozygous mutation in mice results in embryonic lethality suggesting a role in cardiovascular development. Effects of KLF6 on pathological regulation of the heart were investigated in the present study. Methods and results Mice heterozygous for Klf6 resulted in significantly diminished levels of cardiac fibrosis in response to angiotensin II infusion. Intriguingly, a similar phenotype was seen in cardiomyocyte-specific Klf6 knockout mice, but not in cardiac fibroblast-specific knockout mice. Microarray analysis revealed increased levels of the extracellular matrix factor, thrombospondin 4 (TSP4), in the Klf6-ablated heart. Mechanistically, KLF6 directly suppressed Tsp4 expression levels, and cardiac TSP4 regulated the activation of cardiac fibroblasts to regulate cardiac fibrosis. Conclusion Our present studies on the cardiac function of KLF6 show a new mechanism whereby cardiomyocytes regulate cardiac fibrosis through transcriptional control of the extracellular matrix factor, TSP4, which, in turn, modulates activation of cardiac fibroblasts. PMID:25987545

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

Modulation of Ca2+ Activity in Cardiomyocytes through Caveolae-Gαq Interactions

PubMed Central

Guo, Yuanjian; Golebiewska, Urszula; Scarlata, Suzanne

2011-01-01

Cardiomyocytes have a complex Ca2+ behavior and changes in this behavior may underlie certain disease states. Intracellular Ca2+ activity can be regulated by the phospholipase Cβ–Gαq pathway localized on the plasma membrane. The plasma membranes of cardiomycoytes are rich in caveolae domains organized by caveolin proteins. Caveolae may indirectly affect cell signals by entrapping and localizing specific proteins. Recently, we found that caveolin may specifically interact with activated Gαq, which could affect Ca2+ signals. Here, using fluorescence imaging and correlation techniques we show that Gαq-Gβγ subunits localize to caveolae in adult ventricular canine cardiomyoctyes. Carbachol stimulation releases Gβγ subunits from caveolae with a concurrent stabilization of activated Gαq by caveolin-3 (Cav3). These cells show oscillating Ca2+ waves that are not seen in neonatal cells that do not contain Cav3. Microinjection of a peptide that disrupts Cav3-Gαq association, but not a control peptide, extinguishes the waves. Furthermore, these waves are unchanged with rynaodine treatment, but not seen with treatment of a phospholipase C inhibitor, implying that Cav3-Gαq is responsible for this Ca2+ activity. Taken together, these studies show that caveolae play a direct and active role in regulating basal Ca2+ activity in cardiomyocytes. PMID:21463572

β3-adrenergic receptor activation induces TGFβ1 expression in cardiomyocytes via the PKG/JNK/c-Jun pathway.

PubMed

Xu, Zhongcheng; Wu, Jimin; Xin, Junzhou; Feng, Yenan; Hu, Guomin; Shen, Jing; Li, Mingzhe; Zhang, Youyi; Xiao, Han; Wang, Li

2018-06-05

In heart failure, the expression of cardiac β 3 -adrenergic receptors (β 3 -ARs) increases. However, the precise role of β 3 -AR signaling within cardiomyocytes remains unclear. Transforming growth factor β1 (TGFβ1) is a crucial cytokine mediating the cardiac remodeling that plays a causal role in the progression of heart failure. Here, we set out to determine the effect of β 3 -AR activation on TGFβ1 expression in rat cardiomyocytes and examine the underlying mechanism. The selective β 3 -AR agonist BRL37344 induced an increase in TGFβ1 expression and the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun in β 3 -AR-overexpressing cardiomyocytes. Those effects of BRL37344 were suppressed by a β 3 -AR antagonist. Moreover, the inhibition of JNK and c-Jun activity by a JNK inhibitor and c-Jun siRNA blocked the increase in TGFβ1 expression upon β 3 -AR activation. A protein kinase G (PKG) inhibitor also attenuated β 3 -AR-agonist-induced TGFβ1 expression and the phosphorylation of JNK and c-Jun. In conclusion, the β 3 -AR activation in cardiomyocytes increases the expression of TGFβ1 via the PKG/JNK/c-Jun pathway. These results help us further understand the role of β 3 -AR signaling in heart failure. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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.

Cardiomyocyte specific expression of Acyl-coA thioesterase 1 attenuates sepsis induced cardiac dysfunction and mortality

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

Xia, Congying; Dong, Ruolan; Chen, Chen

Compromised cardiac fatty acid oxidation (FAO) induced energy deprivation is a critical cause of cardiac dysfunction in sepsis. Acyl-CoA thioesterase 1 (ACOT1) is involved in regulating cardiac energy production via altering substrate metabolism. This study aims to clarify whether ACOT1 has a potency to ameliorate septic myocardial dysfunction via enhancing cardiac FAO. Transgenic mice with cardiomyocyte specific expression of ACOT1 (αMHC-ACOT1) and their wild type (WT) littermates were challenged with Escherichia coli lipopolysaccharide (LPS; 5 mg/kg i.p.) and myocardial function was assessed 6 h later using echocardiography and hemodynamics. Deteriorated cardiac function evidenced by reduction of the percentage of left ventricular ejectionmore » fraction and fractional shortening after LPS administration was significantly attenuated by cardiomyocyte specific expression of ACOT1. αMHC-ACOT1 mice exhibited a markedly increase in glucose utilization and cardiac FAO compared with LPS-treated WT mice. Suppression of cardiac peroxisome proliferator activated receptor alpha (PPARa) and PPARγ-coactivator-1α (PGC1a) signaling observed in LPS-challenged WT mice was activated by the presence of ACOT1. These results suggest that ACOT1 has potential therapeutic values to protect heart from sepsis mediated dysfunction, possibly through activating PPARa/PGC1a signaling. - Highlights: • ACOT1 has potential therapeutic values to protect heart from sepsis mediated dysfunction. • ACOT1 can regulate PPARa/PGC1a signaling pathway. • We first generate the transgenic mice with cardiomyocyte specific expression of ACOT1.« less

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.

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).

skNAC, a Smyd1-interacting transcription factor, is involved in cardiac development and skeletal muscle growth and regeneration.

PubMed

Park, Chong Yon; Pierce, Stephanie A; von Drehle, Morgan; Ivey, Kathryn N; Morgan, Jayson A; Blau, Helen M; Srivastava, Deepak

2010-11-30

Cardiac and skeletal muscle development and maintenance require complex interactions between DNA-binding proteins and chromatin remodeling factors. We previously reported that Smyd1, a muscle-restricted histone methyltransferase, is essential for cardiogenesis and functions with a network of cardiac regulatory proteins. Here we show that the muscle-specific transcription factor skNAC is the major binding partner for Smyd1 in the developing heart. Targeted deletion of skNAC in mice resulted in partial embryonic lethality by embryonic day 12.5, with ventricular hypoplasia and decreased cardiomyocyte proliferation that were similar but less severe than in Smyd1 mutants. Expression of Irx4, a ventricle-specific transcription factor down-regulated in hearts lacking Smyd1, also depended on the presence of skNAC. Viable skNAC(-/-) adult mice had reduced postnatal skeletal muscle growth and impaired regenerative capacity after cardiotoxin-induced injury. Satellite cells isolated from skNAC(-/-) mice had impaired survival compared with wild-type littermate satellite cells. Our results indicate that skNAC plays a critical role in ventricular cardiomyocyte expansion and regulates postnatal skeletal muscle growth and regeneration in mice.

[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.

Brief Report: Oxidative Stress Mediates Cardiomyocyte Apoptosis in a Human Model of Danon Disease and Heart Failure

PubMed Central

Hashem, Sherin I.; Perry, Cynthia N.; Bauer, Matthieu; Han, Sangyoon; Clegg, Stacey D.; Ouyang, Kunfu; Deacon, Dekker C.; Spinharney, Mary; Panopoulos, Athanasia D.; Belmonte, Juan Carlos Izpisua; Frazer, Kelly A.; Chen, Ju; Gong, Qiuming; Zhou, Zhengfeng; Chi, Neil C.; Adler, Eric D.

2015-01-01

Danon disease is a familial cardiomyopathy associated with impaired autophagy due to mutations in the gene encoding lysosomal-associated membrane protein type 2 (LAMP-2). Emerging evidence has highlighted the importance of autophagy in regulating cardiomyocyte bioenergetics, function, and survival. However, the mechanisms responsible for cellular dysfunction and death in cardiomyocytes with impaired autophagic flux remain unclear. To investigate the molecular mechanisms responsible for Danon disease, we created induced pluripotent stem cells (iPSCs) from two patients with different LAMP-2 mutations. Danon iPSC-derived cardiomyocytes (iPSC-CMs) exhibited impaired autophagic flux and key features of heart failure such as increased cell size, increased expression of natriuretic peptides, and abnormal calcium handling compared to control iPSC-CMs. Additionally, Danon iPSC-CMs demonstrated excessive amounts of mitochondrial oxidative stress and apoptosis. Using the sulfhydryl antioxidant N-acetylcysteine to scavenge free radicals resulted in a significant reduction in apoptotic cell death in Danon iPSC-CMs. In summary, we have modeled Danon disease using human iPSC-CMs from patients with mutations in LAMP-2, allowing us to gain mechanistic insight into the pathogenesis of this disease. We demonstrate that LAMP-2 deficiency leads to an impairment in autophagic flux, which results in excessive oxidative stress, and subsequent cardiomyocyte apoptosis. Scavenging excessive free radicals with antioxidants may be beneficial for patients with Danon disease. In vivo studies will be necessary to validate this new treatment strategy. PMID:25826782

Gab1 Is Modulated by Chronic Hypoxia in Children with Cyanotic Congenital Heart Defect and Its Overexpression Reduces Apoptosis in Rat Neonatal Cardiomyocytes

PubMed Central

Cherif, Myriam; Nakaoka, Yoshikazu; Angelini, Gianni D.; Ghorbel, Mohamed T.

2015-01-01

Gab1 (Grb2 associated binding protein 1) is a member of the scaffolding/docking proteins (Gab1, Gab2, and Gab3). It is required for fibroblast cell survival and maintaining cardiac function. Very little is known about human Gab1 expression in response to chronic hypoxia. The present study examined the hypothesis that hypoxia regulates Gab1 expression in human paediatric myocardium and cultured rat cardiomyocytes. Here we showed that Gab1 is expressed in myocardial tissue in acyanotic and cyanotic children with congenital heart defects. Gab1 protein was upregulated in cyanotic compared to acyanotic hearts suggesting that Gab1 upregulation is a component of the survival program initiated by hypoxia in cyanotic children. The expression of other Gab1 interacting partners was not affected by hypoxia and Gab1 regulation. Additionally, using an in vitro model, we demonstrated that overexpressing Gab1 in neonatal cardiomyocytes, under hypoxic condition, resulted in the reduction of apoptosis suggesting a role for this protein in cardiomyocyte survival. Altogether, our data provide strong evidence that Gab1 is important for heart cell survival following hypoxic stress. PMID:26090437

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

Resveratrol protects the loss of connexin 43 induced by ethanol exposure in neonatal mouse cardiomyocytes.

PubMed

Tu, Su; Cao, Fu-Tao; Fan, Xiao-Chun; Yang, Cheng-Jian

2017-06-01

Excessive alcohol consumption provides risk to cardiomyopathy with unknown mechanisms. Resveratrol, a plant polyphenol, is widely reported for its cardiovascular benefits, while its effect on alcohol-induced impairments in cardiomyocytes largely remains unknown. Effects of resveratrol on the cardiomyocytes under ethanol insult were studied in vitro. Ethanol exposure in mouse neonatal cardiomyocytes increased cell death and induced a specific loss of tight junction protein, connexin 43. In spite of adverse effects at higher concentrations, resveratrol at 10 μM improved cell viability of cardiomyocytes in the presence of a deleterious dose of ethanol. Importantly, the co-treatment of resveratrol with ethanol exhibited the restoration of connexin 43 protein. Further assays showed that these effects were likely associated with the antioxidative actions of resveratrol, and correlated with the alleviation of MAP kinase activation in cultured cardiomyocytes in response to ethanol. Our data suggests a novel mechanism of cardiomyocyte cell loss under ethanol exposure and provides new evidence of protective effects of resveratrol in the cardiomyocytes.

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.

Investigation of the cardiomyocyte dysfunction in bradykinin type 2 receptor knockout mice.

PubMed

Roman-Campos, Danilo; Duarte, Hugo Leonardo; Gomes, Enéas Ricardo; Castro, Carlos Henrique; Guatimosim, Silvia; Natali, Antonio José; Almeida, Alvair Pinto; Pesquero, João Bosco; Pesquero, Jorge Luiz; Cruz, Jader Santos

2010-12-18

Bradykinin type 2 receptor (B(2)R) is the key component to trigger the intracellular signaling pathway in response to bradykinin under physiological conditions. The present study sought to investigate whether the B(2)R gene deletion will have an impact on myocardial function. Isolated cell shortening, patch-clamp technique, Western blot and confocal microscopy. Isolated cell shortening measurements showed significant reduction in B(2)R knockout (B(2)R(-/-)) left ventricular cardiac myocytes' shortening. Whole-cell recordings were used to study the electrophysiological aspects of the left ventricular B(2)R(-/-) cardiomyocytes. Results showed: 1) action potential lengthening; 2) unchanged inwardly rectifying K(+) current; 3) reduced transient outward K(+) (I(to)) and L-type Ca(2+) current densities; 5) changes in kinetic properties related to I(to) and I(Ca,L). In addition, transient sarcoplasmic reticulum (SR) Ca(2+) release was found to be smaller in B(2)R(-/-) cardiomyocytes. Importantly, evidence is provided that NO constitutive production is, at least in part, responsible for the reported electrophysiological modifications observed in cardiomyocytes from B(2)R(-/-) mice. Surprisingly, NO is not involved in the SR Ca(2+) release reduction as demonstrated in the present study. Taken together, our findings indicate that B(2)R plays a fundamental role in the regulation of cardiac function and Ca(2+) homeostasis, probably through a NO dependent pathway. These results may contribute to our understanding of the kinins participation in the control of cardiac function. Copyright © 2010 Elsevier Inc. All rights reserved.

Do Binucleate Cardiomyocytes Have A Role in Myocardial Repair? Insights Using Isolated Rodent Myocytes and Cell Culture

PubMed Central

Stephen, Michael J; Poindexter, Brian J; Moolman, Johan A; Sheikh-Hamad, David; Bick, Roger J

2009-01-01

Neonatal and adult cardiomyocytes were isolated from rat hearts. Some of the adult myocytes were cultured to allow for cell dedifferentiation, a phenomenon thought to mimic cell changes that occur in stressed myocardium, with myocytes regressing to a fetal pattern of metabolism and stellate neonatal shape. Using fluorescence deconvolution microscopy, cells were probed with fluorescent markers and scanned for a number of proteins associated with ion control, calcium movements and cardiac function. Image analysis of deconvoluted image stacks and sequential real-time image recordings of calcium transients of cells were made. All three myocyte groups were predominantly comprised of binucleate cells. Clustering of proteins to a single nucleus was a common observation, suggesting that one nucleus is active in protein synthesis pathways, while the other nucleus assumes a ‘dormant’ or different role and that cardiomyocytes might be mitotically active even in late development, or specific protein syntheses could be targeted and regulated for reintroduction into the cell cycle. Such possibilities would extend cardiac disease associated stem cell research and therapy options, while producing valuable insights into developmental and death pathways of binucleate cardiomyocytes (word count 183). PMID:19430572

Soluble CD14 inhibits contractile function and insulin action in primary adult rat cardiomyocytes.

PubMed

Overhagen, Sabrina; Blumensatt, Marcel; Fahlbusch, Pia; Herzfeld de Wiza, Daniella; Müller, Heidi; Maxhera, Bujar; Akhyari, Payam; Ouwens, D Margriet

2017-02-01

Epicardial adipose tissue (EAT) from patients with type 2 diabetes (T2D) is characterized by monocyte infiltrations and displays an elevated release of the monocyte marker soluble cluster of differentiation 14 (sCD14) versus EAT from patients without T2D. We propose that an increased abundance of sCD14 in EAT from patients with T2D may impair the function and insulin sensitivity of the adjacent cardiomyocytes. To examine this, primary adult rat cardiomyocytes were incubated with increasing concentrations of sCD14 in the presence and absence of the co-receptor lipopolysaccharide (LPS), and analyzed for effects on determinants of contractile function, activation of inflammation signalling and insulin action. Exposing cardiomyocytes to sCD14 increased the phosphorylation of the stress kinases p38 and extracellular-signal regulated kinase (ERK). In contrast, insulin-mediated phosphorylation of Akt on Thr308 and Ser473 was inhibited. Furthermore, sCD14 impaired sarcomere shortening and cytosolic Ca 2+ -fluxes. All responses were concentration-dependent and became significant at 1ng/ml sCD14. LPS, either alone or in complex with sCD14, did not affect contractile function or the activation of stress kinases and insulin signalling pathways. Similar data on protein phosphorylation were obtained when exposing human umbilical vein endothelial cells to sCD14. Finally, pharmacological inhibition of p38 reversed the detrimental effects of sCD14 on contractile function, but not on sCD14-induced insulin resistance. Collectively, these data show that sCD14 impairs the function and insulin sensitivity of cardiomyocytes, suggesting that an enhanced sCD14 release from EAT in patients with T2D may contribute to the pathogenesis of diabetes-related cardiometabolic complications. Copyright © 2016 Elsevier B.V. All rights reserved.

iPSC-derived cardiomyocytes reveal abnormal TGFβ signaling in left ventricular non-compaction cardiomyopathy

PubMed Central

Kodo, Kazuki; Ong, Sang-Ging; Jahanbani, Fereshteh; Termglinchan, Vittavat; Hirono, Keiichi; InanlooRahatloo, Kolsoum; Ebert, Antje D.; Shukla, Praveen; Abilez, Oscar J.; Churko, Jared M.; Karakikes, Ioannis; Jung, Gwanghyun; Ichida, Fukiko; Wu, Sean M.; Snyder, Michael P.; Bernstein, Daniel; Wu, Joseph C.

2016-01-01

Left ventricular non-compaction (LVNC) is the third most prevalent cardiomyopathy in children and its pathogenesis has been associated with the developmental defect of the embryonic myocardium. We show that patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from LVNC patients carrying a mutation in the cardiac transcription factor TBX20 recapitulate a key aspect of the pathological phenotype at the single-cell level and was associated with perturbed transforming growth factor beta (TGFβ) signaling. LVNC iPSC-CMs have decreased proliferative capacity due to abnormal activation of TGFβ signaling. TBX20 regulates the expression of TGFβ signaling modifiers including a known genetic cause of LVNC, PRDM16, and genome editing of PRDM16 caused proliferation defects in iPSC-CMs. Inhibition of TGFβ signaling and genome correction of the TBX20 mutation were sufficient to reverse the disease phenotype. Our study demonstrates that iPSC-CMs are a useful tool for the exploration of pathological mechanisms underlying poorly understood cardiomyopathies including LVNC. PMID:27642787

Direct Interaction of CaVβ with Actin Up-regulates L-type Calcium Currents in HL-1 Cardiomyocytes*

PubMed Central

Stölting, Gabriel; de Oliveira, Regina Campos; Guzman, Raul E.; Miranda-Laferte, Erick; Conrad, Rachel; Jordan, Nadine; Schmidt, Silke; Hendriks, Johnny; Gensch, Thomas; Hidalgo, Patricia

2015-01-01

Expression of the β-subunit (CaVβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVβ binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVβ2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVβ2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVβ mediated L-type up-regulation, and CaVβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVβ that is directly involved in vesicular trafficking. We propose a model in which CaVβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane. PMID:25533460

Regulated expression and role of c-Myb in the cardiovascular-directed differentiation of mouse embryonic stem cells.

PubMed

Ishida, Masayoshi; El-Mounayri, Omar; Kattman, Steven; Zandstra, Peter; Sakamoto, Hiroshi; Ogawa, Minetaro; Keller, Gordon; Husain, Mansoor

2012-01-20

c-myb null (knockout) embryonic stem cells (ESC) can differentiate into cardiomyocytes but not contractile smooth muscle cells (SMC) in embryoid bodies (EB). To define the role of c-Myb in SMC differentiation from ESC. In wild-type (WT) EB, high c-Myb levels on days 0-2 of differentiation undergo ubiquitin-mediated proteosomal degradation on days 2.5-3, resurging on days 4-6, without changing c-myb mRNA levels. Activin-A and bone morphogenetic protein 4-induced cardiovascular progenitors were isolated by FACS for expression of vascular endothelial growth factor receptor (VEGFR)2 and platelet-derived growth factor receptor (PDGFR)α. By day 3.75, hematopoesis-capable VEGFR2+ cells were fewer, whereas cardiomyocyte-directed VEGFR2+/PDGFRα+ cells did not differ in abundance in knockout versus WT EB. Importantly, highest and lowest levels of c-Myb were observed in VEGFR2+ and VEGFR2+/PDGFRα+ cells, respectively. Proteosome inhibitor MG132 and lentiviruses enabling inducible expression or knockdown of c-myb were used to regulate c-Myb in WT and knockout EB. These experiments showed that c-Myb promotes expression of VEGFR2 over PDGFRα, with chromatin immunopreciptation and promoter-reporter assays defining specific c-Myb-responsive binding sites in the VEGFR2 promoter. Next, FACS-sorted VEGFR2+ cells expressed highest and lowest levels of SMC- and fibroblast-specific markers, respectively, at days 7-14 after retinoic acid (RA) as compared with VEGFR2+/PDGFRα+ cells. By contrast, VEGFR2+/PDGFRα+ cells cultured without RA beat spontaneously, like cardiomyocytes between days 7 and 14, and expressed cardiac troponin. Notably, RA was required to more fully differentiate SMC from VEGFR2+ cells and completely blocked differentiation of cardiomyocytes from VEGFR2+/PDGFRα+ cells. c-Myb is tightly regulated by proteosomal degradation during cardiovascular-directed differentiation of ESC, expanding early-stage VEGFR2+ progenitors capable of RA-responsive SMC formation.

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.

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

Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.

PubMed

Willey, Christopher D; Balasubramanian, Sundaravadivel; Rodríguez Rosas, María C; Ross, Robert S; Kuppuswamy, Dhandapani

2003-06-01

In pressure-overloaded myocardium, our recent study demonstrated cytoskeletal assembly of c-Src and other signaling proteins which was partially mimicked in vitro using adult feline cardiomyocytes embedded in three-dimensional (3D) collagen matrix and stimulated with an integrin-binding Arg-Gly-Asp (RGD) peptide. In the present study, we improved this model further to activate c-Src and obtain a full assembly of the focal adhesion complex (FAC), and characterized c-Src localization and integrin subtype(s) involved. RGD dose response experiments revealed that c-Src activation occurs subsequent to its cytoskeletal recruitment and is accompanied by p130Cas cytoskeletal binding and focal adhesion kinase (FAK) Tyr925 phosphorylation. When cardiomyocytes expressing hexahistidine-tagged c-Src via adenoviral gene delivery were used for RGD stimulation, the expressed c-Src exhibited relocation: (i) biochemical analysis revealed c-Src movement from the detergent-soluble to the -insoluble cytoskeletal fraction and (ii) confocal microscopic analysis showed c-Src movement from a nuclear/perinuclear to a sarcolemmal region. RGD treatment also caused sarcolemmal co-localization of FAK and vinculin. Characterization of integrin subtypes revealed that beta3, but not beta1, integrin plays a predominant role: (i) expression of cytoplasmic domain of beta1A integrin did not affect the RGD-stimulated FAC formation and (ii) both pressure-overloaded myocardium and RGD-stimulated cardiomyocytes exhibited phosphorylation of beta3 integrin at Tyr773/785 sites but not beta1 integrin at Thr788/789 sites. Together these data indicate that RGD treatment in cardiomyocytes causes beta3 integrin activation and c-Src sarcolemmal localization, that subsequent c-Src activation is accompanied by p130Cas binding and FAK Tyr925 phosphorylation, and that these events might be crucial for growth and remodeling of hypertrophying adult cardiomyocytes.

Myostatin regulates energy homeostasis in the heart and prevents heart failure.

PubMed

Biesemann, Nadine; Mendler, Luca; Wietelmann, Astrid; Hermann, Sven; Schäfers, Michael; Krüger, Marcus; Boettger, Thomas; Borchardt, Thilo; Braun, Thomas

2014-07-07

Myostatin is a major negative regulator of skeletal muscle mass and initiates multiple metabolic changes, including enhanced insulin sensitivity. However, the function of myostatin in the heart is barely understood, although it is upregulated in the myocardium under several pathological conditions. Here, we aimed to decipher the role of myostatin and myostatin-dependent signaling pathways for cardiac function and cardiac metabolism in adult mice. To avoid potential counterregulatory mechanisms occurring in constitutive and germ-line-based myostatin mutants, we generated a mouse model that allows myostatin inactivation in adult cardiomyocytes. Cardiac MRI revealed that genetic inactivation of myostatin signaling in the adult murine heart caused cardiac hypertrophy and heart failure, partially recapitulating effects of the age-dependent decline of the myostatin paralog growth and differentiation factor 11. We found that myostatin represses AMP-activated kinase activation in the heart via transforming growth factor-β-activated kinase 1, thereby preventing a metabolic switch toward glycolysis and glycogen accumulation. Furthermore, myostatin stimulated expression of regulator of G-protein signaling 2, a GTPase-activating protein that restricts Gaq and Gas signaling and thereby protects against cardiac failure. Inhibition of AMP-activated kinase in vivo rescued cardiac hypertrophy and prevented enhanced glycolytic flow and glycogen accumulation after inactivation of myostatin in cardiomyocytes. Our results uncover an important role of myostatin in the heart for maintaining cardiac energy homeostasis and preventing cardiac hypertrophy. © 2014 American Heart Association, Inc.

MicroRNA-363 negatively regulates the left ventricular determining transcription factor HAND1 in human embryonic stem cell-derived cardiomyocytes.

PubMed

Wagh, Vilas; Pomorski, Alexander; Wilschut, Karlijn J; Piombo, Sebastian; Bernstein, Harold S

2014-06-06

Posttranscriptional control of mRNA by microRNA (miRNA) has been implicated in the regulation of diverse biologic processes from directed differentiation of stem cells through organism development. We describe a unique pathway by which miRNA regulates the specialized differentiation of cardiomyocyte (CM) subtypes. We differentiated human embryonic stem cells (hESCs) to cardiac progenitor cells and functional CMs, and characterized the regulated expression of specific miRNAs that target transcriptional regulators of left/right ventricular-subtype specification. From >900 known human miRNAs in hESC-derived cardiac progenitor cells and functional CMs, a subset of differentially expressed cardiac miRNAs was identified, and in silico analysis predicted highly conserved binding sites in the 3'-untranslated regions (3'UTRs) of Hand-and-neural-crest-derivative-expressed (HAND) genes 1 and 2 that are involved in left and right ventricular development. We studied the temporal and spatial expression patterns of four miRNAs in differentiating hESCs, and found that expression of miRNA (miR)-363, miR-367, miR-181a, and miR-181c was specific for stage and site. Further analysis showed that miR-363 overexpression resulted in downregulation of HAND1 mRNA and protein levels. A dual luciferase reporter assay demonstrated functional interaction of miR-363 with the full-length 3'UTR of HAND1. Expression of anti-miR-363 in-vitro resulted in enrichment for HAND1-expressing CM subtype populations. We also showed that BMP4 treatment induced the expression of HAND2 with less effect on HAND1, whereas miR-363 overexpression selectively inhibited HAND1. These data show that miR-363 negatively regulates the expression of HAND1 and suggest that suppression of miR-363 could provide a novel strategy for generating functional left-ventricular CMs.

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.

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

PubMed

Naito, Daisuke; Ogata, Takehiro; Hamaoka, Tetsuro; Nakanishi, Naohiko; Miyagawa, Kotaro; Maruyama, Naoki; Kasahara, Takeru; Taniguchi, Takuya; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

2015-12-15

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function. Copyright © 2015 the American Physiological Society.

Impedimetric Analysis of the Effect of Decellularized Porcine Heart Scaffold on Human Fibrosarcoma, Endothelial, and Cardiomyocyte Cell Lines

PubMed Central

Bäcker, Henrik; Polgár, Livia; Soós, Pal; Lajkó, Eszter; Láng, Orsolya; Merkely, Bela; Szabó, Gabor; Dohmen, Pascal M.; Weymann, Alexander; Kőhidai, Laszlo

2017-01-01

Background Experiments on porcine heart scaffold represent significant assays in development of immunoneutral materials for cardiac surgery. Characterization of cell-cell and cell-scaffold interactions is essential to understand the homing process of cardiac cells into the scaffolds. Material/Methods In the present study, the highly sensitive and real-time impedimetric technique of xCELLigence SP was used to monitor cell adhesion, which is the key process of recellularization in heart scaffolds. Our objectives were: (i) to characterize the effect of decellularized porcine heart scaffold on cell adhesion of human cardiovascular cells potentially used in the recellularization process; and (ii) to investigate cell-extracellular matrix element interactions for building artificial multi-layer systems, applied as cellular models of recellularization experiments. Human fibrosarcoma, endothelial, and cardiomyocyte cells were investigated and the effect of decellularized porcine heart scaffold (HS) and fibronectin on cell adhesion was examined. Adhesion was quantified as slope of curves. Results Heart scaffold had neutral effect on cardiomyocytes as well as on endothelial cells. Adhesion of cardiomyocytes was increased by fibronectin (1.480±0.021) compared to control (0.745±0.029). The combination of fibronectin and HS induced stronger adhesion of cardiomyocytes (2.407±0.634) than fibronectin alone. Endothelial and fibrosarcoma cells showed similarly strong adhesion profiles with marked enhancer effect by fibronectin. Conclusions Decellularized porcine HS does not inhibit adhesion of human cardiovascular cells at the cell biological level, while fibronectin has strong cell adhesion-inducer effect, as well as an enhancer effect on activity of HS. Consequently, decellularized porcine hearts could be used as scaffolds for recellularization with cardiomyocytes and endothelial cells with fibronectin acting as a regulator, leading to construction of working bioartificial

Translational Control of FOG-2 Expression in Cardiomyocytes by MicroRNA-130a

PubMed Central

Kim, Gene H.; Samant, Sadhana A.; Earley, Judy U.; Svensson, Eric C.

2009-01-01

MicroRNAs are increasingly being recognized as regulators of embryonic development; however, relatively few microRNAs have been identified to regulate cardiac development. FOG-2 (also known as zfpm2) is a transcriptional co-factor that we have previously shown is critical for cardiac development. In this report, we demonstrate that FOG-2 expression is controlled at the translational level by microRNA-130a. We identified a conserved region in the FOG-2 3′ untranslated region predicted to be a target for miR-130a. To test the functional significance of this site, we generated an expression construct containing the luciferase coding region fused with the 3′ untranslated region of FOG-2 or a mutant version lacking this microRNA binding site. When these constructs were transfected into NIH 3T3 fibroblasts (which are known to express miR-130a), we observed a 3.3-fold increase in translational efficiency when the microRNA target site was disrupted. Moreover, knockdown of miR-130a in fibroblasts resulted in a 3.6-fold increase in translational efficiency. We also demonstrate that cardiomyocytes express miR-130a and can attenuate translation of mRNAs with a FOG-2 3′ untranslated region. Finally, we generated transgenic mice with cardiomyocyte over-expression of miR-130a. In the hearts of these mice, FOG-2 protein levels were reduced by as much as 80%. Histological analysis of transgenic embryos revealed ventricular wall hypoplasia and ventricular septal defects, similar to that seen in FOG-2 deficient hearts. These results demonstrate the importance of miR-130a for the regulation of FOG-2 protein expression and suggest that miR-130a may also play a role in the regulation of cardiac development. PMID:19582148

Cardiomyocyte binucleation is associated with aberrant mitotic microtubule distribution, mislocalization of RhoA and IQGAP3, as well as defective actomyosin ring anchorage and cleavage furrow ingression.

PubMed

Leone, Marina; Musa, Gentian; Engel, Felix Benedikt

2018-03-07

After birth mammalian cardiomyocytes initiate a last cell cycle which results in binucleation due to cytokinesis failure. Despite its importance for cardiac regenerative therapies, this process is poorly understood. Here, we aimed at a better understanding of the difference between cardiomyocyte proliferation and binucleation, and providing a new tool to distinguish these two processes. Monitoring of cell division by time-lapse imaging revealed that rat cardiomyocyte binucleation stems from a failure to properly ingress the cleavage furrow. Astral microtubule required for actomyosin ring anchorage and thus furrow ingression were not symmetrically distributed at the periphery of the equatorial region during anaphase in binucleating cardiomyocytes. Consequently, RhoA, the master regulator of actomyosin ring formation and constriction, non-muscle myosin IIB, a central component of the actomyosin ring, as well as IQGAP3 were abnormally localized during cytokinesis. In agreement with improper furrow ingression, binucleation in vitro as well as in vivo was associated with a failure of RhoA as well as IQGAP3 to localize to the stembody of the midbody. Taken together, these results indicate that naturally occurring cytokinesis failure in primary cardiomyocytes is due to an aberrant mitotic microtubule apparatus resulting in inefficient anchorage of the actomyosin ring to the plasma cell membrane. Thus, cardiomyocyte binucleation and division can be discriminated by the analysis of RhoA as well as IQGAP3 localization.

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

Disturbance of cardiac gene expression and cardiomyocyte structure predisposes Mecp2-null mice to arrhythmias

PubMed Central

Hara, Munetsugu; Takahashi, Tomoyuki; Mitsumasu, Chiaki; Igata, Sachiyo; Takano, Makoto; Minami, Tomoko; Yasukawa, Hideo; Okayama, Satoko; Nakamura, Keiichiro; Okabe, Yasunori; Tanaka, Eiichiro; Takemura, Genzou; Kosai, Ken-ichiro; Yamashita, Yushiro; Matsuishi, Toyojiro

2015-01-01

Methyl-CpG-binding protein 2 (MeCP2) is an epigenetic regulator of gene expression that is essential for normal brain development. Mutations in MeCP2 lead to disrupted neuronal function and can cause Rett syndrome (RTT), a neurodevelopmental disorder. Previous studies reported cardiac dysfunction, including arrhythmias in both RTT patients and animal models of RTT. In addition, recent studies indicate that MeCP2 may be involved in cardiac development and dysfunction, but its role in the developing and adult heart remains unknown. In this study, we found that Mecp2-null ESCs could differentiate into cardiomyocytes, but the development and further differentiation of cardiovascular progenitors were significantly affected in MeCP2 deficiency. In addition, we revealed that loss of MeCP2 led to dysregulation of endogenous cardiac genes and myocardial structural alterations, although Mecp2-null mice did not exhibit obvious cardiac functional abnormalities. Furthermore, we detected methylation of the CpG islands in the Tbx5 locus, and showed that MeCP2 could target these sequences. Taken together, these results suggest that MeCP2 is an important regulator of the gene-expression program responsible for maintaining normal cardiac development and cardiomyocyte structure. PMID:26073556

In vivo cardiomyocyte response to YTX- and AZA-1-induced damage: autophagy versus apoptosis.

PubMed

Ferreiro, Sara F; Vilariño, Natalia; Carrera, Cristina; Louzao, M Carmen; Santamarina, Germán; Cantalapiedra, Antonio G; Cifuentes, J Manuel; Crespo, Andrés; Botana, Luis M

2017-04-01

Yessotoxins (YTX) and azaspiracids (AZAs) are marine toxins produced by phytoplanktonic dinoflagellates that get accumulated in filter feeding shellfish and finally reach human consumers through the food web. Both toxin classes are worldwide distributed, and food safety authorities have regulated their content in shellfish in many countries. Recently, YTXs and AZAs have been described as compounds with subacute cardiotoxic potential in rats owed to alterations of the cardiovascular function and ultrastructural heart damage. These molecules are also well known in vitro inducers of cell death. The aim of this study was to explore the presence of cardiomyocyte death after repeated subacute exposure of rats to AZA-1 and YTX for 15 days. Because autophagy and apoptosis are often found in dying cardiomyocytes, several autophagic and apoptotic markers were determined by western blot in heart tissues of these rats. The results showed that hearts from YTX-treated rats presented increased levels of the autophagic markers microtubule-associated protein light chain 3-II (LC3-II) and beclin-1, nevertheless AZA-1-treated hearts evidenced increased levels of the apoptosis markers cleaved caspase-3 and -8, cleaved PARP and Fas ligand. Therefore, while YTX-induced damage to the heart triggers autophagic processes, apoptosis activation occurs in the case of AZA-1. For the first time, activation of cell death signals in cardiomyocytes is demonstrated for these toxins with in vivo experiments, which may be related to alterations of the cardiovascular function.

Myostatin induces interstitial fibrosis in the heart via TAK1 and p38.

PubMed

Biesemann, Nadine; Mendler, Luca; Kostin, Sawa; Wietelmann, Astrid; Borchardt, Thilo; Braun, Thomas

2015-09-01

Myostatin, a member of the TGF-β superfamily of secreted growth factors, is a negative regulator of skeletal muscle growth. In the heart, it is expressed at lower levels compared to skeletal muscle but up-regulated under disease conditions. Cre recombinase-mediated inactivation of myostatin in adult cardiomyocytes leads to heart failure and increased mortality but cardiac function of surviving mice is restored after several weeks probably due to compensatory expression in non-cardiomyocytes. To study long-term effects of increased myostatin expression in the heart and to analyze the putative crosstalk between cardiomyocytes and fibroblasts, we overexpressed myostatin in cardiomyocytes. Increased expression of myostatin in heart muscle cells caused interstitial fibrosis via activation of the TAK-1-MKK3/6-p38 signaling pathway, compromising cardiac function in older mice. Our results uncover a novel role of myostatin in the heart and highlight the necessity for tight regulation of myostatin to maintain normal heart function.

Selenium deficiency-induced thioredoxin suppression and thioredoxin knock down disbalanced insulin responsiveness in chicken cardiomyocytes through PI3K/Akt pathway inhibition.

PubMed

Yang, Jie; Hamid, Sattar; Cai, Jingzeng; Liu, Qi; Xu, Shiwen; Zhang, Ziwei

2017-10-01

Thioredoxin (Txn) system is the most crucial antioxidant defense mechanism in cell consisting of Txn, thioredoxin reductase (TR) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH). Perturbations in Txn system may compromise cell survival through oxidative stress induction. Metabolic activity of insulin plays important roles in fulfilling the stable and persistent demands of heart through glucose metabolism. However, the roles of Txn and Txn system in insulin modulated cardiac energy metabolism have been less reported. Therefore, to investigate the role of Txn in myocardial metabolism, we developed a Se-deficient chicken model (0.033mg/kg) for in-vivo and Txn knock down cardiomyocytes culture model (siRNA) for in-vitro studies. Quantitative real time PCR and western blotting was performed. Se deficiency suppressed Txn and TR in cardiac tissues. Significant increases in ROS (P<0.05) levels signify the onset of oxidative stress and in both models. Se deficiency-induced Txn suppression model and Txn knock down cardiomyocytes models significantly decreased (P<0.05), the mRNA and protein levels of insulin-like growth factors (IGF1, IGF2), IGF-binding proteins (IGFBP2, IGFBP4), insulin receptor (IR), insulin receptor substrates (IRS1, IRS2), and glucose transporters (GLUT1, GLUT3, GLUT8), however, IGFBP3 expression increased in Txn knock down cardiomyocytes. In addition, in contrast to their respective controls, Se deficiency-induced Txn depleted tissues and Txn deleted cardiomyocytes showed suppression in mRNA and protein levels of PI3K, AKT, P-PI3K, and repression in FOX, P-FOX JNK genes. Combing the in vitro and in vivo experiments, we demonstrate that Txn gene suppression can cause dysfunction of insulin-modulated cardiac energy metabolism and increase insulin resistance through PI3K-Akt pathway inhibition. Herein, we conclude that inactivation of Txn system can alter cellular insulin response through IRS/PI3K/Akt pathway repression and JNK and FOX expression

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.

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.

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

HSF1 phosphorylation by ERK/GSK3 suppresses RNF126 to sustain IGF-IIR expression for hypertension-induced cardiomyocyte hypertrophy.

PubMed

Huang, Chih-Yang; Lee, Fa-Lun; Peng, Shu-Fen; Lin, Kuan-Ho; Chen, Ray-Jade; Ho, Tsung-Jung; Tsai, Fu-Jen; Padma, Vijaya V; Kuo, Wei-Wen; Huang, Chih-Yang

2018-02-01

Hypertension-induced cardiac hypertrophy and apoptosis are major characteristics of early-stage heart failure (HF). Inhibition of extracellular signal-regulated kinases (ERK) efficaciously suppressed angiotensin II (ANG II)-induced cardiomyocyte hypertrophy and apoptosis by blocking insulin-like growth factor II receptor (IGF-IIR) signaling. However, the detailed mechanism by which ANG II induces ERK-mediated IGF-IIR signaling remains elusive. Here, we found that ANG II activated ERK to upregulate IGF-IIR expression via the angiotensin II type I receptor (AT 1 R). ERK activation subsequently phosphorylates HSF1 at serine 307, leading to a secondary phosphorylation by glycogen synthase kinase III (GSK3) at serine 303. Moreover, we found that ANG II mediated ERK/GSK3-induced IGF-IIR protein stability by downregulating the E3 ubiquitin ligase of IGF-IIR RING finger protein CXXVI (RNF126). The expression of RNF126 decreased following ANG II-induced HSF1 S303 phosphorylation, resulting in IGF-IIR protein stability and increased cardiomyocyte injury. Inhibition of GSK3 significantly alleviated ANG II-induced cardiac hypertrophy in vivo and in vitro. Taken together, these results suggest that HSF1 phosphorylation stabilizes IGF-IIR protein stability by downregulating RNF126 during cardiac hypertrophy. ANG II activates ERK/GSK3 to phosphorylate HSF1, resulting in RNF126 degradation, which stabilizes IGF-IIR protein expression and eventually results in cardiac hypertrophy. HSF1 could be a valuable therapeutic target for cardiac diseases among hypertensive patients. © 2017 Wiley Periodicals, Inc.

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.

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.

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.

Coupling of SK channels, L-type Ca2+ channels, and ryanodine receptors in cardiomyocytes.

PubMed

Zhang, Xiao-Dong; Coulibaly, Zana A; Chen, Wei Chun; Ledford, Hannah A; Lee, Jeong Han; Sirish, Padmini; Dai, Gu; Jian, Zhong; Chuang, Frank; Brust-Mascher, Ingrid; Yamoah, Ebenezer N; Chen-Izu, Ye; Izu, Leighton T; Chiamvimonvat, Nipavan

2018-03-16

Small-conductance Ca 2+ -activated K + (SK) channels regulate the excitability of cardiomyocytes by integrating intracellular Ca 2+ and membrane potentials on a beat-to-beat basis. The inextricable interplay between activation of SK channels and Ca 2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca 2+ microdomains necessary for SK channel activation. SK currents coupled with Ca 2+ influx via L-type Ca 2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca 2+ store, suggesting that LTCCs provide the immediate Ca 2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Ca v 1.2 Ca 2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Ca v 1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca 2+ signaling.

A polysaccharide of Dendrobium officinale ameliorates H2O2-induced apoptosis in H9c2 cardiomyocytes via PI3K/AKT and MAPK pathways.

PubMed

Zhang, Jing-Yi; Guo, Ying; Si, Jin-Ping; Sun, Xiao-Bo; Sun, Gui-Bo; Liu, Jing-Jing

2017-11-01

Dendrobium officinale is one valuable traditional Chinese medicine, which has skyscraping medicinal value. Polysaccharide is the main active ingredient in D. officinale; its antioxidant activity is a hot research topic nowadays. Oxidative stress plays an important role in the pathological progress of a variety of cardiovascular disease, as one of key factors of cardiomyocyte apoptosis. This research adopts a model of H 2 O 2 induction-H9c2 cardiomyocytes apoptosis, aiming to study the effect of Dendrobium officinale Polysaccharide (DOP-GY) for cardiomyocyte apoptosis caused by oxidative stress and its possible mechanism. Our results showed that pretreatment of DOP-GY (low dose: 6.25μg/mL, medium dose: 12.5μg/mL, high dose: 25μg/mL) followed by a 2h incubation with 200μM H 2 O 2 elevated the survival rate, cutted the LDH leakage, reduced lipid peroxidation damage, improved the activity of the endogenous antioxidant enzymes. In addition, the pretreatment of DOP-GY significantly inhibited the production of ROS, declined of the mitochondrial membrane potential, down-regulated pro-apoptosis protein and up-regulated anti-apoptosis protein. The protective effect was correlated with the PI3K/Akt and MAPK signal pathway. Collectively, these observations suggest that DOY-GY has the potential to exert cardioprotective effects against H 2 O 2 -induced H9c2 cardiomyocyte apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct interaction of CaVβ with actin up-regulates L-type calcium currents in HL-1 cardiomyocytes.

PubMed

Stölting, Gabriel; de Oliveira, Regina Campos; Guzman, Raul E; Miranda-Laferte, Erick; Conrad, Rachel; Jordan, Nadine; Schmidt, Silke; Hendriks, Johnny; Gensch, Thomas; Hidalgo, Patricia

2015-02-20

Expression of the β-subunit (CaVβ) is required for normal function of cardiac L-type calcium channels, and its up-regulation is associated with heart failure. CaVβ binds to the α1 pore-forming subunit of L-type channels and augments calcium current density by facilitating channel opening and increasing the number of channels in the plasma membrane, by a poorly understood mechanism. Actin, a key component of the intracellular trafficking machinery, interacts with Src homology 3 domains in different proteins. Although CaVβ encompasses a highly conserved Src homology 3 domain, association with actin has not yet been explored. Here, using co-sedimentation assays and FRET experiments, we uncover a direct interaction between CaVβ and actin filaments. Consistently, single-molecule localization analysis reveals streaklike structures composed by CaVβ2 that distribute over several micrometers along actin filaments in HL-1 cardiomyocytes. Overexpression of CaVβ2-N3 in HL-1 cells induces an increase in L-type current without altering voltage-dependent activation, thus reflecting an increased number of channels in the plasma membrane. CaVβ mediated L-type up-regulation, and CaVβ-actin association is prevented by disruption of the actin cytoskeleton with cytochalasin D. Our study reveals for the first time an interacting partner of CaVβ that is directly involved in vesicular trafficking. We propose a model in which CaVβ promotes anterograde trafficking of the L-type channels by anchoring them to actin filaments in their itinerary to the plasma membrane. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Differentiation of mouse embryonic stem cells into cardiomyocytes via the hanging-drop and mass culture methods.

PubMed

Fuegemann, Christopher J; Samraj, Ajoy K; Walsh, Stuart; Fleischmann, Bernd K; Jovinge, Stefan; Breitbach, Martin

2010-12-01

Herein, we describe two protocols for the in vitro differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. mESCs are pluripotent and can be differentiated into cells of all three germ layers, including cardiomyocytes. The methods described here facilitate the differentiation of mESCs into the different cardiac subtypes (atrial-, ventricular-, nodal-like cells). The duration of cell culture determines whether preferentially early- or late-developmental stage cardiomyocytes can be obtained preferentially. This approach allows the investigation of cardiomyocyte development and differentiation in vitro, and also allows for the enrichment and isolation of physiologically intact cardiomyocytes for transplantation purposes. © 2010 by John Wiley & Sons, Inc.

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.

Rapid effects of aldosterone in primary cultures of cardiomyocytes - do they suggest the existence of a membrane-bound receptor?

PubMed

Araujo, Carolina Morais; Hermidorff, Milla Marques; Amancio, Gabriela de Cassia Sousa; Lemos, Denise da Silveira; Silva, Marcelo Estáquio; de Assis, Leonardo Vinícius Monteiro; Isoldi, Mauro César

2016-10-01

Aldosterone acts on its target tissue through a classical mechanism or through the rapid pathway through a putative membrane-bound receptor. Our goal here was to better understand the molecular and biochemical rapid mechanisms responsible for aldosterone-induced cardiomyocyte hypertrophy. We have evaluated the hypertrophic process through the levels of ANP, which was confirmed by the analysis of the superficial area of cardiomyocytes. Aldosterone increased the levels of ANP and the cellular area of the cardiomyocytes; spironolactone reduced the aldosterone-increased ANP level and cellular area of cardiomyocytes. Aldosterone or spironolactone alone did not increase the level of cyclic 3',5'-adenosine monophosphate (cAMP), but aldosterone plus spironolactone led to increased cAMP level; the treatment with aldosterone + spironolactone + BAPTA-AM reduced the levels of cAMP. These data suggest that aldosterone-induced cAMP increase is independent of mineralocorticoid receptor (MR) and dependent on Ca(2+). Next, we have evaluated the role of A-kinase anchor proteins (AKAP) in the aldosterone-induced hypertrophic response. We have found that St-Ht31 (AKAP inhibitor) reduced the increased level of ANP which was induced by aldosterone; in addition, we have found an increase on protein kinase C (PKC) and extracellular signal-regulated kinase 5 (ERK5) activity when cells were treated with aldosterone alone, spironolactone alone and with a combination of both. Our data suggest that PKC could be responsible for ERK5 aldosterone-induced phosphorylation. Our study suggests that the aldosterone through its rapid effects promotes a hypertrophic response in cardiomyocytes that is controlled by an AKAP, being dependent on ERK5 and PKC, but not on cAMP/cAMP-dependent protein kinase signaling pathways. Lastly, we provide evidence that the targeting of AKAPs could be relevant in patients with aldosterone-induced cardiac hypertrophy and heart failure.

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

DOT1L regulates dystrophin expression and is critical for cardiac function

PubMed Central

Nguyen, Anh T.; Xiao, Bin; Neppl, Ronald L.; Kallin, Eric M.; Li, Juan; Chen, Taiping; Wang, Da-Zhi; Xiao, Xiao; Zhang, Yi

2011-01-01

Histone methylation plays an important role in regulating gene expression. One such methylation occurs at Lys 79 of histone H3 (H3K79) and is catalyzed by the yeast DOT1 (disruptor of telomeric silencing) and its mammalian homolog, DOT1L. Previous studies have demonstrated that germline disruption of Dot1L in mice resulted in embryonic lethality. Here we report that cardiac-specific knockout of Dot1L results in increased mortality rate with chamber dilation, increased cardiomyocyte cell death, systolic dysfunction, and conduction abnormalities. These phenotypes mimic those exhibited in patients with dilated cardiomyopathy (DCM). Mechanistic studies reveal that DOT1L performs its function in cardiomyocytes through regulating Dystrophin (Dmd) transcription and, consequently, stability of the Dystrophin–glycoprotein complex important for cardiomyocyte viability. Importantly, expression of a miniDmd can largely rescue the DCM phenotypes, indicating that Dmd is a major target mediating DOT1L function in cardiomyocytes. Interestingly, analysis of available gene expression data sets indicates that DOT1L is down-regulated in idiopathic DCM patient samples compared with normal controls. Therefore, our study not only establishes a critical role for DOT1L-mediated H3K79 methylation in cardiomyocyte function, but also reveals the mechanism underlying the role of DOT1L in DCM. In addition, our study may open new avenues for the diagnosis and treatment of human heart disease. PMID:21289070

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

Growth factor transgenes interactively regulate articular chondrocytes.

PubMed

Shi, Shuiliang; Mercer, Scott; Eckert, George J; Trippel, Stephen B

2013-04-01

Adult articular chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. Multiple growth factor genes regulate these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2), and bone morphogenetic protien-7 (BMP-7) to articular chondrocytes and measured changes in the production of DNA, glycosaminoglycan, and collagen. The transgenes differentially regulated all these chondrocyte activities. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2, and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate an approach to articular chondrocyte regulation that may be tailored to stimulate specific cell functions, and suggest that certain growth factor gene combinations have potential value for cell-based articular cartilage repair. Copyright © 2012 Wiley Periodicals, Inc.

Expression of Inflammation-related Intercellular Adhesion Molecules in Cardiomyocytes In Vitro and Modulation by Pro-inflammatory Agents.

PubMed

El-Battrawy, Ibrahim; Tülümen, Erol; Lang, Siegfried; Akin, Ibrahim; Behnes, Michael; Zhou, Xiabo; Mavany, Martin; Bugert, Peter; Bieback, Karen; Borggrefe, Martin; Elmas, Elif

2016-01-01

Cell-surface adhesion molecules regulate multiple intercellular and intracellular processes and play important roles in inflammation by facilitating leukocyte endothelial transmigration. Whether cardiomyocytes express surface-adhesion molecules related to inflammation and the effect of pro-inflammatory mediators remain unknown. In the present study, the expression of different cell-adhesion molecules (CD11a, CD11b, CD31, CD62P, CD162, F11 receptor and mucosal vascular addressin cell adhesion molecule 1 (MADCAM1)) and the effect of pro-inflammatory mediators were investigated in an in vitro model of human cardiomyocytes. Cells were supplied as a primary culture of cardiac alpha actin-positive cells from human heart tissue. The cells were incubated for 24 h with 1 U/ml thrombin or 700 ng/ml lipopolysaccharide (LPS) or with a combination of both. The expression of the cell adhesion molecules was measured by flow cytometry. In cultured human cardiomyocytes, 22.8% of cells expressed CD31, 7.1% MADCAM1 and 2.6% F11R. CD11a, CD11b, CD62P and CD162 were expressed by fewer than 2% of the cells at baseline. CD31 expression increased on incubation of cardiomyocytes with thrombin by 26% (p<0.05) and with LPS by 26% (p=0.06). The combination of thrombin and LPS did not result in increased levels of CD31 (p>0.10). The pro-inflammatory agents LPS and thrombin had no effect on the expression of MADCAM1 and F11R. Inflammation-related cell-adhesion molecules CD31, MADCAM1 and F11R were shown to be expressed on the surface of human cardiomyocytes in an in vitro model. Incubation with LPS or thrombin resulted in increased expression of CD31, however, it did not modify the expression of the cell adhesion molecules MADCAM1 and F11R. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

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

Cardiomyocyte proliferation and progenitor cell recruitment underlie therapeutic regeneration after myocardial infarction in the adult mouse heart

PubMed Central

Malliaras, Konstantinos; Zhang, Yiqiang; Seinfeld, Jeffrey; Galang, Giselle; Tseliou, Eleni; Cheng, Ke; Sun, Baiming; Aminzadeh, Mohammad; Marbán, Eduardo

2013-01-01

Cardiosphere-derived cells (CDCs) have been shown to regenerate infarcted myocardium in patients after myocardial infarction (MI). However, whether the cells of the newly formed myocardium originate from the proliferation of adult cardiomyocytes or from the differentiation of endogenous stem cells remains unknown. Using genetic fate mapping to mark resident myocytes in combination with long-term BrdU pulsing, we investigated the origins of postnatal cardiomyogenesis in the normal, infarcted and cell-treated adult mammalian heart. In the normal mouse heart, cardiomyocyte turnover occurs predominantly through proliferation of resident cardiomyocytes at a rate of ∼1.3–4%/year. After MI, new cardiomyocytes arise from both progenitors as well as pre-existing cardiomyocytes. Transplantation of CDCs upregulates host cardiomyocyte cycling and recruitment of endogenous progenitors, while boosting heart function and increasing viable myocardium. The observed phenomena cannot be explained by cardiomyocyte polyploidization, bi/multinucleation, cell fusion or DNA repair. Thus, CDCs induce myocardial regeneration by differentially upregulating two mechanisms of endogenous cell proliferation. PMID:23255322

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

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

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

Chemical Growth Regulators for Guayule Plants

NASA Technical Reports Server (NTRS)

Dastoor, M. N.; Schubert, W. W.; Petersen, G. R.

1982-01-01

Test Tubes containing Guayule - tissue cultures were used in experiments to test effects of chemical-growth regulators. The shoots grew in response to addition of 2-(3,4-dichlorophenoxy)-triethylamine (triethylamine (TEA) derivative) to agar medium. Preliminary results indicate that a class of compounds that promotes growth in soil may also promote growth in a culture medium. Further experiments are needed to define the effect of the TEA derivative.

Interaction of gonadal steroids and the glucocorticoid corticosterone in the regulation of the L-type Ca(2+) current in rat left ventricular cardiomyocytes.

PubMed

Wagner, M; Moritz, A; Volk, T

2011-08-01

Gonadal steroids as well as glucocorticoids have been shown to regulate the cardiac L-type Ca(2+) current (I(CaL) ). Herein, we compare the effects of the gonadal steroids testosterone and 17β-estradiol with the glucocorticoid corticosterone on I(CaL) , and investigate the interaction between the gonadal steroids and corticosterone. Myocytes were isolated from the left ventricular free wall of female and male Wistar rats and investigated using the ruptured-patch whole-cell patch-clamp technique. In myocytes isolated from female rats, 24 h incubation with 100 nm testosterone led to a 33% increase in I(CaL) compared with control (-8.8 ± 0.5 pA pF(-1) , n = 25 vs. -6.6 ± 0.4 pA pF(-1) , n = 26, P < 0.01, V(Pip) = 0 mV). Incubation with 1 μm corticosterone resulted in a 79% increase in I(CaL) (-11.8 ± 0.7 pA pF(-1) , n = 29, P < 0.001). However, the combination of testosterone and corticosterone did not have any additional effect compared with corticosterone alone (-11.7 ± 0.6 pA pF(-1) , n = 25, ns). In cardiomyocytes from male rats, I(CaL) was not affected by testosterone, whereas the effect of corticosterone was preserved (P < 0.05). 24 h incubation with 17β-estradiol increased I(CaL) by 32% from -7.6 ± 0.5 pA pF(-1) (n = 15) to 10.0 ± 0.9 pA pF(-1) (n = 15, P < 0.05). 17β-estradiol did not exert an additional effect upon co-incubation with corticosterone and did not have an effect on I(CaL) in cardiomyocytes from female rats. Higher concentrations of the gonadal steroids did not result in increased effects. When compared with corticosterone, the in vitro effects of the gonadal steroids are small. However, under conditions in which I(CaL) is not fully activated by glucocorticoids, gonadal steroids may significantly contribute to I(CaL) regulation. © 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.

The transcription factor MEF2C mediates cardiomyocyte hypertrophy induced by IGF-1 signaling

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

Munoz, Juan Pablo; Collao, Andres; Chiong, Mario

2009-10-09

Myocyte enhancer factor 2C (MEF2C) plays an important role in cardiovascular development and is a key transcription factor for cardiac hypertrophy. Here, we describe MEF2C regulation by insulin-like growth factor-1 (IGF-1) and its role in IGF-1-induced cardiac hypertrophy. We found that IGF-1 addition to cultured rat cardiomyocytes activated MEF2C, as evidenced by its increased nuclear localization and DNA binding activity. IGF-1 stimulated MEF2 dependent-gene transcription in a time-dependent manner, as indicated by increased MEF2 promoter-driven reporter gene activity; IGF-1 also induced p38-MAPK phosphorylation, while an inhibitor of p38-MAPK decreased both effects. Additionally, inhibitors of phosphatidylinositol 3-kinase and calcineurin prevented IGF-1-inducedmore » MEF2 transcriptional activity. Via MEF2C-dependent signaling, IGF-1 also stimulated transcription of atrial natriuretic factor and skeletal {alpha}-actin but not of fos-lux reporter genes. These novel data suggest that MEF2C activation by IGF-1 mediates the pro-hypertrophic effects of IGF-1 on cardiac gene expression.« less

Overexpressed connective tissue growth factor in cardiomyocytes attenuates left ventricular remodeling induced by angiotensin II perfusion.

PubMed

Zhang, Ying; Yan, Hua; Guang, Gong-Chang; Deng, Zheng-Rong

2017-01-01

To evaluate the improving effects of specifically overexpressed connective tissue growth factor (CTGF) in cardiomyocytes on mice with hypertension induced by angiotensin II (AngII) perfusion, 24 transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) were divided into two equal groups that were perfused with acetic acid and AngII, respectively, for 7 days. Another 24 cage-control wild-type C57BL/6 mice (NLC) were divided and treated identically. Blood pressure was detected by caudal artery cannulation. Cardiac structural and functional changes were observed by echocardiography. Cardiac fibrosis was detected by Masson staining. After AngII perfusion, blood pressures of NLC and Tg-CTGF mice, especially those of the formers, significantly increased. Compared with NLC + AngII group, Tg-CTGF + AngII group had significantly lower left ventricular posterior wall thickness at end-diastole and left ventricular posterior wall thickness at end-systole as well as significantly higher left ventricular end-systolic diameter and left ventricular end-diastolic diameter (P < 0.05). Reverse transcription-polymerase chain reaction (RT-PCR) showed that Tg-CTGF + AngII group had significantly lower collagen I, α-SMA, and TGF-β mRNA expressions in cardiac tissues (P < 0.05). Tg-CTGF can protect AngII-induced cardiac remodeling of mice with hypertension by mitigating inflammatory response. CTGF may be a therapy target for hypertension-induced myocardial fibrosis, but the detailed mechanism still needs in-depth studies.

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

5-Azacytidine delivered by mesoporous silica nanoparticles regulates the differentiation of P19 cells into cardiomyocytes

NASA Astrophysics Data System (ADS)

Cheng, Jin; Ding, Qian; Wang, Jia; Deng, Lin; Yang, Lu; Tao, Lei; Lei, Haihong; Lu, Shaoping

2016-01-01

Heart disease is one of the deadliest diseases causing mortality due to the limited regenerative capability of highly differentiated cardiomyocytes. Stem cell-based therapy in tissue engineering is one of the most exciting and rapidly growing areas and raises promising prospects for cardiac repair. In this study, we have synthesized FITC-mesoporous silica nanoparticles (FMSNs) based on a sol-gel method (known as Stöber's method) as a drug delivery platform to transport 5-azacytidine in P19 embryonic carcinoma stem cells. The surfactant CTAB is utilized as a liquid crystal template to self-aggregate into micelles, resulting in the synthesis of MSNs. Based on the cell viability assay, treatment with FMSNs + 5-azacytidine resulted in much more significant inhibition of the proliferation than 5-azacytidine alone. To study the mechanism, we have tested the differentiation genes and cardiac marker genes in P19 cells and found that these genes have been up-regulated in P19 embryonic carcinoma stem cells treated with FMSNs + 5-azacytidine + poly(allylamine hydrochloride) (PAH), with the changes of histone modifications on the regulatory region. In conclusion, with FMSNs as drug delivery platforms, 5-azacytidine can be more efficiently delivered into stem cells and can be used to monitor and track the transfection process in situ to clarify their effects on stem cell functions and the differentiation process, which can serve as a promising tool in tissue engineering and other biomedical fields.

Induction of HO-1 by carbon monoxide releasing molecule-2 attenuates thrombin-induced COX-2 expression and hypertrophy in primary human cardiomyocytes

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

Chien, Peter Tzu-Yu; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan; Lin, Chih-Chung

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE{sub 2} release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression andmore » cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes. - Graphical abstract: In summary, CORM-2 treatment induces Pyk2 transactivated PDGFR, which induces PI3K/Akt/MAPK activation, and then recruits Sp1/Foxo1 transcriptional factors to regulate HO-1 gene expression in primary human cardiomyocytes. - Highlights: • CORM-2 induces HO-1 expression. • Pyk2-dependent PDGFR activates PI3K/Akt/MAPK pathway in CORM-2

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.

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.

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

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.

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress

PubMed Central

Judge, Luke M.; Perez-Bermejo, Juan A.; Truong, Annie; Ribeiro, Alexandre J.S.; Yoo, Jennie C.; Jensen, Christina L.; Mandegar, Mohammad A.; Huebsch, Nathaniel; Kaake, Robyn M.; So, Po-Lin; Srivastava, Deepak; Krogan, Nevan J.

2017-01-01

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity. PMID:28724793

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

PubMed

Judge, Luke M; Perez-Bermejo, Juan A; Truong, Annie; Ribeiro, Alexandre Js; Yoo, Jennie C; Jensen, Christina L; Mandegar, Mohammad A; Huebsch, Nathaniel; Kaake, Robyn M; So, Po-Lin; Srivastava, Deepak; Pruitt, Beth L; Krogan, Nevan J; Conklin, Bruce R

2017-07-20

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.

TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation

PubMed Central

Kirschmer, Nadine; Bandleon, Sandra; von Ehrlich-Treuenstätt, Viktor; Hartmann, Sonja; Schaaf, Alice; Lamprecht, Anna-Karina; Miranda-Laferte, Erick; Langsenlehner, Tanja; Ritter, Oliver; Eder, Petra

2016-01-01

The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an

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.

β-Adrenergic receptor stimulation inhibits proarrhythmic alternans in postinfarction border zone cardiomyocytes: a computational analysis.

PubMed

Tomek, Jakub; Rodriguez, Blanca; Bub, Gil; Heijman, Jordi

2017-08-01

The border zone (BZ) of the viable myocardium adjacent to an infarct undergoes extensive autonomic and electrical remodeling and is prone to repolarization alternans-induced cardiac arrhythmias. BZ remodeling processes may promote or inhibit Ca 2+ and/or repolarization alternans and may differentially affect ventricular arrhythmogenesis. Here, we used a detailed computational model of the canine ventricular cardiomyocyte to study the determinants of alternans in the BZ and their regulation by β-adrenergic receptor (β-AR) stimulation. The BZ model developed Ca 2+ transient alternans at slower pacing cycle lengths than the control model, suggesting that the BZ may promote spatially heterogeneous alternans formation in an infarcted heart. β-AR stimulation abolished alternans. By evaluating all combinations of downstream β-AR stimulation targets, we identified both direct (via ryanodine receptor channels) and indirect [via sarcoplasmic reticulum (SR) Ca 2+ load] modulation of SR Ca 2+ release as critical determinants of Ca 2+ transient alternans. These findings were confirmed in a human ventricular cardiomyocyte model. Cell-to-cell coupling indirectly modulated the likelihood of alternans by affecting the action potential upstroke, reducing the trigger for SR Ca 2+ release in one-dimensional strand simulations. However, β-AR stimulation inhibited alternans in both single and multicellular simulations. Taken together, these data highlight a potential antiarrhythmic role of sympathetic hyperinnervation in the BZ by reducing the likelihood of alternans and provide new insights into the underlying mechanisms controlling Ca 2+ transient and repolarization alternans. NEW & NOTEWORTHY We integrated, for the first time, postmyocardial infarction electrical and autonomic remodeling in a detailed, validated computer model of β-adrenergic stimulation in ventricular cardiomyocytes. Here, we show that β-adrenergic stimulation inhibits alternans and provide novel insights

Three-dimensional extracellular matrix scaffolds by microfluidic fabrication for long-term spontaneously contracted cardiomyocyte culture.

PubMed

Mei, Jeng-Chun; Wu, Aden Yuan Kun; Wu, Po-Chen; Cheng, Nai-Chen; Tsai, Wei-Bor; Yu, Jiashing

2014-11-01

To repair damaged cardiac tissue, the important principle of in vitro cell culture is to mimic the in vivo cell growth environment. Thus, micro-sized cells are more suitably cultured in three-dimensional (3D) than in two-dimensional (2D) microenvironments (ex: culture dish). With the matching dimensions of works produced by microfluidic technology, chemical engineering and biochemistry applications have used this technology extensively in cellular works. The 3D scaffolds produced in our investigation has essential properties, such has high mass transfer efficiency, and variable pore sizes, to adapt to various needs of different cell types. In addition to the malleability of these innovative scaffolds, fabrication procedure was effortless and fast. Primary neonatal mice cardiomyocytes were successfully harvested and cultured in 3D scaffolds made of gelatin and collagen. Gelatin and gelatin-collagen scaffold were produced by the formation of microbubbles through a microfluidic device, and the mechanical properties of gelatin scaffold and gelatin-collagen scaffold were measured. Cellular properties in the microbubbles were also monitored. Fluorescence staining results assured that cardiomyocytes could maintain in vivo morphology in 3D gelatin scaffold. In addition, it was found that 3D scaffold could prolong the contraction behavior of cardiomyocytes compared with a conventional 2D culture dish. Spontaneously contracted behavior was maintained for the longest (about 1 month) in the 3D gelatin scaffold, about 19 days in the 3D gelatin-collagen scaffold. To sum up, this 3D platform for cell culture has promising potential for myocardial tissue engineering.

Axon growth regulation by a bistable molecular switch.

PubMed

Padmanabhan, Pranesh; Goodhill, Geoffrey J

2018-04-25

For the brain to function properly, its neurons must make the right connections during neural development. A key aspect of this process is the tight regulation of axon growth as axons navigate towards their targets. Neuronal growth cones at the tips of developing axons switch between growth and paused states during axonal pathfinding, and this switching behaviour determines the heterogeneous axon growth rates observed during brain development. The mechanisms controlling this switching behaviour, however, remain largely unknown. Here, using mathematical modelling, we predict that the molecular interaction network involved in axon growth can exhibit bistability, with one state representing a fast-growing growth cone state and the other a paused growth cone state. Owing to stochastic effects, even in an unchanging environment, model growth cones reversibly switch between growth and paused states. Our model further predicts that environmental signals could regulate axon growth rate by controlling the rates of switching between the two states. Our study presents a new conceptual understanding of growth cone switching behaviour, and suggests that axon guidance may be controlled by both cell-extrinsic factors and cell-intrinsic growth regulatory mechanisms. © 2018 The Author(s).

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

Regulation of fish growth hormone transcription.

PubMed

Farchi-Pisanty, O; Hackett, P B; Moav, B

1995-09-01

Regulation of endogenous fish growth hormone transcription was studied using carp pituitaries in vitro. It was demonstrated that thyroid hormone (T3) and 9-cis retinoic acid have increased the steady state levels of growth hormone messenger RNA in pituitary cells, as compared with beta-actin messenger RNA levels. In contrast, estrogen failed to increase growth hormone mRNA levels. The possible involvement of thyroid hormone receptor in pituitary gene expression was demonstrated by in situ localization of both growth hormone mRNA and thyroid hormone receptor mRNA in the pituitaries as early as 4 days after fertilization.

Reverse engineering life: physical and chemical mimetics for controlled stem cell differentiation into cardiomyocytes.

PubMed

Skuse, Gary R; Lamkin-Kennard, Kathleen A

2013-01-01

Our ability to manipulate stem cells in order to induce differentiation along a desired developmental pathway has improved immeasurably in recent years. That is in part because we have a better understanding of the intracellular and extracellular signals that regulate differentiation. However, there has also been a realization that stem cell differentiation is not regulated only by chemical signals but also by the physical milieu in which a particular stem cell exists. In this regard we are challenged to mimic both chemical and physical environments. Herein we describe a method to induce stem cell differentiation into cardiomyocytes using a combination of chemical and physical cues. This method can be applied to produce differentiated cells for research and potentially for cell-based therapy of cardiomyopathies.

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

Nicotine plus a high-fat diet triggers cardiomyocyte apoptosis.

PubMed

Sinha-Hikim, Indrani; Friedman, Theodore C; Falz, Mark; Chalfant, Victor; Hasan, Mohammad Kamrul; Espinoza-Derout, Jorge; Lee, Desean L; Sims, Carl; Tran, Peter; Mahata, Sushil K; Sinha-Hikim, Amiya P

2017-04-01

Cigarette smoking is an important risk factor for diabetes, cardiovascular disease and non-alcoholic fatty liver disease. The health risk associated with smoking can be aggravated by obesity. Smoking might also trigger cardiomyocyte (CM) apoptosis. Given that CM apoptosis has been implicated as a potential mechanism in the development of cardiomyopathy and heart failure, we characterize the key signaling pathways in nicotine plus high-fat diet (HFD)-induced CM apoptosis. Adult C57BL6 male mice were fed a normal diet (ND) or HFD and received twice-daily intraperitoneal (IP) injections of nicotine (0.75 mg/kg body weight [BW]) or saline for 16 weeks. An additional group of nicotine-treated mice on HFD received twice-daily IP injections of mecamylamine (1 mg/kg BW), a non-selective nicotinic acetylcholine receptor antagonist, for 16 weeks. Nicotine when combined with HFD led to a massive increase in CM apoptosis that was fully prevented by mecamylamine treatment. Induction of CM apoptosis was associated with increased oxidative stress and activation of caspase-2-mediated intrinsic pathway signaling coupled with inactivation of AMP-activated protein kinase (AMPK). Furthermore, nicotine treatment significantly (P < 0.05) attenuated the HFD-induced decrease in fibroblast growth factor 21 (FGF21) and silent information regulator 1 (SIRT1). We conclude that nicotine, when combined with HFD, triggers CM apoptosis through the generation of oxidative stress and inactivation of AMPK together with the activation of caspase-2-mediated intrinsic apoptotic signaling independently of FGF21 and SIRT1.

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

Actinidia chinensis planch polysaccharide protects against hypoxia‑induced apoptosis of cardiomyocytes in vitro.

PubMed

Wang, Qiang; Xu, Yunfa; Gao, Ying; Wang, Qi

2018-05-03

Cardiac hypertrophy is frequently accompanied by ischemic heart disease. Actinidia chinensis planch polysaccharide (ACP) is the main active compound from Actinidia chinensis planch. In the present study, a cardiac hypertrophy model was produced by treating cells with Angiotensin II (Ang II), which was used to investigate whether ACP protected against cardiac hypertrophy in vitro. It was demonstrated that ACP alleviated Ang II‑induced cardiac hypertrophy. In addition, pretreatment with ACP prior to hypoxic culture reduced the disruption of the mitochondrial membrane potential as investigated by flow cytometry. Cell Counting kit‑8 analysis demonstrated that ACP maintained the cell viability of cardiomyocytes. The flow cytometric analysis revealed that ACP inhibited hypoxia‑induced apoptosis in cardiomyocytes treated with Ang II. Additionally, reverse transcription‑polymerase chain reaction and western blotting assays demonstrated that ACP decreased the expression of apoptosis‑associated genes including apoptosis‑inducing factor mitochondria associated 1, the cysteinyl aspartate specific proteinases caspases‑3/8/9, and cleaved caspases‑3/8/9. The results of the present study also demonstrated that ACP inhibited the activation of the extracellular signal‑regulated kinase 1/2 (ERK1/2) and phosphoinositide 3‑kinase/protein kinase B (PI3K/AKT) signaling pathways. Furthermore, the specific activation of ERK1/2 and PI3K/AKT reversed the apoptotic‑inhibitory effect of ACP. In conclusion, the protective effects of ACP against hypoxia‑induced apoptosis may depend on depressing the ERK1/2 and PI3K/AKT signaling pathways in cardiomyocytes treated with Ang II.

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

Anti-aging effects of vitamin C on human pluripotent stem cell-derived cardiomyocytes.

PubMed

Kim, Yoon Young; Ku, Seung-Yup; Huh, Yul; Liu, Hung-Ching; Kim, Seok Hyun; Choi, Young Min; Moon, Shin Yong

2013-10-01

Human pluripotent stem cells (hPSCs) have arisen as a source of cells for biomedical research due to their developmental potential. Stem cells possess the promise of providing clinicians with novel treatments for disease as well as allowing researchers to generate human-specific cellular metabolism models. Aging is a natural process of living organisms, yet aging in human heart cells is difficult to study due to the ethical considerations regarding human experimentation as well as a current lack of alternative experimental models. hPSC-derived cardiomyocytes (CMs) bear a resemblance to human cardiac cells and thus hPSC-derived CMs are considered to be a viable alternative model to study human heart cell aging. In this study, we used hPSC-derived CMs as an in vitro aging model. We generated cardiomyocytes from hPSCs and demonstrated the process of aging in both human embryonic stem cell (hESC)- and induced pluripotent stem cell (hiPSC)-derived CMs. Aging in hESC-derived CMs correlated with reduced membrane potential in mitochondria, the accumulation of lipofuscin, a slower beating pattern, and the downregulation of human telomerase RNA (hTR) and cell cycle regulating genes. Interestingly, the expression of hTR in hiPSC-derived CMs was not significantly downregulated, unlike in hESC-derived CMs. In order to delay aging, vitamin C was added to the cultured CMs. When cells were treated with 100 μM of vitamin C for 48 h, anti-aging effects, specifically on the expression of telomere-related genes and their functionality in aging cells, were observed. Taken together, these results suggest that hPSC-derived CMs can be used as a unique human cardiomyocyte aging model in vitro and that vitamin C shows anti-aging effects in this model.

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

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

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

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

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.

GSK3 controls axon growth via CLASP-mediated regulation of growth cone microtubules

PubMed Central

Hur, Eun-Mi; Saijilafu; Lee, Byoung Dae; Kim, Seong-Jin; Xu, Wen-Lin; Zhou, Feng-Quan

2011-01-01

Suppression of glycogen synthase kinase 3 (GSK3) activity in neurons yields pleiotropic outcomes, causing both axon growth promotion and inhibition. Previous studies have suggested that specific GSK3 substrates, such as adenomatous polyposis coli (APC) and collapsin response mediator protein 2 (CRMP2), support axon growth by regulating the stability of axonal microtubules (MTs), but the substrate(s) and mechanisms conveying axon growth inhibition remain elusive. Here we show that CLIP (cytoplasmic linker protein)-associated protein (CLASP), originally identified as a MT plus end-binding protein, displays both plus end-binding and lattice-binding activities in nerve growth cones, and reveal that the two MT-binding activities regulate axon growth in an opposing manner: The lattice-binding activity mediates axon growth inhibition induced by suppression of GSK3 activity via preventing MT protrusion into the growth cone periphery, whereas the plus end-binding property supports axon extension via stabilizing the growing ends of axonal MTs. We propose a model in which CLASP transduces GSK3 activity levels to differentially control axon growth by coordinating the stability and configuration of growth cone MTs. PMID:21937714

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

Redox signaling in the cardiomyocyte: From physiology to failure.

PubMed

Santos, Celio X C; Raza, Sadaf; Shah, Ajay M

2016-05-01

The specific effect of oxygen and reactive oxygen species (ROS) in mediating post-translational modification of protein targets has emerged as a key mechanism regulating signaling components, a process termed redox signaling. ROS act in the post-translational modification of multiple target proteins including receptors, kinases, phosphatases, ion channels and transcription factors. Both O2 and ROS are major source of electrons in redox reactions in aerobic organisms. Because the heart has the highest O2 consumption among body organs, it is not surprising that redox signaling is central to heart function and pathophysiology. In this article, we review some of the main cardiac redox signaling pathways and their roles in the cardiomyocyte and in heart failure, with particular focus on the specific molecular targets of ROS in the heart. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

Differentiation of Rat bone marrow Mesenchymal stem cells into Adipocytes and Cardiomyocytes after treatment with platelet lysate.

PubMed

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Barzegar, Kazem

2016-01-01

Mesenchymal stem cells (MSCs) are multipotential cells and their therapeutic potency is under intense investigation. Studying the effect of different induction factors on MSCs could increase our knowledge about the differentiation potency of these cells. One of the most important sources of these factors in mammalian body is platelet. Platelet lysate (PL) contains many growth factors and therefore, it can be used as a differentiation inducer. In the present study, the effect of PL on differentiation of rat bone marrow MSCs into cardiomyocytes was studied. To study the differentiation-inducing effect of PL, MSCs were treated with 2.5, 5 and 10% PL. Early results of this study showed that PL in high concentrations (10%) induces adipogenic differentiation of MSCs. Therefore, to evaluate differentiation to cardiomyocytes, MSCs were cultured in media containing lower levels of PL (2.5% and 5%) and then cardiomyogenic differentiation was induced by treatment with 5-azacytidine. Differentiation of MSCs was evaluated using direct observation of beating cells, immunostaining and real-time PCR techniques. The results of qPCR showed that treatment with PL alone increased the expression of cardiac alpha actinin (CAA) being predictable by earlier observation of beating cells in PL-treated groups. The results of staining assays against cardiac alpha actinin also showed that there were stained cells in PL-treated groups. The results of the present study showed that PL is a powerful induction factor for differentiation of MSCs into different cell lines such as cardiomyocytes and adipocytes.

Wnt/β-catenin signaling directs the regional expansion of first and second heart field-derived ventricular cardiomyocytes

PubMed Central

Buikema, Jan Willem; Mady, Ahmed S.; Mittal, Nikhil V.; Atmanli, Ayhan; Caron, Leslie; Doevendans, Pieter A.; Sluijter, Joost P. G.; Domian, Ibrahim J.

2013-01-01

In mammals, cardiac development proceeds from the formation of the linear heart tube, through complex looping and septation, all the while increasing in mass to provide the oxygen delivery demands of embryonic growth. The developing heart must orchestrate regional differences in cardiomyocyte proliferation to control cardiac morphogenesis. During ventricular wall formation, the compact myocardium proliferates more vigorously than the trabecular myocardium, but the mechanisms controlling such regional differences among cardiomyocyte populations are not understood. Control of definitive cardiomyocyte proliferation is of great importance for application to regenerative cell-based therapies. We have used murine and human pluripotent stem cell systems to demonstrate that, during in vitro cellular differentiation, early ventricular cardiac myocytes display a robust proliferative response to β-catenin-mediated signaling and conversely accelerate differentiation in response to inhibition of this pathway. Using gain- and loss-of-function murine genetic models, we show that β-catenin controls ventricular myocyte proliferation during development and the perinatal period. We further demonstrate that the differential activation of the Wnt/β-catenin signaling pathway accounts for the observed differences in the proliferation rates of the compact versus the trabecular myocardium during normal cardiac development. Collectively, these results provide a mechanistic explanation for the differences in localized proliferation rates of cardiac myocytes and point to a practical method for the generation of the large numbers of stem cell-derived cardiac myocytes necessary for clinical applications. PMID:24026118

Recent Insights into the Regulation of the Growth Plate

PubMed Central

Lui, Julian C.; Nilsson, Ola; Baron, Jeffrey

2014-01-01

For most bones, elongation is driven primarily by chondrogenesis at the growth plates. This process results from chondrocyte proliferation, hypertrophy, and extracellular matrix secretion and is carefully orchestrated by complex networks of local paracrine factors and modulated by endocrine factors. We review here recent advances in the understanding of growth plate physiology. These advances include new approaches to study expression patterns of large numbers of genes in the growth plate, using microdissection followed by microarray. This approach has been combined with genome-wide association studies to provide insights into the regulation of the human growth plate. We also review recent studies elucidating the roles of bone morphogenetic proteins, fibroblast growth factors, C-type natriuretic peptide, and suppressor of cytokine signaling in the local regulation of growth plate chondrogenesis and longitudinal bone growth. PMID:24740736

In situ reprogramming to transdifferentiate fibroblasts into cardiomyocytes using adenoviral vectors: Implications for clinical myocardial regeneration.

PubMed

Mathison, Megumi; Singh, Vivek P; Chiuchiolo, Maria J; Sanagasetti, Deepthi; Mao, Yun; Patel, Vivekkumar B; Yang, Jianchang; Kaminsky, Stephen M; Crystal, Ronald G; Rosengart, Todd K

2017-02-01

The reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells improves ventricular function in myocardial infarction models. Only integrating persistent expression vectors have thus far been used to induce reprogramming, potentially limiting its clinical applicability. We therefore tested the reprogramming potential of nonintegrating, acute expression adenoviral (Ad) vectors. Ad or lentivirus vectors encoding Gata4 (G), Mef2c (M), and Tbx5 (T) were validated in vitro. Sprague-Dawley rats then underwent coronary ligation and Ad-mediated administration of vascular endothelial growth factor to generate infarct prevascularization. Three weeks later, animals received Ad or lentivirus encoding G, M, or T (AdGMT or LentiGMT) or an equivalent dose of a null vector (n = 11, 10, and 10, respectively). Outcomes were analyzed by echocardiography, magnetic resonance imaging, and histology. Ad and lentivirus vectors provided equivalent G, M, and T expression in vitro. AdGMT and LentiGMT both likewise induced expression of the cardiomyocyte marker cardiac troponin T in approximately 6% of cardiac fibroblasts versus <1% cardiac troponin T expression in AdNull (adenoviral vector that does not encode a transgene)-treated cells. Infarcted myocardium that had been treated with AdGMT likewise demonstrated greater density of cells expressing the cardiomyocyte marker beta myosin heavy chain 7 compared with AdNull-treated animals. Echocardiography demonstrated that AdGMT and LentiGMT both increased ejection fraction compared with AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; P < .05). Ad vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into induced cardiomyocyte-like cells and improving cardiac function in postinfarct rat hearts. Short-term expression Ad vectors may represent an important means to induce cardiac cellular reprogramming in humans. Copyright © 2016 The American

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.

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.

Regulation of zebrafish heart regeneration by miR-133.

PubMed

Yin, Viravuth P; Lepilina, Alexandra; Smith, Ashley; Poss, Kenneth D

2012-05-15

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. Using pharmacological inhibition and EGFP sensor interaction studies, we found that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation. Copyright © 2012. Published by Elsevier Inc.

Regulation of zebrafish heart regeneration by miR-133

PubMed Central

Yin, Viravuth P.; Lepilina, Alexandra; Smith, Ashley; Poss, Kenneth D.

2012-01-01

Zebrafish regenerate cardiac muscle after severe injuries through the activation and proliferation of spared cardiomyocytes. Little is known about factors that control these events. Here we investigated the extent to which miRNAs regulate zebrafish heart regeneration. Microarray analysis identified many miRNAs with increased or reduced levels during regeneration. miR-133, a miRNA with known roles in cardiac development and disease, showed diminished expression during regeneration. Induced transgenic elevation of miR-133 levels after injury inhibited myocardial regeneration, while transgenic miR-133 depletion enhanced cardiomyocyte proliferation. Expression analyses indicated that cell cycle factors mps1, cdc37, and PA2G4, and cell junction components cx43 and cldn5, are miR-133 targets during regeneration. With pharmacological inhibition and EGFP sensor interaction studies, we demonstrated that cx43 is a new miR-133 target and regeneration gene. Our results reveal dynamic regulation of miRNAs during heart regeneration, and indicate that miR-133 restricts injury-induced cardiomyocyte proliferation. PMID:22374218

Cardiac microRNA-133 is down-regulated in thyroid hormone-mediated cardiac hypertrophy partially via Type 1 Angiotensin II receptor.

PubMed

Diniz, Gabriela Placoná; Lino, Caroline Antunes; Guedes, Elaine Castilho; Moreira, Luana do Nascimento; Barreto-Chaves, Maria Luiza Morais

2015-09-01

Elevated thyroid hormone (TH) levels induce cardiac hypertrophy partially via type 1 Angiotensin II receptor (AT1R). MicroRNAs (miRNAs) are key regulators of cardiac homeostasis, and miR-133 has been shown to be involved in cardiac hypertrophy. However, the potential role of miR-133 in cardiac growth induced by TH is unknown. Thus, we aimed to investigate the miR-133 expression, as well as its potential role in cardiac hypertrophy in response to TH. Wistar rats were subjected to hyperthyroidism combined or not with the AT1R blocker. T3 serum levels were assessed to confirm the hyperthyroid status. TH induced cardiac hypertrophy, as evidenced by higher cardiac weight/tibia length ratio and α-actin mRNA levels, which was prevented by AT1R blocker. miR-133 expression was decreased in TH-induced cardiac hypertrophy in part through the AT1R. Additionally, the cardiac mRNA levels of miR-133 targets, SERCA2a and calcineurin were increased in hyperthyroidism partially via AT1R, as evaluated by real-time RT-PCR. Interestingly, miR-133 levels were unchanged in T3-induced cardiomyocyte hypertrophy in vitro. However, a gain-of-function study revealed that miR-133 mimic blunted the T3-induced cardiomyocyte hypertrophy in vitro. Together, our data indicate that miR-133 expression is reduced in TH-induced cardiac hypertrophy partially by the AT1R and that miR-133 mimic prevents the cardiomyocyte hypertrophy in response to T3 in vitro. These findings provide new insights regarding the mechanisms involved in the cardiac growth mediated by TH, suggesting that miR-133 plays a key role in TH-induced cardiomyocyte hypertrophy.

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

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

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.

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

PubMed

Benz, Peter M; Merkel, Carla J; Offner, Kristin; Abeßer, Marco; Ullrich, Melanie; Fischer, Tobias; Bayer, Barbara; Wagner, Helga; Gambaryan, Stepan; Ursitti, Jeanine A; Adham, Ibrahim M; Linke, Wolfgang A; Feller, Stephan M; Fleming, Ingrid; Renné, Thomas; Frantz, Stefan; Unger, Andreas; Schuh, Kai

2013-08-12

In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

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.

Fibroblast Growth Factors and Vascular Endothelial Growth Factor Promote Cardiac Reprogramming under Defined Conditions

PubMed Central

Yamakawa, Hiroyuki; Muraoka, Naoto; Miyamoto, Kazutaka; Sadahiro, Taketaro; Isomi, Mari; Haginiwa, Sho; Kojima, Hidenori; Umei, Tomohiko; Akiyama, Mizuha; Kuishi, Yuki; Kurokawa, Junko; Furukawa, Tetsushi; Fukuda, Keiichi; Ieda, Masaki

2015-01-01

Summary Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors, including Gata4, Mef2c, and Tbx5; however, this process is inefficient under serum-based culture conditions, in which conversion of partially reprogrammed cells into fully reprogrammed functional iCMs has been a major hurdle. Here, we report that a combination of fibroblast growth factor (FGF) 2, FGF10, and vascular endothelial growth factor (VEGF), termed FFV, promoted cardiac reprogramming under defined serum-free conditions, increasing spontaneously beating iCMs by 100-fold compared with those under conventional serum-based conditions. Mechanistically, FFV activated multiple cardiac transcriptional regulators and converted partially reprogrammed cells into functional iCMs through the p38 mitogen-activated protein kinase and phosphoinositol 3-kinase/AKT pathways. Moreover, FFV enabled cardiac reprogramming with only Mef2c and Tbx5 through the induction of cardiac reprogramming factors, including Gata4. Thus, defined culture conditions promoted the quality of cardiac reprogramming, and this finding provides new insight into the mechanism of cardiac reprogramming. PMID:26626177

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

Caffeine exposure alters cardiac gene expression in embryonic cardiomyocytes

PubMed Central

Fang, Xiefan; Mei, Wenbin; Barbazuk, William B.; Rivkees, Scott A.

2014-01-01

Previous studies demonstrated that in utero caffeine treatment at embryonic day (E) 8.5 alters DNA methylation patterns, gene expression, and cardiac function in adult mice. To provide insight into the mechanisms, we examined cardiac gene and microRNA (miRNA) expression in cardiomyocytes shortly after exposure to physiologically relevant doses of caffeine. In HL-1 and primary embryonic cardiomyocytes, caffeine treatment for 48 h significantly altered the expression of cardiac structural genes (Myh6, Myh7, Myh7b, Tnni3), hormonal genes (Anp and BnP), cardiac transcription factors (Gata4, Mef2c, Mef2d, Nfatc1), and microRNAs (miRNAs; miR208a, miR208b, miR499). In addition, expressions of these genes were significantly altered in embryonic hearts exposed to in utero caffeine. For in utero experiments, pregnant CD-1 dams were treated with 20–60 mg/kg of caffeine, which resulted in maternal circulation levels of 37.3–65.3 μM 2 h after treatment. RNA sequencing was performed on embryonic ventricles treated with vehicle or 20 mg/kg of caffeine daily from E6.5-9.5. Differential expression (DE) analysis revealed that 124 genes and 849 transcripts were significantly altered, and differential exon usage (DEU) analysis identified 597 exons that were changed in response to prenatal caffeine exposure. Among the DE genes identified by RNA sequencing were several cardiac structural genes and genes that control DNA methylation and histone modification. Pathway analysis revealed that pathways related to cardiovascular development and diseases were significantly affected by caffeine. In addition, global cardiac DNA methylation was reduced in caffeine-treated cardiomyocytes. Collectively, these data demonstrate that caffeine exposure alters gene expression and DNA methylation in embryonic cardiomyocytes. PMID:25354728

Persistence of neoangiogenesis and cardiomyocyte divisions in right ventricular myocardium of rats born and raised in hypoxic conditions.

PubMed

Moravec, Mireille; Turek, Zdenek; Moravec, Josef

2002-03-01

Effects of chronic hypoxia on capillary and myocyte growth were examined in rats born and raised in a low pressure chamber (equivalent of 3500 m a.s.l.). The animals were sacrificed at the age of 3 months and their hearts were used to study right ventricular growth and vascularization. The results of our cytological and morphometric analysis suggest the persistence of capillary neogenesis in this particular model of cardiac hypertrophy. Under the optical microscope, we observed significant changes in capillary spatial patterns such as the presence of sinusoids and irregular capillary sprouts. This resulted in a significant shortening of the effective diffusion distance and in a slight decrease in the calculated diameter of the Krogh cylinder. Concomitant to the remodeling of the terminal capillary network, the right ventricular myocardium of hypoxic rats exhibited peculiar changes in myocyte cytology. The principal alteration consisted in the ectopic subsarcolemmal location of some of muscle cell nuclei which appeared enlarged and rounded, sometimes irregularly folded. At the E. M. level, they presented chromatine condensation, nucleolemmal folding and, occasionally, nuclear splitting. Irregular chromatin densifications at the equatorial position were also encountered but we never observed nucleolemmal dissolution or typical metaphase plaques which excludes the presence of mitotic division. Some of the marginalized nuclei were progressively excluded from original binucleate cells into small cytoplasmic processes that invaded the adjacent neo-formed pericapillar spaces and gave rise to small well-organized cardiomyocytes. This apparent fragmentation of cardiomyocytes may evoke the description of the apoptotic process which is believed to be stimulated in hypoxic tissues. However, we could not confirm that myocyte fragmentation that we describe is followed by shrinkage necrosis or by any mobilization of adjacent resident cells. Nuclear exclusions into pericapillary

Cited2 Gene Controls Pluripotency and Cardiomyocyte Differentiation of Murine Embryonic Stem Cells through Oct4 Gene*

PubMed Central

Li, Qiang; Ramírez-Bergeron, Diana L.; Dunwoodie, Sally L.; Yang, Yu-Chung

2012-01-01

Cited2 (CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail 2) is a transcriptional modulator critical for the development of multiple organs. Although many Cited2-mediated phenotypes and molecular events have been well characterized using in vivo genetic murine models, Cited2-directed cell fate decision in embryonic stem cells (ESCs) remains elusive. In this study, we examined the role of Cited2 in the maintenance of stemness and pluripotency of murine ESCs by a gene-targeting approach. Cited2 knock-out (Cited2Δ/−, KO) ESCs display defective differentiation. Loss of Cited2 in differentiating ESCs results in delayed silencing of the genes involved in the maintenance of pluripotency and self-renewal of stem cells (Oct4, Klf4, Sox2, and c-Myc) and the disturbance in cardiomyocyte, hematopoietic, and neuronal differentiation. In addition, Cited2 KO ESCs experience a delayed induction of cardiomyocyte differentiation-associated proteins, NFAT3 (along with the reduced expression of NFAT3 target genes, Nkx2.5 and β-MHC), N-cadherin, and smooth muscle actin. CITED2 is recruited to the Oct4 promoter to regulate its expression during early ESC differentiation. This is the first demonstration that Cited2 controls ESC pluripotency and differentiation via direct regulation of Oct4 gene expression. PMID:22761414

In vitro particulate matter exposure causes direct and lung-mediated indirect effects on cardiomyocyte function.

PubMed

Gorr, Matthew W; Youtz, Dane J; Eichenseer, Clayton M; Smith, Korbin E; Nelin, Timothy D; Cormet-Boyaka, Estelle; Wold, Loren E

2015-07-01

Particulate matter (PM) exposure induces a pathological response from both the lungs and the cardiovascular system. PM is capable of both manifestation into the lung epithelium and entrance into the bloodstream. Therefore, PM has the capacity for both direct and lung-mediated indirect effects on the heart. In the present studies, we exposed isolated rat cardiomyocytes to ultrafine particulate matter (diesel exhaust particles, DEP) and examined their contractile function and calcium handling ability. In another set of experiments, lung epithelial cells (16HBE14o- or Calu-3) were cultured on permeable supports that allowed access to both the basal (serosal) and apical (mucosal) media; the basal media was used to culture cardiomyocytes to model the indirect, lung-mediated effects of PM on the heart. Both the direct and indirect treatments caused a reduction in contractility as evidenced by reduced percent sarcomere shortening and reduced calcium handling ability measured in field-stimulated cardiomyocytes. Treatment of cardiomyocytes with various anti-oxidants before culture with DEP was able to partially prevent the contractile dysfunction. The basal media from lung epithelial cells treated with PM contained several inflammatory cytokines, and we found that monocyte chemotactic protein-1 was a key trigger for cardiomyocyte dysfunction. These results indicate the presence of both direct and indirect effects of PM on cardiomyocyte function in vitro. Future work will focus on elucidating the mechanisms involved in these separate pathways using in vivo models of air pollution exposure. Copyright © 2015 the American Physiological Society.

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.

Regulation of cell growth by redox-mediated extracellular proteolysis of platelet-derived growth factor receptor beta.

PubMed

Okuyama, H; Shimahara, Y; Kawada, N; Seki, S; Kristensen, D B; Yoshizato, K; Uyama, N; Yamaoka, Y

2001-07-27

Redox-regulated processes are important elements in various cellular functions. Reducing agents, such as N-acetyl-l-cysteine (NAC), are known to regulate signal transduction and cell growth through their radical scavenging action. However, recent studies have shown that reactive oxygen species are not always involved in ligand-stimulated intracellular signaling. Here, we report a novel mechanism by which NAC blocks platelet-derived growth factor (PDGF)-induced signaling pathways in hepatic stellate cells, a fibrogenic player in the liver. Unlike in vascular smooth muscle cells, we found that reducing agents, including NAC, triggered extracellular proteolysis of PDGF receptor-beta, leading to desensitization of hepatic stellate cells toward PDGF-BB. This effect was mediated by secreted mature cathepsin B. In addition, type II transforming growth factor-beta receptor was also down-regulated. Furthermore, these events seemed to cause a dramatic improvement of rat liver fibrosis. These results indicated that redox processes impact the cell's response to growth factors by regulating the turnover of growth factor receptors and that "redox therapy" is promising for fibrosis-related disease.

In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes.

PubMed

van Deel, Elza D; Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão-Pires, Inês; van der Velden, Jolanda

2017-07-15

This paper describes a novel model that allows exploration of matrix-induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function. Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca 2+ handling and myofilament function. Cell shortening and Ca 2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix. Matrix stiffness-impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness. Matrix stiffness-induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte-matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix-induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca 2+ handling but does not alter myofilament-generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness-induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness-induced changes in cardiomyocyte

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

Bioreactor cultivation enhances NTEB formation and differentiation of NTES cells into cardiomyocytes.

PubMed

Lü, Shuanghong; Liu, Sheng; He, Wenjun; Duan, Cuimi; Li, Yanmin; Liu, Zhiqiang; Zhang, Ye; Hao, Tong; Wang, Yanmeng; Li, Dexue; Wang, Changyong; Gao, Shaorong

2008-09-01

Autogenic embryonic stem cells established from somatic cell nuclear transfer (SCNT) embryos have been proposed as unlimited cell sources for cell transplantation-based treatment of many genetic and degenerative diseases, which can eliminate the immune rejection that occurs after transplantation. In the present study, pluripotent nuclear transfer ES (NTES) cell lines were successfully established from different strains of mice. One NTES cell line, NT1, with capacity of germline transmission, was used to investigate in vitro differentiation into cardiomyocytes. To optimize differentiation conditions for mass production of embryoid bodies (NTEBs) from NTES cells, a slow-turning lateral vessel (STLV) rotating bioreactor was used for culturing the NTES cells to produce NTEBs compared with a conventional static cultivation method. Our results demonstrated that the NTEBs formed in STLV bioreactor were more uniform in size, and no large necrotic centers with most of the cells in NTEBs were viable. Differentiation of the NTEBs formed in both the STLV bioreactor and static culture into cardiomyocytes was induced by ascorbic acid, and the results demonstrated that STLV-produced NTEBs differentiated into cardiomyocytes more efficiently. Taken together, our results suggested that STLV bioreactor provided a more ideal culture condition, which can facilitate the formation of better quality NTEBs and differentiation into cardiomyocytes more efficiently in vitro.

Coculturing with endothelial cells promotes in vitro maturation and electrical coupling of human embryonic stem cell-derived cardiomyocytes.

PubMed

Pasquier, Jennifer; Gupta, Renuka; Rioult, Damien; Hoarau-Véchot, Jessica; Courjaret, Raphael; Machaca, Khaled; Al Suwaidi, Jassim; Stanley, Edouard G; Rafii, Shahin; Elliott, David A; Abi Khalil, Charbel; Rafii, Arash

2017-06-01

Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is, containing both endothelial cells (ECs) and cardiomyocytes. We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP), and an Akt-activated EC line (E4 + ECs). We quantified spontaneous beating rates, synchrony, and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. After 8 days in culture, 94% ± 6% of the NKX2-5GFP + cells were beating when hESCs embryonic bodies were plated on E4 + ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP + cardiomyocytes were close to the E4 + ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network, as illustrated by the loss of synchronization upon the disruption of endothelial bridges. The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. Copyright © 2017. Published by Elsevier Inc.

Analysis of beat fluctuations and oxygen consumption in cardiomyocytes by scanning electrochemical microscopy.

PubMed

Hirano, Yu; Kodama, Mikie; Shibuya, Masahiro; Maki, Yoshiyuki; Komatsu, Yasuo

2014-02-15

The contractile behavior of cardiomyocytes can be monitored by measuring their action potentials, and the analysis is essential for screening the safety of potential drugs. However, immobilizing cardiac cells on a specific electrode is considerably complicated. In this study, we demonstrate that scanning electrochemical microscopy (SECM) can be used to analyze rapid topographic changes in beating cardiomyocytes in a standard culture dish. Various cardiomyocyte contraction parameters and oxygen consumption based on cell respiration could be determined from SECM data. We also confirmed that cellular changes induced by adding the cardiotonic agent digoxin were conveniently monitored by this SECM system. These results show that SECM can be a potentially powerful tool for use in drug development for cardiovascular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

Manipulation-free cultures of human iPSC-derived cardiomyocytes offer a novel screening method for cardiotoxicity.

PubMed

Rajasingh, Sheeja; Isai, Dona Greta; Samanta, Saheli; Zhou, Zhi-Gang; Dawn, Buddhadeb; Kinsey, William H; Czirok, Andras; Rajasingh, Johnson

2018-04-05

Induced pluripotent stem cell (iPSC)-based cardiac regenerative medicine requires the efficient generation, structural soundness and proper functioning of mature cardiomyocytes, derived from the patient's somatic cells. The most important functional property of cardiomyocytes is the ability to contract. Currently available methods routinely used to test and quantify cardiomyocyte function involve techniques that are labor-intensive, invasive, require sophisticated instruments or can adversely affect cell vitality. We recently developed optical flow imaging method analyses and quantified cardiomyocyte contractile kinetics from video microscopic recordings without compromising cell quality. Specifically, our automated particle image velocimetry (PIV) analysis of phase-contrast video images captured at a high frame rate yields statistical measures characterizing the beating frequency, amplitude, average waveform and beat-to-beat variations. Thus, it can be a powerful assessment tool to monitor cardiomyocyte quality and maturity. Here we demonstrate the ability of our analysis to characterize the chronotropic responses of human iPSC-derived cardiomyocytes to a panel of ion channel modulators and also to doxorubicin, a chemotherapy agent with known cardiotoxic side effects. We conclude that the PIV-derived beat patterns can identify the elongation or shortening of specific phases in the contractility cycle, and the obtained chronotropic responses are in accord with known clinical outcomes. Hence, this system can serve as a powerful tool to screen the new and currently available pharmacological compounds for cardiotoxic effects.

Differential effects of saturated and unsaturated fatty acid diets on cardiomyocyte apoptosis, adipose distribution, and serum leptin.

PubMed

Okere, Isidore C; Chandler, Margaret P; McElfresh, Tracy A; Rennison, Julie H; Sharov, Victor; Sabbah, Hani N; Tserng, Kou-Yi; Hoit, Brian D; Ernsberger, Paul; Young, Martin E; Stanley, William C

2006-07-01

Fatty acids are the primary fuel for the heart and are ligands for peroxisome proliferator-activated receptors (PPARs), which regulate the expression of genes encoding proteins involved in fatty acid metabolism. Saturated fatty acids, particularly palmitate, can be converted to the proapoptotic lipid intermediate ceramide. This study assessed cardiac function, expression of PPAR-regulated genes, and cardiomyocyte apoptosis in rats after 8 wk on either a low-fat diet [normal chow control (NC); 10% fat calories] or high-fat diets composed mainly of either saturated (Sat) or unsaturated fatty acids (Unsat) (60% fat calories) (n = 10/group). The Sat group had lower plasma insulin and leptin concentrations compared with the NC or Unsat groups. Cardiac function and mass and body mass were not different. Cardiac triglyceride content was increased in the Sat and Unsat groups compared with NC (P < 0.05); however, ceramide content was higher in the Sat group compared with the Unsat group (2.9 +/- 0.2 vs. 1.4 +/- 0.2 nmol/g; P < 0.05), whereas the NC group was intermediate (2.3 +/- 0.3 nmol/g). The number of apoptotic myocytes, assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining, was higher in the Sat group compared with the Unsat group (0.28 +/- 0.05 vs. 0.17 +/- 0.04 apoptotic cells/1,000 nuclei; P < 0.04) and was positively correlated to ceramide content (P < 0.02). Both high-fat diets increased the myocardial mRNA expression of the PPAR-regulated genes encoding uncoupling protein-3 and pyruvate dehydrogenase kinase-4, but only the Sat diet upregulated medium-chain acyl-CoA dehydrogenase. In conclusion, dietary fatty acid composition affects cardiac ceramide accumulation, cardiomyocyte apoptosis, and expression of PPAR-regulated genes independent of cardiac mass or function.

In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes

PubMed Central

Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão‐Pires, Inês; van der Velden, Jolanda

2017-01-01

Key points This paper describes a novel model that allows exploration of matrix‐induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function.Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca2+ handling and myofilament function.Cell shortening and Ca2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix.Matrix stiffness‐impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness.Matrix stiffness‐induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Abstract Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte–matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix‐induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca2+ handling but does not alter myofilament‐generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness‐induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness�

BDNF-mediates Down-regulation of MicroRNA-195 Inhibits Ischemic Cardiac Apoptosis in Rats

PubMed Central

Hang, Pengzhou; Sun, Chuan; Guo, Jing; Zhao, Jing; Du, Zhimin

2016-01-01

Background: Our previous studies suggested that brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) axis inhibited cardiomyocyte apoptosis in myocardial infarction (MI). However, the relationship between BDNF and microRNA (miRNA) in cardiomyocytes are unclear. The present study was performed to investigate the role of miR-195 and the interplay between BDNF and miR-195 in ischemic cardiomyocyte apoptosis. Methods: Male Wistar rats were subjected to coronary artery ligation, and primary neonatal rat ventricular myocytes were treated with hypoxia or hydrogen peroxide (H2O2). BDNF level in rat ventricles was measured by enzyme linked immunosorbent assay (ELISA). miR-195 mimic, inhibitor or negative control was transfected into the cardiomyocytes. Cell viability and apoptosis were detected by MTT assay and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. Cardiac function and apoptosis were detected in MI rats intravenously injected with antagomiR-195. Luciferase assay, Western blot and Real-time RT-PCR were employed to clarify the interplay between miR-195 and BDNF. Results: miR-195 level was dynamically regulated in response to MI and significantly increased in ischemic regions 24 h post-MI as well as in hypoxic or H2O2-treated cardiomyocytes. Meanwhile, BDNF protein level was rapidly increased in MI rats and H2O2-treated cardiomyocytes. Apoptosis in both hypoxic and H2O2-treated cardiomyocytes were markedly reduced and cell viability was increased by miR-195 inhibitor. Moreover, inhibition of miR-195 significantly improved cardiac function of MI rats. Bcl-2 but not BDNF was validated as the direct target of miR-195. Furthermore, BDNF abolished the pro-apoptotic role of miR-195, which was reversed by its scavenger TrkB-Fc. Conclusion: Up-regulation of miR-195 in ischemic cardiomyocytes promotes ischemic apoptosis by targeting Bcl-2. BDNF mitigated the pro-apoptotic effect of miR-195 in rat cardiomyocytes. These findings may

Inflammatory and mitochondrial gene expression data in GPER-deficient cardiomyocytes from male and female mice.

PubMed

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

2017-02-01

We previously showed that cardiomyocyte-specific G protein-coupled estrogen receptor (GPER) gene deletion leads to sex-specific adverse effects on cardiac structure and function; alterations which may be due to distinct differences in mitochondrial and inflammatory processes between sexes. Here, we provide the results of Gene Set Enrichment Analysis (GSEA) based on the DNA microarray data from GPER-knockout versus GPER-intact (intact) cardiomyocytes. This article contains complete data on the mitochondrial and inflammatory response-related gene expression changes that were significant in GPER knockout versus intact cardiomyocytes from adult male and female mice. The data are supplemental to our original research article "Cardiomyocyte-specific deletion of the G protein-coupled estrogen receptor (GPER) leads to left ventricular dysfunction and adverse remodeling: a sex-specific gene profiling" (Wang et al., 2016) [1]. Data have been deposited to the Gene Expression Omnibus (GEO) database repository with the dataset identifier GSE86843.

3D aggregate culture improves metabolic maturation of human pluripotent stem cell derived cardiomyocytes.

PubMed

Correia, Cláudia; Koshkin, Alexey; Duarte, Patrícia; Hu, Dongjian; Carido, Madalena; Sebastião, Maria J; Gomes-Alves, Patrícia; Elliott, David A; Domian, Ibrahim J; Teixeira, Ana P; Alves, Paula M; Serra, Margarida

2018-03-01

Three-dimensional (3D) cultures of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) hold great promise for drug discovery, providing a better approximation to the in vivo physiology over standard two-dimensional (2D) monolayer cultures. However, the transition of CM differentiation protocols from 2D to 3D cultures is not straightforward. In this work, we relied on the aggregation of hPSC-derived cardiac progenitors and their culture under agitated conditions to generate highly pure cardiomyocyte aggregates. Whole-transcriptome analysis and 13 C-metabolic flux analysis allowed to demonstrate at both molecular and fluxome levels that such 3D culture environment enhances metabolic maturation of hiPSC-CMs. When compared to 2D, 3D cultures of hiPSC-CMs displayed down-regulation of genes involved in glycolysis and lipid biosynthesis and increased expression of genes involved in OXPHOS. Accordingly, 3D cultures of hiPSC-CMs had lower fluxes through glycolysis and fatty acid synthesis and increased TCA-cycle activity. Importantly, we demonstrated that the 3D culture environment reproducibly improved both CM purity and metabolic maturation across different hPSC lines, thereby providing a robust strategy to derive enriched hPSC-CMs with metabolic features closer to that of adult CMs. © 2017 Wiley Periodicals, Inc.

Differentiation of Rat bone marrow Mesenchymal stem cells into Adipocytes and Cardiomyocytes after treatment with platelet lysate

PubMed Central

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Barzegar, Kazem

2016-01-01

Background: Mesenchymal stem cells (MSCs) are multipotential cells and their therapeutic potency is under intense investigation. Studying the effect of different induction factors on MSCs could increase our knowledge about the differentiation potency of these cells. One of the most important sources of these factors in mammalian body is platelet. Platelet lysate (PL) contains many growth factors and therefore, it can be used as a differentiation inducer. In the present study, the effect of PL on differentiation of rat bone marrow MSCs into cardiomyocytes was studied. Materials and Methods: To study the differentiation-inducing effect of PL, MSCs were treated with 2.5, 5 and 10% PL. Early results of this study showed that PL in high concentrations (10%) induces adipogenic differentiation of MSCs. Therefore, to evaluate differentiation to cardiomyocytes, MSCs were cultured in media containing lower levels of PL (2.5% and 5%) and then cardiomyogenic differentiation was induced by treatment with 5-azacytidine. Differentiation of MSCs was evaluated using direct observation of beating cells, immunostaining and real-time PCR techniques. Results: The results of qPCR showed that treatment with PL alone increased the expression of cardiac alpha actinin (CAA) being predictable by earlier observation of beating cells in PL-treated groups. The results of staining assays against cardiac alpha actinin also showed that there were stained cells in PL-treated groups. Conclusion: The results of the present study showed that PL is a powerful induction factor for differentiation of MSCs into different cell lines such as cardiomyocytes and adipocytes. PMID:27047647

The cell-cycle interactome: a source of growth regulators?

PubMed

Blomme, Jonas; Inzé, Dirk; Gonzalez, Nathalie

2014-06-01

When plants develop, cell proliferation and cell expansion are tightly controlled in order to generate organs with a determinate final size such as leaves. Several studies have demonstrated the importance of the cell proliferation phase for leaf growth, illustrating that cell-cycle regulation is crucial for correct leaf development. A large and complex set of interacting proteins that constitute the cell-cycle interactome controls the transition from one cell-cycle phase to another. Here, we review the current knowledge on cell-cycle regulators from this interactome affecting final leaf size when their expression is altered, mainly in Arabidopsis. In addition to the description of mutants of CYCLIN-DEPENDENT KINASES (CDKs), CYCLINS (CYCs), and their transcriptional and post-translational regulators, a phenotypic analysis of gain- and loss-of-function mutants for 27 genes encoding proteins that interact with cell-cycle proteins is presented. This compilation of information shows that when cell-cycle-related genes are mis-expressed, leaf growth is often altered and that, seemingly, three main trends appear to be crucial in the regulation of final organ size by cell-cycle-related genes: (i) cellular compensation; (ii) gene dosage; and (iii) correct transition through the G2/M phase by ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) activation. In conclusion, this meta-analysis shows that the cell-cycle interactome is enriched in leaf growth regulators, and illustrates the potential to identify new leaf growth regulators among putative new cell-cycle regulators. © The Author 2013. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair.

PubMed

Hartman, Matthew E; Dai, Dao-Fu; Laflamme, Michael A

2016-01-15

Human pluripotent stem cells (PSCs) represent an attractive source of cardiomyocytes with potential applications including disease modeling, drug discovery and safety screening, and novel cell-based cardiac therapies. Insights from embryology have contributed to the development of efficient, reliable methods capable of generating large quantities of human PSC-cardiomyocytes with cardiac purities ranging up to 90%. However, for human PSCs to meet their full potential, the field must identify methods to generate cardiomyocyte populations that are uniform in subtype (e.g. homogeneous ventricular cardiomyocytes) and have more mature structural and functional properties. For in vivo applications, cardiomyocyte production must be highly scalable and clinical grade, and we will need to overcome challenges including graft cell death, immune rejection, arrhythmogenesis, and tumorigenic potential. Here we discuss the types of human PSCs, commonly used methods to guide their differentiation into cardiomyocytes, the phenotype of the resultant cardiomyocytes, and the remaining obstacles to their successful translation. Copyright © 2015 Elsevier B.V. All rights reserved.

Cardiac peroxisome proliferator-activated receptor-γ expression is modulated by oxidative stress in acutely infrasound-exposed cardiomyocytes.

PubMed

Pei, Zhaohui; Meng, Rongsen; Zhuang, Zhiqiang; Zhao, Yiqiao; Liu, Fangpeng; Zhu, Miao-Zhang; Li, Ruiman

2013-12-01

The aim of the present study was to examine the effects of acute infrasound exposure on oxidative damage and investigate the underlying mechanisms in rat cardiomyocytes. Neonatal rat cardiomyocytes were cultured and exposed to infrasound for several days. In the study, the expression of CAT, GPx, SOD1, and SOD2 and their activities in rat cardiomyocytes in infrasound exposure groups were significantly decreased compared to those in the various time controls, along with significantly higher levels of O2 (-) and H2O2. Decreased cardiac cell viability was not observed in various time controls. A significant reduction in cardiac cell viability was observed in the infrasound group compared to the control, while significantly increased cardiac cell viability was observed in the infrasound exposure and rosiglitazone pretreatment group. Compared to the control, rosiglitazone significantly upregulated CAT, GPx, SOD1, and SOD2 expression and their activities in rat cardiomyocytes exposed to infrasound, while the levels of O2 (-) or H2O2 were significantly decreased. A potential link between a significant downregulation of PPAR-γ expression in rat cardiomyocytes in the infrasound group was compared to the control and infrasound-induced oxidative stress. These findings indicate that infrasound can induce oxidative damage in rat cardiomyocytes by inactivating PPAR-γ.

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.

Iron Overload and Apoptosis of HL-1 Cardiomyocytes: Effects of Calcium Channel Blockade

PubMed Central

Chen, Mei-pian; Cabantchik, Z. Ioav; Chan, Shing; Chan, Godfrey Chi-fung; Cheung, Yiu-fai

2014-01-01

Background Iron overload cardiomyopathy that prevails in some forms of hemosiderosis is caused by excessive deposition of iron into the heart tissue and ensuing damage caused by a raise in labile cell iron. The underlying mechanisms of iron uptake into cardiomyocytes in iron overload condition are still under investigation. Both L-type calcium channels (LTCC) and T-type calcium channels (TTCC) have been proposed to be the main portals of non-transferrinic iron into heart cells, but controversies remain. Here, we investigated the roles of LTCC and TTCC as mediators of cardiac iron overload and cellular damage by using specific Calcium channel blockers as potential suppressors of labile Fe(II) and Fe(III) ingress in cultured cardiomyocytes and ensuing apoptosis. Methods Fe(II) and Fe(III) uptake was assessed by exposing HL-1 cardiomyocytes to iron sources and quantitative real-time fluorescence imaging of cytosolic labile iron with the fluorescent iron sensor calcein while iron-induced apoptosis was quantitatively measured by flow cytometry analysis with Annexin V. The role of calcium channels as routes of iron uptake was assessed by cell pretreatment with specific blockers of LTCC and TTCC. Results Iron entered HL-1 cardiomyocytes in a time- and dose-dependent manner and induced cardiac apoptosis via mitochondria-mediated caspase-3 dependent pathways. Blockade of LTCC but not of TTCC demonstrably inhibited the uptake of ferric but not of ferrous iron. However, neither channel blocker conferred cardiomyocytes with protection from iron-induced apoptosis. Conclusion Our study implicates LTCC as major mediators of Fe(III) uptake into cardiomyocytes exposed to ferric salts but not necessarily as contributors to ensuing apoptosis. Thus, to the extent that apoptosis can be considered a biological indicator of damage, the etiopathology of cardiosiderotic damage that accompanies some forms of hemosiderosis would seem to be unrelated to LTCC or TTCC, but rather to other

Cyp26b1 within the growth plate regulates bone growth in juvenile mice

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

Minegishi, Yoshiki; Department of Plastic and Reconstructive Surgery, University of Fukui Hospital, 23-3 Matsuokashimoaizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193; Department of Plastic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871

Highlights: • Retinoic acid and Cyp26b1 were oppositely localized in growth plate cartilage. • Cyp26b1 deletion in chondrocytes decreased bone growth in juvenile mice. • Cyp26b1 deletion reduced chondrocyte proliferation and growth plate height. • Vitamin A-depletion partially reversed growth plate abnormalities caused by Cyp26b1 deficiency. • Cyp26b1 regulates bone growth by controlling chondrocyte proliferation. - Abstract: Retinoic acid (RA) is an active metabolite of vitamin A and plays important roles in embryonic development. CYP26 enzymes degrade RA and have specific expression patterns that produce a RA gradient, which regulates the patterning of various structures in the embryo. However, itmore » has not been addressed whether a RA gradient also exists and functions in organs after birth. We found localized RA activities in the diaphyseal portion of the growth plate cartilage were associated with the specific expression of Cyp26b1 in the epiphyseal portion in juvenile mice. To disturb the distribution of RA, we generated mice lacking Cyp26b1 specifically in chondrocytes (Cyp26b1{sup Δchon} cKO). These mice showed reduced skeletal growth in the juvenile stage. Additionally, their growth plate cartilage showed decreased proliferation rates of proliferative chondrocytes, which was associated with a reduced height in the zone of proliferative chondrocytes, and closed focally by four weeks of age, while wild-type mouse growth plates never closed. Feeding the Cyp26b1 cKO mice a vitamin A-deficient diet partially reversed these abnormalities of the growth plate cartilage. These results collectively suggest that Cyp26b1 in the growth plate regulates the proliferation rates of chondrocytes and is responsible for the normal function of the growth plate and growing bones in juvenile mice, probably by limiting the RA distribution in the growth plate proliferating zone.« less

Effect of Plant Growth Regulators on Phytoremediation of Hexachlorocyclohexane-Contaminated Soil.

PubMed

Chouychai, Waraporn; Kruatrachue, Maleeya; Lee, Hung

2015-01-01

The influence of three plant growth regulators, indolebutyric acid (IBA), thidiazuron (TDZ) and gibberellic acid (GA3), either individually or in pair-wise combinations, on the ability of waxy corn plant to remove hexachlorocyclohexane (HCH) from contaminated soil was studied. Waxy corn seeds were immersed for 3 h in solutions of 1.0 mg/l IBA, 0.01 mg/l TDZ, 0.1 mg/l GA3, or a mixture of two of the growth regulators, and then inoculated in soil contaminated with 46.8 mg/kg HCH for 30 days. Pretreatment of corn seeds with the plant growth regulators did not enhance corn growth when compared with those immersed in distilled water (control), but the pretreatment enhanced HCH removal significantly. On day 30, HCH concentration in the bulk soil planted with corn seeds pretreated with GA3 or TDZ+GA3 decreased by 97.4% and 98.4%, respectively. In comparison, HCH removal in soil planted with non-pretreated control waxy corn seeds was only 35.7%. The effect of several growth regulator application methods was tested with 0.01 mg/l TDZ. The results showed that none of the methods, which ranged from seed immersion, watering in soil, or spraying on shoots, affected HCH removal from soil. However, the method of applying the growth regulators may affect corn growth. Watering the corn plant with TDZ in soil led to higher root fresh weight (2.2 g) and higher root dried weight (0.57 g) than the other treatments (0.2-1.7 g root fresh weight and 0.02-0.43 g root dried weight) on day 30. Varying the concentrations of GA3 did not affect the enhancement of corn growth and HCH removal on day 30. The results showed that plant growth regulators may have potential for use to enhance HCH phytoremediation.

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.

Tmem88a mediates GATA-dependent specification of cardiomyocyte progenitors by restricting WNT signaling

PubMed Central

Novikov, Natasha; Evans, Todd

2013-01-01

Biphasic control of WNT signaling is essential during cardiogenesis, but how the pathway switches from promoting cardiac mesoderm to restricting cardiomyocyte progenitor fate is unknown. We identified genes expressed in lateral mesoderm that are dysregulated in zebrafish when both gata5 and gata6 are depleted, causing a block to cardiomyocyte specification. This screen identified tmem88a, which is expressed in the early cardiac progenitor field and was previously implicated in WNT modulation by overexpression studies. Depletion of tmem88a results in a profound cardiomyopathy, secondary to impaired cardiomyocyte specification. In tmem88a morphants, activation of the WNT pathway exacerbates the cardiomyocyte deficiency, whereas WNT inhibition rescues progenitor cells and cardiogenesis. We conclude that specification of cardiac fate downstream of gata5/6 involves activation of the tmem88a gene to constrain WNT signaling and expand the number of cardiac progenitors. Tmem88a is a novel component of the regulatory mechanism controlling the second phase of biphasic WNT activity essential for embryonic cardiogenesis. PMID:23903195

Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy

PubMed Central

Ferguson, Bradley S.; Harrison, Brooke C.; Jeong, Mark Y.; Reid, Brian G.; Wempe, Michael F.; Wagner, Florence F.; Holson, Edward B.; McKinsey, Timothy A.

2013-01-01

Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy. PMID:23720316

Signal-dependent repression of DUSP5 by class I HDACs controls nuclear ERK activity and cardiomyocyte hypertrophy.

PubMed

Ferguson, Bradley S; Harrison, Brooke C; Jeong, Mark Y; Reid, Brian G; Wempe, Michael F; Wagner, Florence F; Holson, Edward B; McKinsey, Timothy A

2013-06-11

Cardiac hypertrophy is a strong predictor of morbidity and mortality in patients with heart failure. Small molecule histone deacetylase (HDAC) inhibitors have been shown to suppress cardiac hypertrophy through mechanisms that remain poorly understood. We report that class I HDACs function as signal-dependent repressors of cardiac hypertrophy via inhibition of the gene encoding dual-specificity phosphatase 5 (DUSP5) DUSP5, a nuclear phosphatase that negatively regulates prohypertrophic signaling by ERK1/2. Inhibition of DUSP5 by class I HDACs requires activity of the ERK kinase, mitogen-activated protein kinase kinase (MEK), revealing a self-reinforcing mechanism for promotion of cardiac ERK signaling. In cardiac myocytes treated with highly selective class I HDAC inhibitors, nuclear ERK1/2 signaling is suppressed in a manner that is absolutely dependent on DUSP5. In contrast, cytosolic ERK1/2 activation is maintained under these same conditions. Ectopic expression of DUSP5 in cardiomyocytes results in potent inhibition of agonist-dependent hypertrophy through a mechanism involving suppression of the gene program for hypertrophic growth. These findings define unique roles for class I HDACs and DUSP5 as integral components of a regulatory signaling circuit that controls cardiac hypertrophy.

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer.

PubMed

Lopes de Campos, Walter R; Chirwa, Nthato; London, Grace; Rotherham, Lia S; Morris, Lynn; Mayosi, Bongani M; Khati, Makobetsa

2014-01-01

HIV-associated cardiomyopathy (HIVCM) is of clinical concern in developing countries because of a high HIV-1 prevalence, especially subtype C, and limited access to highly active antiretroviral therapy (HAART). For these reasons, we investigated the direct and indirect effects of HIV-1 subtype C infection of cultured human cardiomyocytes and the mechanisms leading to cardiomyocytes damage; as well as a way to mitigate the damage. We evaluated a novel approach to mitigate HIVCM using a previously reported gp120 binding and HIV-1 neutralizing aptamer called UCLA1. We established a cell-based model of HIVCM by infecting human cardiomyocytes with cell-free HIV-1 or co-culturing human cardiomyocytes with HIV-infected monocyte derived macrophages (MDM). We discovered that HIV-1 subtype C unproductively (i.e. its life cycle is arrested after reverse transcription) infects cardiomyocytes. Furthermore, we found that HIV-1 initiates apoptosis of cardiomyocytes through caspase-9 activation, preferentially via the intrinsic or mitochondrial initiated pathway. CXCR4 receptor-using viruses were stronger inducers of apoptosis than CCR5 utilizing variants. Importantly, we discovered that HIV-1 induced apoptosis of cardiomyocytes was mitigated by UCLA1. However, UCLA1 had no protective effective on cardiomyocytes when apoptosis was triggered by HIV-infected MDM. When HIV-1 was treated with UCLA1 prior to infection of MDM, it failed to induce apoptosis of cardiomyocytes. These data suggest that HIV-1 causes a mitochondrial initiated apoptotic cascade, which signal through caspase-9, whereas HIV-1 infected MDM causes apoptosis predominantly via the death-receptor pathway, mediated by caspase-8. Furthermore the data suggest that UCLA1 protects cardiomyocytes from caspase-mediated apoptosis, directly by binding to HIV-1 and indirectly by preventing infection of MDM.

Disruption of the circadian clock within the cardiomyocyte influences mycardial contractile function, metabolism, and gene expression

USDA-ARS?s Scientific Manuscript database

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variatio...

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy

PubMed Central

2013-01-01

Background In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. Results We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Conclusions Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities. PMID:23937664

Vitamin E Reversed Apoptosis of Cardiomyocytes Induced by Exposure to High Dose Formaldehyde During Mice Pregnancy.

PubMed

Wu, Dongyuan; Jiang, Zhirong; Gong, Bing; Dou, Yue; Song, Mingxuan; Song, Xiaoxia; Tian, Yu

2017-10-21

In this study, we investigated the protection effect of Vitamin E (Vit E) on formaldehyde (FA) exposure during pregnancy induced apoptosis of cardiomyocytes, and used an HL-1 cell line to confirmed the findings in vivo.Pregnant mice received different doses of FA (0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 0.1 μg Vit E, or 1.5 mg/kg + 0.1 μg Vit E). TUNEL staining was used to reveal the apoptosis in cardiomyocytes, and SOD, MDA, GSH, Livin, and Caspase-3 in cardiomyocytes were detected by ELISA, RT-PCR, and Western blot. For in vitro study, HL-1 cells were treated with vehicle, 5 μmol/L FA, 25 μmol/L FA, 50 μmol/L FA, 10 mg/L Vit. E, and 50 μmol/L FA+ 10 mg/L Vit E, respectively. CCK-8 assay and flow cytometry were used to evaluate cell vitality and apoptosis. A high dose of FA exposure led to cytotoxicity in both pregnant mice and offspring, as TUNEL staining revealed a significant apoptosis of cardiomyocytes, and the alternation in SOD, GSH, MDA, Livin, and Caspase-3 was found in cardiomyocytes. 0.1 μg Vit. E could reverse high doses of FA exposure induced apoptosis of cardiomyocytes in both pregnant mice and offspring. The in vitro study revealed that FA exposure induced a decrease of cell viability and increased cell apoptosis, as well as oxidative stress in HL-1 cells with alternation in SOD, GSH, MDA, Livin, and Caspase-3.This study revealed a high dose of FA induced oxidative stress and apoptosis of cardiomyocytes in both pregnant mice and offspring, and Vit E supplement during pregnancy reversed the systemic and myocardial toxicity of FA.

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

Regulation of intestinal mucosal growth by amino acids.

PubMed

Ray, Ramesh M; Johnson, Leonard R

2014-03-01

Amino acids, especially glutamine (GLN) have been known for many years to stimulate the growth of small intestinal mucosa. Polyamines are also required for optimal mucosal growth, and the inhibition of ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, blocks growth. Certain amino acids, primarily asparagine (ASN) and GLN stimulate ODC activity in a solution of physiological salts. More importantly, their presence is also required before growth factors and hormones such as epidermal growth factor and insulin are able to increase ODC activity. ODC activity is inhibited by antizyme-1 (AZ) whose synthesis is stimulated by polyamines, thus, providing a negative feedback regulation of the enzyme. In the absence of amino acids mammalian target of rapamycin complex 1 (mTORC1) is inhibited, whereas, mTORC2 is stimulated leading to the inhibition of global protein synthesis but increasing the synthesis of AZ via a cap-independent mechanism. These data, therefore, explain why ASN or GLN is essential for the activation of ODC. Interestingly, in a number of papers, AZ has been shown to inhibit cell proliferation, stimulate apoptosis, or increase autophagy. Each of these activities results in decreased cellular growth. AZ binds to and accelerates the degradation of ODC and other proteins shown to regulate proliferation and cell death, such as Aurora-A, Cyclin D1, and Smad1. The correlation between the stimulation of ODC activity and the absence of AZ as influenced by amino acids is high. Not only do amino acids such as ASN and GLN stimulate ODC while inhibiting AZ synthesis, but also amino acids such as lysine, valine, and ornithine, which inhibit ODC activity, increase the synthesis of AZ. The question remaining to be answered is whether AZ inhibits growth directly or whether it acts by decreasing the availability of polyamines to the dividing cells. In either case, evidence strongly suggests that the regulation of AZ synthesis is the

Evaluation of electrical propagation delay with cardiomyocytes by photosensitization reaction in vitro

NASA Astrophysics Data System (ADS)

Doi, Marika; Ogawa, Emiyu; Arai, Tsunenori

2017-02-01

In order to study cardiomyocyte electrical conduction damage by a photosensitization reaction (PR) mostly comes from outside of the cells in a few minutes after the PR, we studied propagation delay of contact action potential with cardiomyocyte by the PR. To determine appropriate PR condition for tachyarrhythmia ablation, a precise electrophysiological experiment in vitro has been preferable. We measured the contact action potential using a microelectrode array system of which information may be correct than conventional Ca2+ measurement. We investigated the propagation delays of an evoked potential to evaluate the electrical conduction damage by the PR. Rat cardiomyocytes were cultivated for 5-7 days on a dish with which 64 electrodes were patterned, in an incubator controlled to 37°C, 5% CO2. The following conditions were used for the PR: 40 μg/ml talapordfin sodium and 290 mW/cm2, 40-78 J/cm2 for an irradiation. A 2D map was obtained to visualize the propagation delays of the evoked potential. The propagation speed, which was calculated based on the measured propagation delays, was decreased by about 30-50% on average of all electrodes after the PR. Therefore, we think 2D propagation delays measurement of the evoked potential with contact action potential measuring system might be available to evaluate the acute electrical conduction damage of cardiomyocyte by the PR.

Nuclear magnetic resonance spectroscopy reveals metabolic changes in living cardiomyocytes after low doses of ionizing radiation.

PubMed

Gramatyka, Michalina; Skorupa, Agnieszka; Sokół, Maria

2018-01-01

Several lines of evidence indicate that exposure of heart to ionizing radiation increases the risk of cardiotoxicity manifested by heart dysfunction and cardiovascular diseases. It was initially believed that the heart is an organ relatively resistant to radiation. Currently, however, it is suspected that even low doses of radiation (< 2 Gy) may have a negative impact on the cardiovascular system. Cardiotoxicity of ionizing radiation is associated with metabolic changes observed in cardiac cells injured by radiation. In this study, we used human cardiomyocytes as a model system, and studied their metabolic response to radiation using high-resolution magic angle spinning nuclear magnetic resonance techniques (HR-MAS NMR). Human cardiomyocytes cultured in vitro were exposed to ionizing radiation and their survival was assessed by clonogenic assay. Changes in apoptosis intensity and cell cycle distribution after the irradiation were measured as well. NMR spectra of cardiomyocytes were acquired using Bruker Avance 400 MHz spectrometer at a spinning rate of 3200 Hz. Survival of cardiomyocytes after NMR experiments was assessed by the Trypan blue exclusion assay. Exposure of cardiomyocytes to small doses of ionizing radiation had no effect on cell proliferation potential and intensity of cell death. However, analysis of metabolic profiles revealed changes in lipids, threonine, glycine, glycerophosphocholine, choline, valine, isoleucine, glutamate, reduced glutathione and taurine metabolism. The results of this study showed that ionizing radiation affects metabolic profiles of cardiomyocytes even at low doses, which potentially have no effect on cell viability.

Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

PubMed

Wang, Jiaxian; Cui, Chang; Nan, Haiyan; Yu, Yuanfang; Xiao, Yini; Poon, Ellen; Yang, Gang; Wang, Xijie; Wang, Chenchen; Li, Lingsong; Boheler, Kenneth Richard; Ma, Xu; Cheng, Xin; Ni, Zhenhua; Chen, Minglong

2017-08-09

Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease modeling, drug screening and regenerative medicine. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate, which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity, and enhanced both the Ca 2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast, and scalable maturation of regenerated cardiomyocytes.

Temporal Impact of Substrate Mechanics on Differentiation of Human Embryonic Stem Cells to Cardiomyocytes

PubMed Central

Hazeltine, Laurie B.; Badur, Mehmet G.; Lian, Xiaojun; Das, Amritava; Han, Wenqing; Palecek, Sean P.

2014-01-01

A significant clinical need exists to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, enabling tissue modeling for in vitro discovery of new drugs or cell-based therapies for heart repair in vivo. Chemical and mechanical microenvironmental factors are known to impact efficiency of stem cell differentiation, but cardiac differentiation protocols in hPSCs are typically performed on rigid tissue culture polystyrene (TCPS) surfaces which do not present a physiological mechanical setting. To investigate the temporal effects of mechanics on cardiac differentiation, we cultured human embryonic stem cells (hESCs) and their derivatives on polyacrylamide hydrogel substrates with a physiologically relevant range of stiffnesses. In directed differentiation and embryoid body culture systems, differentiation of hESCs to cardiac Troponin T-expressing (cTnT+) cardiomyocytes peaked on hydrogels of intermediate stiffness. Brachyury expression also peaked on intermediate stiffness hydrogels at day 1 of directed differentiation, suggesting that stiffness impacted the initial differentiation trajectory of hESCs to mesendoderm. To investigate the impact of substrate mechanics during cardiac specification of mesodermal progenitors, we initiated directed cardiomyocyte differentiation on TCPS and transferred cells to hydrogels at the Nkx2.5/Isl1+ cardiac progenitor cell stage. No differences in cardiomyocyte purity with stiffness were observed on day 15. These experiments indicate that differentiation of hESCs is sensitive to substrate mechanics at early stages of mesodermal induction, and proper application of substrate mechanics can increase the propensity of hESCs to differentiate to cardiomyocytes. PMID:24200714

NOX4 regulates autophagy during energy deprivation.

PubMed

Sciarretta, Sebastiano; Volpe, Massimo; Sadoshima, Junichi

2014-04-01

NADPH oxidase is a cellular enzyme devoted to the production of reactive oxygen species (ROS). NOX4 and NOX2 are the main isoforms of NADPH oxidase in the cardiovascular system. In our recent study, we demonstrated that NOX4, but not NOX2, is a critical mediator of the cardiomyocyte adaptive response to energy stress. NOX4 activity and protein levels are increased in the endoplasmic reticulum (ER) but not in mitochondria of cardiomyocytes during the early phase of energy deprivation. NOX4-derived production of ROS in the ER is a critical event that activates autophagy through stimulation of the EIF2AK3/PERK-EIF2S1/eIF-2α-ATF4 pathway. NOX4-dependent autophagy is an important mechanism to preserve cellular energy and limit cell death in energy-deprived cardiomyocytes. Aside from elucidating a crucial physiological function of NOX4 during cellular energy stress, our study dissects a novel signaling mechanism that regulates autophagy under this condition.

NOX4 regulates autophagy during energy deprivation

PubMed Central

Sciarretta, Sebastiano; Volpe, Massimo; Sadoshima, Junichi

2014-01-01

NADPH oxidase is a cellular enzyme devoted to the production of reactive oxygen species (ROS). NOX4 and NOX2 are the main isoforms of NADPH oxidase in the cardiovascular system. In our recent study, we demonstrated that NOX4, but not NOX2, is a critical mediator of the cardiomyocyte adaptive response to energy stress. NOX4 activity and protein levels are increased in the endoplasmic reticulum (ER) but not in mitochondria of cardiomyocytes during the early phase of energy deprivation. NOX4-derived production of ROS in the ER is a critical event that activates autophagy through stimulation of the EIF2AK3/PERK-EIF2S1/eIF-2α-ATF4 pathway. NOX4-dependent autophagy is an important mechanism to preserve cellular energy and limit cell death in energy-deprived cardiomyocytes. Aside from elucidating a crucial physiological function of NOX4 during cellular energy stress, our study dissects a novel signaling mechanism that regulates autophagy under this condition. PMID:24492492

Muscle FBPase binds to cardiomyocyte mitochondria under glycogen synthase kinase-3 inhibition or elevation of cellular Ca2+ level.

PubMed

Gizak, Agnieszka; Pirog, Michal; Rakus, Dariusz

2012-01-02

A growing body of research suggests that fructose 1,6-bisphosphatase (FBPase) might be involved in regulation of cell mortality/survival. However, the precise role of FBPase in the process remains unknown. Here, we show for the first time that in HL-1 cardiomyocytes, inhibition of glycogen synthase kinase-3 results in translocation of FBPase to mitochondria. In vitro experiments demonstrate that FBPase reduces the rate of calcium-induced mitochondrial swelling, affects ATP synthesis and interacts with mitochondrial proteins involved in regulation of volume and energy homeostasis. We suggest that FBPase might be engaged in a regulation of cell survival by influencing mitochondrial function. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Trimetazidine prevents palmitate-induced mitochondrial fission and dysfunction in cultured cardiomyocytes.

PubMed

Kuzmicic, Jovan; Parra, Valentina; Verdejo, Hugo E; López-Crisosto, Camila; Chiong, Mario; García, Lorena; Jensen, Michael D; Bernlohr, David A; Castro, Pablo F; Lavandero, Sergio

2014-10-01

Metabolic and cardiovascular disease patients have increased plasma levels of lipids and, specifically, of palmitate, which can be toxic for several tissues. Trimetazidine (TMZ), a partial inhibitor of lipid oxidation, has been proposed as a metabolic modulator for several cardiovascular pathologies. However, its mechanism of action is controversial. Given the fact that TMZ is able to alter mitochondrial metabolism, we evaluated the protective role of TMZ on mitochondrial morphology and function in an in vitro model of lipotoxicity induced by palmitate. We treated cultured rat cardiomyocytes with BSA-conjugated palmitate (25 nM free), TMZ (0.1-100 μM), or a combination of both. We evaluated mitochondrial morphology and lipid accumulation by confocal fluorescence microscopy, parameters of mitochondrial metabolism (mitochondrial membrane potential, oxygen consumption rate [OCR], and ATP levels), and ceramide production by mass spectrometry and indirect immunofluorescence. Palmitate promoted mitochondrial fission evidenced by a decrease in mitochondrial volume (50%) and an increase in the number of mitochondria per cell (80%), whereas TMZ increased mitochondrial volume (39%), and decreased mitochondrial number (56%), suggesting mitochondrial fusion. Palmitate also decreased mitochondrial metabolism (ATP levels and OCR), while TMZ potentiated all the metabolic parameters assessed. Moreover, pretreatment with TMZ protected the cardiomyocytes from palmitate-induced mitochondrial fission and dysfunction. TMZ also increased lipid accumulation in cardiomyocytes, and prevented palmitate-induced ceramide production. Our data show that TMZ protects cardiomyocytes by changing intracellular lipid management. Thus, the beneficial effects of TMZ on patients with different cardiovascular pathologies can be related to modulation of the mitochondrial morphology and function. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

Reduced ACTC1 expression might play a role in the onset of congenital heart disease by inducing cardiomyocyte apoptosis.

PubMed

Jiang, Hong-Kun; Qiu, Guang-Rong; Li-Ling, Jesse; Xin, Na; Sun, Kai-Lai

2010-11-01

The Cardiac α actin 1 gene (ACTC1) has been related to familial atrial septal defects. This study was set to explore a potential role of this gene in the formation of sporadic congenital heart disease (CHD). Assessment of cardiac tissue samples from 33 patients with sporadic CHD (gestational age (GA) 18 weeks-49 months) with real-time RT-PCR, Western blotting and immunohistochemistry has revealed a markedly decreased ACTC1 expression in the majority of samples (78.8%) compared with autopsied normal heart tissue from aged-matched subjects (GA 17 weeks-36 months). Also, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, the proportion of apoptotic cardiomyocytes in samples featuring down-regulated ACTC1 expression (Group 1) was significantly greater than those with normal expression (Group 2) and the controls (P<0.01). The proportion of apoptotic cells strongly correlated with the expression of ACTC1 (r=-0.918, P<0.01). A study of 2 essential genes involved in apoptosis, Caspase-3 and Bcl-2, confirmed that the former has significantly increased expression, whilst the latter has decreased expression in Group 1 than in the other groups (P<0.01). Transfection of a small interfering RNA targeting, Actc1 (Actc1-siRNA), to a cardiomyocyte cell line, H9C2, also detected more apoptotic cells. Reduced ACTC1 expression might play a role in the onset of CHD through induction of cardiomyocyte apoptosis.

Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN.

PubMed

Enugutti, Balaji; Kirchhelle, Charlotte; Oelschner, Maxi; Torres Ruiz, Ramón Angel; Schliebner, Ivo; Leister, Dario; Schneitz, Kay

2012-09-11

The spatial coordination of growth is of central importance for the regulation of plant tissue architecture. Individual layers, such as the epidermis, are clonally propagated and structurally maintained by symmetric cell divisions that are oriented along the plane of the layer. The developmental control of this process is poorly understood. The simple cellular basis and sheet-like structure of Arabidopsis integuments make them an attractive model system to address planar growth. Here we report on the characterization of the Arabidopsis UNICORN (UCN) gene. Analysis of ucn integuments reveals localized distortion of planar growth, eventually resulting in an ectopic multicellular protrusion. In addition, ucn mutants exhibit ectopic growth in filaments and petals, as well as aberrant embryogenesis. We further show that UCN encodes an active AGC VIII kinase. Genetic, biochemical, and cell biological data suggest that UCN suppresses ectopic growth in integuments by directly repressing the KANADI transcription factor ABERRANT TESTA SHAPE. Our findings indicate that UCN represents a unique plant growth regulator that maintains planar growth of integuments by repressing a developmental regulator involved in the control of early integument growth and polarity.

Symbiotic regulation of plant growth, development and reproduction

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Russell J. Rodriguez; D. Carl Freeman; E. Durant McArthur; Yong Ok Kim; Regina S. Redman