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Sample records for mouse cardiomyocyte injury

  1. Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

    PubMed Central

    Xu, Jiqian; Hu, Houxiang; Chen, Bin; Yue, Rongchuan; Zhou, Zhou; Liu, Yin; Zhang, Shuang; Xu, Lei; Wang, Huan; Yu, Zhengping

    2015-01-01

    Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes. PMID:26291709

  2. Isolation and Culture of Neonatal Mouse Cardiomyocytes

    PubMed Central

    Ehler, Elisabeth; Moore-Morris, Thomas; Lange, Stephan

    2013-01-01

    Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes. The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions. PMID:24056408

  3. Standardized bioenergetic profiling of adult mouse cardiomyocytes.

    PubMed

    Readnower, Ryan D; Brainard, Robert E; Hill, Bradford G; Jones, Steven P

    2012-12-18

    Mitochondria are at the crux of life and death and as such have become ideal targets of intervention in cardiovascular disease. Generally, current methods to measure mitochondrial dysfunction rely on working with the isolated organelle and fail to incorporate mitochondrial function in a cellular context. Extracellular flux methodology has been particularly advantageous in this respect; however, certain primary cell types, such as adult cardiac myocytes, have been difficult to standardize with this technology. Here, we describe methods for using extracellular flux (XF) analysis to measure mitochondrial bioenergetics in isolated, intact, adult mouse cardiomyocytes (ACMs). Following isolation, ACMs were seeded overnight onto laminin-coated (20 μg/ml) microplates, which resulted in high attachment efficiency. After establishing seeding density, we found that a commonly used assay medium (containing a supraphysiological concentration of pyruvate at 1 mmol/l) produced a maximal bioenergetic response. After performing a pyruvate dose-response, we determined that pyruvate titrated to 0.1 mmol/l was optimal for examining alternative substrate oxidation. Methods for measuring fatty acid oxidation were established. These methods lay the framework using XF analysis to profile metabolism of ACMs and will likely augment our ability to understand mitochondrial dysfunction in heart failure and acute myocardial ischemia. This platform could easily be extended to models of diabetes or other metabolic defects.

  4. HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange.

    PubMed

    Andersen, Ann-Dorit; Poulsen, Kristian Arild; Lambert, Ian H; Pedersen, Stine Falsig

    2009-05-01

    The Ca(2+)-independent phospholipase A(2) VI (iPLA(2)-VI) and the Na(+)/H(+) exchanger isoform 1 (NHE1) are highly pH-sensitive proteins that exert both protective and detrimental effects in cardiac ischemia-reperfusion. Here, we investigated the role of extracellular pH (pH(o)) in ischemia-reperfusion injury and death and in regulation and function of iPLA(2)-VI and NHE1 under these conditions. HL-1 cardiomyocytes were exposed to simulated ischemia (SI; 0.5% O(2), 8 mM K(+), and 20 mM lactate) at pH(o) 6.0 and 7.4, with or without 4 or 8 h of reperfusion (SI/R). Cytochrome c release and caspase-3 activation were reduced after acidic compared with neutral SI, whereas necrotic death, estimated as glucose-6-phosphate dehydrogenase release, was similar in the two conditions. Inhibition of iPLA(2)-VI activity by bromoenol lactone (BEL) elicited cardiomyocyte necrosis during normoxia and after acidic, yet not after neutral, SI. The isoform-selective enantiomers R- and S-BEL both mimicked the effect of racemic BEL after acidic SI. In contrast, inhibition of NHE activity by EIPA had no significant effect on necrosis after SI. Both neutral and acidic SI were associated with a reversible loss of F-actin and cortactin integrity. Inhibition of iPLA(2)-VI disrupted F-actin, cortactin, and mitochondrial integrity, whereas inhibition of NHE slightly reduced stress fiber content. iPLA(2)-VIA and NHE1 mRNA levels were reduced during SI and upregulated in a pH(o)-dependent manner during SI/R. This also affected the subcellular localization of iPLA(2)-VIA. Thus, the mode of cell death and the roles and regulation of iPLA(2)-VI and NHE1 are at least in part determined by the pH(o) during SI. In addition to having clinically relevant implications, these findings can in part explain the contradictory results obtained from previous studies of iPLA(2)-VIA and NHE1 during cardiac I/R.

  5. Excitation–Contraction Coupling of the Mouse Embryonic Cardiomyocyte

    PubMed Central

    Rapila, Risto; Korhonen, Topi; Tavi, Pasi

    2008-01-01

    In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca2+ release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9–11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat. PMID:18794377

  6. Excitation-contraction coupling of the mouse embryonic cardiomyocyte.

    PubMed

    Rapila, Risto; Korhonen, Topi; Tavi, Pasi

    2008-10-01

    In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca(2+) release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9-11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

  7. Silencing cardiomyocyte TLR4 reduces injury following hypoxia.

    PubMed

    Avlas, Orna; Srara, Smadar; Shainberg, Asher; Aravot, Dan; Hochhauser, Edith

    2016-11-01

    Toll-like receptor 4 (TLR4), the receptor for lipopolysaccharide (LPS) of gram-negative pathogens expressed in the heart, is activated by several endogenous ligands associated with tissue injury in response to myocardial infarction (MI). The aim of this study was to investigate the involvement of TLR4 signaling in cardiomyocytes dysfunction following hypoxia (90min) using multiple methodologies such as knocking down TLR4 and small interfering RNA (siTLR4). Cardiomyocytes of C57Bl/6 mice (WT) subjected to hypoxic stress showed increased cardiac release of LDH, HMGB1, IκB, TNF-α and myocardial apoptotic and necrotic markers (BAX, PI) compared to TLR4 knock out mice (TLR4KO). Treating these cardiomyocytes with siRNA against TLR4 decreased the damage markers (LDH, IκB, TNF-α). TLR4 silencing during hypoxic stress resulted in the activation of the p-AKT and p-GSK3β (by ∼25%). The latter is an indicator that there is a reduction of mitochondrial permeability transition pore (mPTP) opening following hypoxic myocardial induced injury leading to preserved mitochondrial membrane potential. Silencing TLR4 in cardiomyocytes improved cell survival following hypoxic injury through activation of the AKT/GSK3β pathway, reduced inflammatory and apoptotic signals. These findings suggest that TLR4 may serve as a potential target in the treatment of ischemic myocardial injury. Moreover, RNA interfering targeting TLR4 expression represents a therapeutic strategy.

  8. Direct reprogramming of mouse fibroblasts into cardiomyocytes with chemical cocktails.

    PubMed

    Fu, Yanbin; Huang, Chenwen; Xu, Xinxiu; Gu, Haifeng; Ye, Youqiong; Jiang, Cizhong; Qiu, Zilong; Xie, Xin

    2015-09-01

    The direct conversion, or transdifferentiation, of non-cardiac cells into cardiomyocytes by forced expression of transcription factors and microRNAs provides promising approaches for cardiac regeneration. However, genetic manipulations raise safety concerns and are thus not desirable in most clinical applications. The discovery of full chemically induced pluripotent stem cells suggest the possibility of replacing transcription factors with chemical cocktails. Here, we report the generation of automatically beating cardiomyocyte-like cells from mouse fibroblasts using only chemical cocktails. These chemical-induced cardiomyocyte-like cells (CiCMs) express cardiomyocyte-specific markers, exhibit sarcomeric organization, and possess typical cardiac calcium flux and electrophysiological features. Genetic lineage tracing confirms the fibroblast origin of these CiCMs. Further studies show the generation of CiCMs passes through a cardiac progenitor stage instead of a pluripotent stage. Bypassing the use of viral-derived factors, this proof of concept study lays a foundation for in vivo cardiac transdifferentiation with pharmacological agents and possibly safer treatment of heart failure.

  9. Green tea protects cytoskeleton from oxidative injury in cardiomyocytes.

    PubMed

    Pagnotta, Eleonora; Calonghi, Natalia; Hrelia, Silvana; Masotti, Lanfranco; Biagi, Pierluigi; Angeloni, Cristina

    2006-12-27

    Cardiac ischemia/reperfusion injury results in oxidative stress and poor physiological recovery. Episodes of hypoxia/reoxygenation (H/R) cause some subtle functional and structural alterations in sarcolemma, mithocondria, sarcoplasmic reticulum, nucleus, as well as cytoskeleton. In this report, by using cultured rat cardiomyocytes and laser confocal microscopy we have verified the possibility to counteract cytoskeleton alterations induced by H/R with the supplementation of an antioxidant agent, a green tea extract (GTE), and compared its effects to those of alpha-tocopherol. Moreover the effects of GTE on cell viability and cytosolic antioxidant activity have been evaluated. H/R induced myocardial damage occurs as histological alterations such as degeneration and disorganization of the cytoskeleton and loss of structural integrity of the nucleus. GTE supplementation increases cytosolic antioxidant activity and shows protective effects on cardiomyocyte cytoarchitecture and viability.

  10. Berberine alleviates cardiac ischemia/reperfusion injury by inhibiting excessive autophagy in cardiomyocytes.

    PubMed

    Huang, Zhouqing; Han, Zhihua; Ye, Bozhi; Dai, Zhenyu; Shan, Peiren; Lu, Zhongqiu; Dai, Kezhi; Wang, Changqian; Huang, Weijian

    2015-09-05

    Ischemia/reperfusion (I/R)-induced autophagy increases the severity of cardiomyocyte injury. The aim of this study was to investigate the effects of berberine, a natural extract from Rhizoma coptidis, on the I/R-induced excessive autophagy in in vitro and in vivo models. Autophagy was increased both in H9c2 myocytes during hypoxia/reoxygenation (H/R) injury and in mouse hearts exposed to I/R. And the expression level of p-AMPK and p-mTORC2 (Ser2481) were increased during H/R period. In addition, the increased autophagy level was correlated with reduced cell survival in H9c2 myocytes and increased infarct size in mouse hearts. However, berberine treatment significantly enhanced the H/R-induced cell viability and reduced I/R-induced myocardial infarct size, which was accompanied by improved cardiac function. The beneficial effect of berberine is associated with inhibiting the cellular autophagy level, due to decreasing the expression level of autophagy-related proteins such as SIRT1, BNIP3, and Beclin-1. Furthermore, both the level of p-AMPK and p-mTORC2 (Ser2481) in H9c2 myocytes exposed to H/R were decreased by berberine. In summary, berberine protects myocytes during I/R injury through suppressing autophagy activation. Therefore, berberine may be a promising agent for treating I/R-induced cardiac myocyte injury.

  11. Hypoxia Enhances Direct Reprogramming of Mouse Fibroblasts to Cardiomyocyte-Like Cells.

    PubMed

    Wang, Yanyan; Shi, Shujun; Liu, Huiwen; Meng, Li

    2016-02-01

    Recent work has shown that mouse and human fibroblasts can be reprogrammed to cardiomyocyte-like cells with a combination of transcription factors. Current research has focused on improving the efficiency and mechanisms for fibroblast reprogramming. Previously, it has been reported that hypoxia enhances fibroblast cell reprogramming to pluripotent stem cells. In this study, we observed that 6 h of hypoxic conditions (2% oxygen) on newborn mouse dermal fibroblasts can improve the efficiency of reprogramming to cardiomyocyte-like cells. Expression of cardiac-related genes and proteins increased at 4 weeks after transfer of three transcription factors (Gata4/Mef2c/Tbx5 [GMT]). However, beating cardiomyocyte cells were not detected. The epigenetic mechanism of hypoxia-induced fibroblast reprogramming to cardiomyocyte cells requires further study.

  12. Autophagy protects cardiomyocytes from the myocardial ischaemia-reperfusion injury through the clearance of CLP36

    PubMed Central

    Li, Shiguo; Liu, Chao; Gu, Lei; Wang, Lina; Shang, Yongliang; Liu, Qiong; Wan, Junyi; Shi, Jian; Wang, Fang; Xu, Zhiliang; Ji, Guangju

    2016-01-01

    Cardiovascular disease (CVD) is the leading cause of the death worldwide. An increasing number of studies have found that autophagy is involved in the progression or prevention of CVD. However, the precise mechanism of autophagy in CVD, especially the myocardial ischaemia-reperfusion injury (MI/R injury), is unclear and controversial. Here, we show that the cardiomyocyte-specific disruption of autophagy by conditional knockout of Atg7 leads to severe contractile dysfunction, myofibrillar disarray and vacuolar cardiomyocytes. A negative cytoskeleton organization regulator, CLP36, was found to be accumulated in Atg7-deficient cardiomyocytes. The cardiomyocyte-specific knockout of Atg7 aggravates the MI/R injury with cardiac hypertrophy, contractile dysfunction, myofibrillar disarray and severe cardiac fibrosis, most probably due to CLP36 accumulation in cardiomyocytes. Altogether, this work reveals autophagy may protect cardiomyocytes from the MI/R injury through the clearance of CLP36, and these findings define a novel relationship between autophagy and the regulation of stress fibre in heart. PMID:27512143

  13. Mathematical model of mouse embryonic cardiomyocyte excitation-contraction coupling.

    PubMed

    Korhonen, Topi; Rapila, Risto; Tavi, Pasi

    2008-10-01

    Excitation-contraction (E-C) coupling is the mechanism that connects the electrical excitation with cardiomyocyte contraction. Embryonic cardiomyocytes are not only capable of generating action potential (AP)-induced Ca(2+) signals and contractions (E-C coupling), but they also can induce spontaneous pacemaking activity. The spontaneous activity originates from spontaneous Ca(2+) releases from the sarcoplasmic reticulum (SR), which trigger APs via the Na(+)/Ca(2+) exchanger (NCX). In the AP-driven mode, an external stimulus triggers an AP and activates voltage-activated Ca(2+) intrusion to the cell. These complex and unique features of the embryonic cardiomyocyte pacemaking and E-C coupling have never been assessed with mathematical modeling. Here, we suggest a novel mathematical model explaining how both of these mechanisms can coexist in the same embryonic cardiomyocytes. In addition to experimentally characterized ion currents, the model includes novel heterogeneous cytosolic Ca(2+) dynamics and oscillatory SR Ca(2+) handling. The model reproduces faithfully the experimentally observed fundamental features of both E-C coupling and pacemaking. We further validate our model by simulating the effect of genetic modifications on the hyperpolarization-activated current, NCX, and the SR Ca(2+) buffer protein calreticulin. In these simulations, the model produces a similar functional alteration to that observed previously in the genetically engineered mice, and thus provides mechanistic explanations for the cardiac phenotypes of these animals. In general, this study presents the first model explaining the underlying cellular mechanism for the origin and the regulation of the heartbeat in early embryonic cardiomyocytes.

  14. Mathematical Model of Mouse Embryonic Cardiomyocyte Excitation–Contraction Coupling

    PubMed Central

    Korhonen, Topi; Rapila, Risto; Tavi, Pasi

    2008-01-01

    Excitation–contraction (E–C) coupling is the mechanism that connects the electrical excitation with cardiomyocyte contraction. Embryonic cardiomyocytes are not only capable of generating action potential (AP)-induced Ca2+ signals and contractions (E–C coupling), but they also can induce spontaneous pacemaking activity. The spontaneous activity originates from spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR), which trigger APs via the Na+/Ca2+ exchanger (NCX). In the AP-driven mode, an external stimulus triggers an AP and activates voltage-activated Ca2+ intrusion to the cell. These complex and unique features of the embryonic cardiomyocyte pacemaking and E–C coupling have never been assessed with mathematical modeling. Here, we suggest a novel mathematical model explaining how both of these mechanisms can coexist in the same embryonic cardiomyocytes. In addition to experimentally characterized ion currents, the model includes novel heterogeneous cytosolic Ca2+ dynamics and oscillatory SR Ca2+ handling. The model reproduces faithfully the experimentally observed fundamental features of both E–C coupling and pacemaking. We further validate our model by simulating the effect of genetic modifications on the hyperpolarization-activated current, NCX, and the SR Ca2+ buffer protein calreticulin. In these simulations, the model produces a similar functional alteration to that observed previously in the genetically engineered mice, and thus provides mechanistic explanations for the cardiac phenotypes of these animals. In general, this study presents the first model explaining the underlying cellular mechanism for the origin and the regulation of the heartbeat in early embryonic cardiomyocytes. PMID:18794378

  15. Effects of caulophine on caffeine-induced cellular injury and calcium homeostasis in rat cardiomyocytes.

    PubMed

    Si, Kai-Wei; Liu, Jun-Tian; He, Lang-Chong; Li, Xi-Kuan; Gou, Wei; Liu, Chuan-Hao; Li, Xiao-Qi

    2010-12-01

    Caulophine is a novel fluorenone alkaloid isolated from the radix of Caulophyllum robustum Maxim. Caulophine showed high affinity for the rat myocardial cell membrane as assessed by cell membrane chromatography, suggesting that the compound may exert bioactivity in the heart. It is known that calcium plays an important role in the pathogenesis of ischaemic heart disease, and caffeine can cause calcium overload in cardiomyocytes by inducing calcium release from the sarcoplasmic reticulum. Therefore, the present study evaluated the effects of caulophine on caffeine-induced injury and calcium homeostasis in cardiomyocytes. Cardiomyocytes were pre-treated with caulophine before exposure to caffeine or potassium chloride (KCl). Cell viability was assayed using the MTT method, and lactate dehydrogenase (LDH) and malondialdehyde (MDA) were measured spectrophotometrically. Caulophine-pre-treated cardiomyocytes were incubated with Fluo-3/AM, and then caffeine or KCl was used to induce Ca(2+) overload. The total intracellular Ca(2+) concentration was measured by flow cytometry. Fluorescence densities of single cardiomyocytes were detected using a confocal microscope. Caulophine increased the viability of caffeine-injured cardiomyocytes and decreased LDH activity and MDA level in cardiomyocytes. Furthermore, caulophine significantly decreased the total intracellular free Ca(2+) concentration and intracellular calcium release in cardiomyocytes in response to caffeine. However, the same concentrations of caulophine did not affect KCl-induced calcium influx. Our results suggest that caulophine protects cardiomyocytes from caffeine-induced injury as a result of calcium antagonism. This finding provides a basis for further study and development of caulophine as a new calcium antagonist for treating ischaemic cardiovascular diseases.

  16. Rac1 modulates cardiomyocyte adhesion during mouse embryonic development

    SciTech Connect

    Abu-Issa, Radwan

    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 normally 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.

  17. Functional brown adipose tissue limits cardiomyocyte injury and adverse remodeling in catecholamine-induced cardiomyopathy

    PubMed Central

    Thoonen, Robrecht; Ernande, Laura; Cheng, Juan; Nagasaka, Yasuko; Yao, Vincent; Miranda-Bezerra, Alexandre; Chen, Chan; Chao, Wei; Panagia, Marcello; Sosnovik, David E.; Puppala, Dheeraj; Armoundas, Antonis A.; Hindle, Allyson; Bloch, Kenneth D.; Buys, Emmanuel S.; Scherrer-Crosbie, Marielle

    2015-01-01

    Brown adipose tissue (BAT) has well recognized thermogenic properties mediated by uncoupling protein 1 (UCP1); more recently, BAT has been demonstrated to modulate cardiovascular risk factors. To investigate whether BAT also affects myocardial injury and remodeling, UCP1-deficient (UCP1−/−) mice, which have dysfunctional BAT, were subjected to catecholamine-induced cardiomyopathy. At baseline, there were no differences in echocardiographic parameters, plasma cardiac troponin I (cTnI) or myocardial fibrosis between wild-type (WT) and UCP1−/− mice. Isoproterenol infusion increased cTnI and myocardial fibrosis and induced left ventricular (LV) hypertrophy in both WT and UCP1−/− mice. UCP1−/− mice also demonstrated exaggerated myocardial injury, fibrosis, and adverse remodeling, as well as decreased survival. Transplantation of WT BAT to UCP1−/− mice prevented the isoproterenol-induced cTnI increase and improved survival, whereas UCP1−/− BAT transplanted to either UCP1−/− or WT mice had no effect on cTnI release. After 3 days of isoproterenol treatment, phosphorylated AKT and ERK were lower in the LV's of UCP1−/− mice than in those of WT mice. Activation of BAT was also noted in a model of chronic ischemic cardiomyopathy, and was correlated to LV dysfunction. Deficiency in UCP1, and accompanying BAT dysfunction, increases cardiomyocyte injury and adverse LV remodeling, and decreases survival in a mouse model of catecholamine-induced cardiomyopathy. Myocardial injury and decreased survival are rescued by transplantation of functional BAT to UCP1−/− mice, suggesting a systemic cardioprotective role of functional BAT. BAT is also activated in chronic ischemic cardiomyopathy. PMID:25968336

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

    PubMed

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

    2017-04-10

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

  19. Rac1 modulates cardiomyocyte adhesion during mouse embryonic development.

    PubMed

    Abu-Issa, Radwan

    2015-01-24

    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 normally 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.

  20. Arsenic trioxide alters the differentiation of mouse embryonic stem cell into cardiomyocytes

    PubMed Central

    Rebuzzini, Paola; Cebral, Elisa; Fassina, Lorenzo; Alberto Redi, Carlo; Zuccotti, Maurizio; Garagna, Silvia

    2015-01-01

    Chronic arsenic exposure is associated with increased morbidity and mortality for cardiovascular diseases. Arsenic increases myocardial infarction mortality in young adulthood, suggesting that exposure during foetal life correlates with cardiac alterations emerging later. Here, we investigated the mechanisms of arsenic trioxide (ATO) cardiomyocytes disruption during their differentiation from mouse embryonic stem cells. Throughout 15 days of differentiation in the presence of ATO (0.1, 0.5, 1.0 μM) we analysed: the expression of i) marker genes of mesoderm (day 4), myofibrillogenic commitment (day 7) and post-natal-like cardiomyocytes (day 15); ii) sarcomeric proteins and their organisation; iii) Connexin 43 and iv) the kinematics contractile properties of syncytia. The higher the dose used, the earlier the stage of differentiation affected (mesoderm commitment, 1.0 μM). At 0.5 or 1.0 μM the expression of cardiomyocyte marker genes is altered. Even at 0.1 μM, ATO leads to reduction and skewed ratio of sarcomeric proteins and to a rarefied distribution of Connexin 43 cardiac junctions. These alterations contribute to the dysruption of the sarcomere and syncytium organisation and to the impairment of kinematic parameters of cardiomyocyte function. This study contributes insights into the mechanistic comprehension of cardiac diseases caused by in utero arsenic exposure. PMID:26447599

  1. Arsenic trioxide alters the differentiation of mouse embryonic stem cell into cardiomyocytes.

    PubMed

    Rebuzzini, Paola; Cebral, Elisa; Fassina, Lorenzo; Alberto Redi, Carlo; Zuccotti, Maurizio; Garagna, Silvia

    2015-10-08

    Chronic arsenic exposure is associated with increased morbidity and mortality for cardiovascular diseases. Arsenic increases myocardial infarction mortality in young adulthood, suggesting that exposure during foetal life correlates with cardiac alterations emerging later. Here, we investigated the mechanisms of arsenic trioxide (ATO) cardiomyocytes disruption during their differentiation from mouse embryonic stem cells. Throughout 15 days of differentiation in the presence of ATO (0.1, 0.5, 1.0 μM) we analysed: the expression of i) marker genes of mesoderm (day 4), myofibrillogenic commitment (day 7) and post-natal-like cardiomyocytes (day 15); ii) sarcomeric proteins and their organisation; iii) Connexin 43 and iv) the kinematics contractile properties of syncytia. The higher the dose used, the earlier the stage of differentiation affected (mesoderm commitment, 1.0 μM). At 0.5 or 1.0 μM the expression of cardiomyocyte marker genes is altered. Even at 0.1 μM, ATO leads to reduction and skewed ratio of sarcomeric proteins and to a rarefied distribution of Connexin 43 cardiac junctions. These alterations contribute to the dysruption of the sarcomere and syncytium organisation and to the impairment of kinematic parameters of cardiomyocyte function. This study contributes insights into the mechanistic comprehension of cardiac diseases caused by in utero arsenic exposure.

  2. Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

    SciTech Connect

    Cao, Xueming; Chen, Aihua Yang, Pingzhen; Song, Xudong; Liu, Yingfeng; Li, Zhiliang; Wang, Xianbao; Wang, Lizi; Li, Yunpeng

    2013-11-29

    Highlights: •We observed the cell viability and death subjected to H/R in H9c2 cardiomyocytes. •We observed the degree of autophagy subjected to H/R in H9c2 cardiomyocytes. •LA inhibited the degree of autophagy in parallel to the enhanced cell survival. •LA inhibited the autophagy in parallel to the decreased total cell death. •We concluded that LA protected cardiomyocytes against H/R by inhibiting autophagy. -- Abstract: Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.

  3. Sevoflurane postconditioning attenuates cardiomyocyte hypoxia/reoxygenation injury via restoring mitochondrial morphology

    PubMed Central

    Yu, Jin; Wu, Jianjiang; Xie, Peng; Maimaitili, Yiliyaer; Wang, Jiang; Xia, Zhengyuan; Gao, Feng; Zhang, Xing

    2016-01-01

    Background Anesthetic postconditioning is a cellular protective approach whereby exposure to a volatile anesthetic renders a tissue more resistant to subsequent ischemic/reperfusion event. Sevoflurane postconditioning (SPostC) has been shown to exert cardioprotection against ischemia/reperfusion injury, but the underlying mechanism is unclear. We hypothesized that SPostC protects cardiomyocytes against hypoxia/reoxygenation (H/R) injury by maintaining/restoring mitochondrial morphological integrity, a critical determinant of cell fate. Methods Primary cultures of neonatal rat cardiomyocytes (NCMs) were subjected to H/R injury (3 h of hypoxia followed by 3 h reoxygenation). Intervention with SPostC (2.4% sevoflurane) was administered for 15 min upon the onset of reoxygenation. Cell viability, Lactate dehydrogenase (LDH) level, cell death, mitochondrial morphology, mitochondrial membrane potential and mitochondrial permeability transition pore (mPTP) opening were assessed after intervention. Mitochondrial fusion and fission regulating proteins (Drp1, Fis1, Mfn1, Mfn2 and Opa1) were assessed by immunofluorescence staining and western blotting was performed to determine the level of protein expression. Results Cardiomyocyte H/R injury resulted in significant increases in LDH release and cell death that were concomitant with reduced cell viability and reduced mitochondrial interconnectivity (mean area/perimeter ratio) and mitochondrial elongation, and with reduced mitochondrial membrane potential and increased mPTP opening. All the above changes were significantly attenuated by SPostC. Furthermore, H/R resulted in significant reductions in mitochondrial fusion proteins Mfn1, Mfn2 and Opa1 and significant enhancement of fission proteins Drp1 and Fis1. SPostC significantly enhanced Mfn2 and Opa1 and reduced Drp1, without significant impact on Mfn1 and Fis1. Conclusions Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury (HRI) by restoring

  4. CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart

    PubMed Central

    Matrone, Gianfranco; Wilson, Kathryn S.; Maqsood, Sana; Mullins, John J.; Tucker, Carl S.; Denvir, Martin A.

    2015-01-01

    ABSTRACT Cyclin dependent kinase (Cdk)9 acts through the positive transcription elongation factor-b (P-TEFb) complex to activate and expand transcription through RNA polymerase II. It has also been shown to regulate cardiomyocyte hypertrophy, with recent evidence linking it to cardiomyocyte proliferation. We hypothesised that modification of CDK9 activity could both impair and enhance the cardiac response to injury by modifying cardiomyocyte proliferation. Cdk9 expression and activity were inhibited in the zebrafish (Danio rerio) embryo. We show that dephosphorylation of residue Ser2 on the C-terminal domain of RNA polymerase II is associated with impaired cardiac structure and function, and cardiomyocyte proliferation and also results in impaired functional recovery following cardiac laser injury. In contrast, de-repression of Cdk9 activity, through knockdown of La-related protein (Larp7) increases phosphorylation of Ser2 in RNA polymerase II and increases cardiomyocyte proliferation. Larp7 knockdown rescued the structural and functional phenotype associated with knockdown of Cdk9. The balance of Cdk9 and Larp7 plays a key role in cardiomyocyte proliferation and response to injury. Larp7 represents a potentially novel therapeutic target to promote cardiomyocyte proliferation and recovery from injury. PMID:26542022

  5. 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.

  6. Exogenous Nitric Oxide Protects Human Embryonic Stem Cell-Derived Cardiomyocytes against Ischemia/Reperfusion Injury

    PubMed Central

    Pálóczi, János; Varga, Zoltán V.; Szebényi, Kornélia; Sarkadi, Balázs; Madonna, Rosalinda; De Caterina, Raffaele; Csont, Tamás; Eschenhagen, Thomas; Ferdinandy, Péter; Görbe, Anikó

    2016-01-01

    Background and Aims. Human embryonic stem cell- (hESC-) derived cardiomyocytes are one of the useful screening platforms of potential cardiocytoprotective molecules. However, little is known about the behavior of these cardiomyocytes in simulated ischemia/reperfusion conditions. In this study, we have tested the cytoprotective effect of an NO donor and the brain type natriuretic peptide (BNP) in a screening platform based first on differentiated embryonic bodies (EBs, 6 + 4 days) and then on more differentiated cardiomyocytes (6 + 24 days), both derived from hESCs. Methods. Both types of hESC-derived cells were exposed to 150 min simulated ischemia, followed by 120 min reperfusion. Cell viability was assessed by propidium iodide staining. The following treatments were applied during simulated ischemia in differentiated EBs: the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) (10−7, 10−6, and 10−5 M), BNP (10−9, 10−8, and 10−7 M), and the nonspecific NO synthase inhibitor Nω-nitro-L-arginine (L-NNA, 10−5 M). Results. SNAP (10−6, 10−5 M) significantly attenuated cell death in differentiated EBs. However, simulated ischemia/reperfusion-induced cell death was not affected by BNP or by L-NNA. In separate experiments, SNAP (10−6 M) also protected hESC-derived cardiomyocytes. Conclusions. We conclude that SNAP, but not BNP, protects differentiated EBs or cardiomyocytes derived from hESCs against simulated ischemia/reperfusion injury. The present screening platform is a useful tool for discovery of cardiocytoprotective molecules and their cellular mechanisms. PMID:27403231

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

    PubMed Central

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

    2011-01-01

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

  8. Induced expression of Fndc5 significantly increased cardiomyocyte differentiation rate of mouse embryonic stem cells.

    PubMed

    Rabiee, Farzaneh; Forouzanfar, Mahboobeh; Ghazvini Zadegan, Faezeh; Tanhaei, Somayeh; Ghaedi, Kamran; Motovali Bashi, Majid; Baharvand, Hossein; Nasr-Esfahani, Mohammad Hossein

    2014-11-10

    Fibronectin type III domain-containing 5 protein (Fndc5) is an exercise hormone and its transcript profile in mouse showed high degree of expression in heart, skeletal muscle and brain. Our previous studies indicated a significant increase (approximately 10 fold) in mRNA level of Fndc5 when embryonic stem cells were differentiated into beating bodies. As a step closer to identify the involvement of Fndc5 in the process of cardiomyocyte differentiation, we generated a stably inducible transduced mouse embryonic stem cell (mESC) line that overexpressed Fndc5 following Doxycycline induction. Our results indicated that the overexpression of Fndc5 during spontaneous cardiac differentiation significantly increased not only at RNA levels for mesodermal markers but also at the transcriptional levels for cardiac progenitor and cardiac genes. These data suggest that Fndc5 may be involved in cardiomyocyte differentiation. Therefore, a new hope will be arisen for potential application of this myokine for regeneration of damaged cardiac tissues especially in cardiac failure.

  9. Heart extracellular matrix supports cardiomyocyte differentiation of mouse embryonic stem cells

    PubMed Central

    Higuchi, Sayaka; Lin, Qingsong; Wang, Jigang; Lim, Teck Kwang; Joshi, Shashikant B.; Anand, Ganesh Srinivasan; Chung, Maxey C.M.; Sheetz, Michael P.; Fujita, Hideaki

    2017-01-01

    We have evaluated the effect of heart extracellular matrix (ECM) on the cardiomyocyte differentiation of mouse embryonic stem cells (ES cells) using de-cellularized heart tissue. Several lines of evidence indicate that ECM plays significant roles in cell proliferation, cell death and differentiation, but role of ECM possessing a 3D structure in differentiation has not been studied in detail. We found that there are substantial differences in the quantitative protein profiles of ECM in SDS-treated heart tissue compared to that of liver tissue, as assessed by iTRAQ™ quantitative proteomics analysis. When mouse ES cells were cultured on thin (60 μm) sections of de-cellularized tissue, the expression of cardiac myosin heavy chain (cMHC) and cardiac troponin I (cTnI) was high in ES cells cultured on heart ECM compared with those cultured on liver ECM. In addition, the protein expression of cMHC and cTnI was detected in cells on heart ECM after 2 weeks, which was not detectable in cells on liver ECM. These results indicate that heart ECM plays a critical role in the cardiomyocyte differentiation of ES cells. We propose that tissue-specific ECM induced cell lineage specification through mechano-transduction mediated by the structure, elasticity and components of ECM. PMID:23168383

  10. Liraglutide directly protects cardiomyocytes against reperfusion injury possibly via modulation of intracellular calcium homeostasis

    PubMed Central

    Hu, Shun-Ying; Zhang, Ying; Zhu, Ping-Jun; Zhou, Hao; Chen, Yun-Dai

    2017-01-01

    Background Liraglutide is glucagon-like peptide-1 receptor agonist for treating patients with type 2 diabetes mellitus. Our previous studies have demonstrated that liraglutide protects cardiac function through improving endothelial function in patients with acute myocardial infarction undergoing percutaneous coronary intervention. The present study will investigate whether liraglutide can perform direct protective effects on cardiomyocytes against reperfusion injury. Methods In vitro experiments were performed using H9C2 cells and neonatal rat ventricular cadiomyocytes undergoing simulative hypoxia/reoxygenation (H/R) induction. Cardiomyocytes apoptosis was detected by fluorescence TUNEL. Mitochondrial membrane potential (ΔΨm) and intracellular reactive oxygen species (ROS) was assessed by JC-1 and DHE, respectively. Fura-2/AM was used to measure intracellular Ca2+ concentration and calcium transient. Immunofluorescence staining was used to assess the expression level of sarcoplasmic reticulum Ca2+-ATPase (SERCA2a). In vivo experiments, myocardial apoptosis and expression of SERCA2a were detected by colorimetric TUNEL and by immunofluorescence staining, respectively. Results In vitro liraglutide inhibited cardiomyotes apoptosis against H/R. ΔΨm of cardiomyocytes was higher in liraglutide group than H/R group. H/R increased ROS production in H9C2 cells which was attenuated by liraglutide. Liraglutide significantly lowered Ca2+ overload and improved calcium transient compared with H/R group. Immunofluorescence staining results showed liraglutide promoted SERCA2a expression which was decreased in H/R group. In ischemia/reperfusion rat hearts, apoptosis was significantly attenuated and SERCA2a expression was increased by liraglutide compared with H/R group. Conclusions Liraglutide can directly protect cardiomyocytes against reperfusion injury which is possibly through modulation of intracellular calcium homeostasis. PMID:28270843

  11. The influence of agent delivery mode on cardiomyocyte injury induced by myocardial contrast echocardiography in rats.

    PubMed

    Miller, Douglas L; Dou, Chunyan; Armstrong, William F

    2005-09-01

    Myocardial contrast echocardiography (MCE) can induce bioeffects in rat hearts by local activation of the contrast agent gas bodies. This study was designed to examine the influence of agent delivery mode on the magnitude of cardiomyocyte injury. A total of 69 hairless rats were anesthetized and mounted vertically in a water bath. Evans blue dye was injected as vital stain for cardiomyocyte injury. Definity contrast agent was diluted in saline and injected via tail vein at 20 or 80 microL/kg in bolus or infusion mode. In 12 rats, 0.57 mg/kg dipyridamole was given to simulate a stress test. MCE in a short axis view with 1:4 or 1:16 ECG triggering was performed at 1.5 MHz for 5 or 20 min. The peak rarefactional pressure amplitude was set to 1.1 or 2.0 MPa. Premature beats were counted from the ECG record. Evans blue fluorescent cells were counted on frozen sections from the center of the scan plane of heart samples obtained 24 h postMCE. Infusion of the contrast agent led to more cardiomyocyte injury than did bolus injection. Dipyridamole stress also increased the effect. Varying the infusion rate or trigger interval was less important than the overall dosage during scanning. Exposure at 1.1 MPa and 80 microL/kg yielded significant cell killing relative to shams. Premature beats generally followed the same trends as cell injury, except that lower infusion rates tended to increase this effect. Contrast agent delivery mode, as well as dose and peak rarefactional pressure amplitude, has a significant influence on the bioeffects potential of MCE.

  12. Zinc pyrithione salvages reperfusion injury by inhibiting NADPH oxidase activation in cardiomyocytes.

    PubMed

    Kasi, Viswanath; Bodiga, Sreedhar; Kommuguri, Upendra Nadh; Sankuru, Suneetha; Bodiga, Vijaya Lakshmi

    2011-07-01

    Zinc pyrithione (ZPT), has a strong anti-apoptotic effect when administered just before reperfusion. Because oxidative stress has been proposed to contribute to myocardial reperfusion injury, we tested whether ZPT can reduce the production of reactive oxygen species during reoxygenation in cultured neonatal rat cardiac myocytes and evaluated the role of NADPH oxidase in hypoxia/reoxygenation (H/R) injury. The cells were subjected to 8h of simulated ischemia, followed by either 30 min or 16 h of reoxygenation. ZPT when started just before reoxygenation significantly reduced superoxide generation, LDH release and improved cell survival compared to H/R. Attenuation of the ROS production by ZPT paralleled its capacity to prevent pyknotic nuclei formation. In addition, ZPT reversed the H/R-induced expression of NOX2 and p47(phox) phosphorylation indicating that ZPT directly protects cardiomyocytes from reperfusion injury by a mechanism that attenuates NADPH oxidase mediated intracellular oxidative stress.

  13. Heart valve cardiomyocytes of mouse embryos express the serotonin transporter SERT

    SciTech Connect

    Pavone, Luigi Michele Spina, Anna; Lo Muto, Roberta; Santoro, Dionea; Mastellone, Vincenzo; Avallone, Luigi

    2008-12-12

    Multiple evidence demonstrate a role for serotonin and its transporter SERT in heart valve development and disease. By utilizing a Cre/loxP system driven by SERT gene expression, we recently demonstrated a regionally restricted distribution of SERT-expressing cells in developing mouse heart. In order to characterize the cell types exhibiting SERT expression within the mouse heart valves at early developmental stages, in this study we performed immunohistochemistry for Islet1 (Isl1) and connexin-43 (Cx-43) on heart sections from SERT{sup Cre/+};ROSA26R embryos previously stained with X-gal. We observed the co-localization of LacZ staining with Isl1 labelling in the outflow tract, the right ventricle and the conal region of E11.5 mouse heart. Cx-43 labelled cells co-localized with LacZ stained cells in the forming atrioventricular valves. These results demonstrate the cardiomyocyte phenotype of SERT-expressing cells in heart valves of the developing mouse heart, thus suggesting an active role of SERT in early heart valve development.

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

    PubMed

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

    2014-06-01

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

  15. Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

    SciTech Connect

    Greenberg, Cynthia C.; Connelly, Patricia S.; Daniels, Mathew P.; Horowits, Robert

    2008-03-10

    Krp1, also called sarcosin, is a cardiac and skeletal muscle kelch repeat protein hypothesized to promote the assembly of myofibrils, the contractile organelles of striated muscles, through interaction with N-RAP and actin. To elucidate its role, endogenous Krp1 was studied in primary embryonic mouse cardiomyocytes. While immunofluorescence showed punctate Krp1 distribution throughout the cell, detergent extraction revealed a significant pool of Krp1 associated with cytoskeletal elements. Reduction of Krp1 expression with siRNA resulted in specific inhibition of myofibril accumulation with no effect on cell spreading. Immunostaining analysis and electron microscopy revealed that cardiomyocytes lacking Krp1 contained sarcomeric proteins with longitudinal periodicities similar to mature myofibrils, but fibrils remained thin and separated. These thin myofibrils were degraded by a scission mechanism distinct from the myofibril disassembly pathway observed during cell division in the developing heart. The data are consistent with a model in which Krp1 promotes lateral fusion of adjacent thin fibrils into mature, wide myofibrils and contribute insight into mechanisms of myofibrillogenesis and disassembly.

  16. 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

  17. Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.

    PubMed

    Lee, Kunwoo; Yu, Pengzhi; Lingampalli, Nithya; Kim, Hyun Jin; Tang, Richard; Murthy, Niren

    2015-01-01

    The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.

  18. Protective Effects of Low-Frequency Magnetic Fields on Cardiomyocytes from Ischemia Reperfusion Injury via ROS and NO/ONOO−

    PubMed Central

    Ma, Sai; Zhang, Zhengxun; Yi, Fu; Wang, Yabin; Zhang, Xiaotian; Li, Xiujuan; Yuan, Yuan; Cao, Feng

    2013-01-01

    Background. Cardiac ischemia reperfusion (I/R) injury is associated with overproduction of reactive oxygen species (ROS). Low frequency pulse magnetic fields (LFMFs) have been reported to decrease ROS generation in endothelial cells. Whether LFMFs could assert protective effects on myocardial from I/R injury via ROS regulation remains unclear. Methods. To simulate in vivo cardiac I/R injury, neonatal rat cardiomyocytes were subjected to hypoxia reoxygenation (H/R) with or without exposure to LFMFs. Cell viability, apoptosis index, ROS generation (including O2− and ONOO−), and NO production were measured in control, H/R, and H/R + LFMF groups, respectively. Results. H/R injury resulted in cardiomyocytes apoptosis and decreased cell viability, whereas exposure to LFMFs before or after H/R injury significantly inhibited apoptosis and improved cell viability (P < 0.05). LFMFs treatment could suppress ROS (including O2− and ONOO−) generation induced by H/R injury, combined with decreased NADPH oxidase activity. In addition, LFMFs elevated NO production and enhanced NO/ONOO− balance in cardiomyocytes, and this protective effect was via the phosphorylation of endothelial nitric oxide synthase (eNOS). Conclusion. LFMFs could protect myocardium against I/R injury via regulating ROS generation and NO/ONOO− balance. LFMFs treatment might serve as a promising strategy for cardiac I/R injury. PMID:24312697

  19. Small Interfering RNA Targeting Mitochondrial Calcium Uniporter Improves Cardiomyocyte Cell Viability in Hypoxia/Reoxygenation Injury by Reducing Calcium Overload

    PubMed Central

    Oropeza-Almazán, Yuriana; Vázquez-Garza, Eduardo; Chapoy-Villanueva, Héctor; Torre-Amione, Guillermo

    2017-01-01

    Intracellular Ca2+ mishandling is an underlying mechanism in hypoxia/reoxygenation (H/R) injury that results in mitochondrial dysfunction and cardiomyocytes death. These events are mediated by mitochondrial Ca2+ (mCa2+) overload that is facilitated by the mitochondrial calcium uniporter (MCU) channel. Along this line, we evaluated the effect of siRNA-targeting MCU in cardiomyocytes subjected to H/R injury. First, cardiomyocytes treated with siRNA demonstrated a reduction of MCU expression by 67%, which resulted in significant decrease in mitochondrial Ca2+ transport. siRNA treated cardiomyocytes showed decreased mitochondrial permeability pore opening and oxidative stress trigger by Ca2+ overload. Furthermore, after H/R injury MCU silencing decreased necrosis and apoptosis levels by 30% and 50%, respectively, and resulted in reduction in caspases 3/7, 9, and 8 activity. Our findings are consistent with previous conclusions that demonstrate that MCU activity is partly responsible for cellular injury induced by H/R and support the concept of utilizing siRNA-targeting MCU as a potential therapeutic strategy. PMID:28337252

  20. The Influence of Copper (Cu) Deficiency in a Cardiomyocyte Cell Model (HL-1 Cell) of Ischemia/Reperfusion Injury

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mitochondria are important mediators of cell death and this study examines whether mitochondrial dysfunction caused by Cu deprivation promotes cell death in a cell culture model for ischemia/reperfusion injury in cardiomyocytes. HL-1 cells (kindly donated by Dr. William C. Claycomb, LSU Health Scien...

  1. Prevention of export of anoxia/reoxygenation injury from ischemic to nonischemic cardiomyocytes via inhibition of endocytosis.

    PubMed

    Khaidakov, Magomed; Mercanti, Federico; Wang, Xianwei; Ding, Zufeng; Dai, Yao; Romeo, Francesco; Sawamura, Tatsuya; Mehta, Jawahar L

    2014-06-15

    Myocardial infarct size is determined by the death of nonischemic border zone cardiomyocytes caused by export of injury signals from the infarct zone. The countermeasures to limit infarct size, therefore, should be aimed at nonselective blockade of most, if not all, injury signals from entering nonischemic cells. To test whether inhibition of endocytosis might limit infarct size, HL-1 cardiomyocytes were subjected to anoxia (6 h) and reoxygenation (1 h). Anoxic and reoxygenated cells showed a multifold increase in mitochondrial ROS production accompanied with upregulation of scavenger receptors lectin-like oxidized low-density lipoprotein receptor-1 and CD36 and stimulation of stress signals, including NADPH oxidase subunit p22(phox), SOD2, and beclin-1. Incubation of healthy cardiomyocytes in media from anoxic and reoxygenated cells (conditioned media) resulted in qualitatively similar responses, including increase in the generation of mitochondrial ROS, p22(phox), SOD2, and beclin-1. Anoxia and reoxygenation caused collapse of clathrin-mediated endocytosis and stimulation of macropinocytosis, whereas in cultures exposed to conditioned media, the activity of endocytosis was uniformly higher. Conditioned media also significantly aggravated cytotoxic effects of TNF-α and angiotensin II, and suppression of endocytosis reversed these trends, resulting in an overall increase of metabolic activity. Moreover, inhibition of endocytosis prevented binding of oxidized cellular fragments with greater efficiency than targeted neutralization of the scavenger receptor lectin-like oxidized low-density lipoprotein receptor-1. Many of the observations in HL-1 cardiomyocytes were confirmed in primary cardiomyocyte cultures. Our data suggest that endocytosis is upregulated in border zone cardiomyocytes, and inhibition of endocytosis may be an effective approach to prevent export of injury signals from the infarct zone.

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

    PubMed

    Yu, Zhi-Bin; Wei, Hongguang; Jin, J-P

    2012-07-01

    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 Ca(2+)-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 Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) 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 Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), 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.

  3. MicroRNA-122 regulates caspase-8 and promotes the apoptosis of mouse cardiomyocytes

    PubMed Central

    Zhang, Z.W.; Li, H.; Chen, S.S.; Li, Y.; Cui, Z.Y.; Ma, J.

    2017-01-01

    Cardiomyocyte apoptosis plays key roles in the pathogenesis of heart diseases such as myocardial infarction. MicroRNAs are important regulators of gene expression, which are also involved in the regulation of cardiomyocyte apoptosis. However, cardiomyocyte apoptosis regulated by microRNA (miR)-122 is largely unexplored. The aim of this study focused on the role of miR-122 in cardiomyocyte apoptosis. Cardiomyocytes were isolated from neonatal mice and primarily cultured. MiR-122 mimic and inhibitor were transfected to cardiomyocytes and verified by qRT-PCR. Cell viability and apoptosis post-transfection were assessed by MTT assay and flow cytometry, respectively. Changes in expression of caspase-8 were quantified by qRT-PCR and western blot. Results showed that miR-122 mimic and inhibitor successfully induced changes in miR-122 levels in cultured cardiomyocytes (P<0.01). MiR-122 overexpression suppressed viability and promoted apoptosis of cardiomyocytes (P<0.05), and miR-122 knockdown promoted cell viability and inhibited apoptosis (P<0.05). The mRNA and protein levels of caspase-8 were elevated by miR-122 overexpression (P<0.01) and reduced by miR-122 knockdown (P<0.001). These results suggest an inductive role of miR-122 in cardiomyocyte apoptosis, which may be related to its regulation on caspase-8. PMID:28177059

  4. Role of nonmuscle myosin IIB and N-RAP in cell spreading and myofibril assembly in primary mouse cardiomyocytes.

    PubMed

    Lu, Shajia; Horowits, Robert

    2008-09-01

    We investigated the role of nonmuscle myosin heavy chain (NMHC) IIB in cultured embryonic mouse cardiomyocytes by specific knockdown using RNA interference. NMHC IIB protein levels decreased 90% compared with mock-transfected cells by 3 days post transfection. NMHC IIB knockdown resulted in a slow decrease in N-RAP protein levels over 6 days with no change in N-RAP transcript levels. N-RAP is a scaffold for alpha-actinin and actin assembly during myofibrillogenesis, and we quantitated myofibril accumulation by morphometric analysis of alpha-actinin organization. Between 3 and 6 days, NMHC IIB knockdown was accompanied by the abolishment of cardiomyocyte spreading. During this period the rate of myofibril accumulation steadily decreased, correlating with the slowly decreasing levels of N-RAP. Between 6 and 8 days NMHC IIB and N-RAP protein levels recovered, and cardiomyocyte spreading and myofibril accumulation resumed. Inhibition of proteasome function using MG132 led to accumulation of excess N-RAP, and the secondary decrease in N-RAP that otherwise accompanied NMHC IIB knockdown was abolished. The results show that NMHC IIB knockdown led to decreased N-RAP levels through proteasome-mediated degradation. Furthermore, these proteins have distinct functional roles, with NMHC IIB playing a role in cardiomyocyte spreading and N-RAP functioning in myofibril assembly.

  5. All-Trans Retinoic Acid Ameliorates Myocardial Ischemia/Reperfusion Injury by Reducing Cardiomyocyte Apoptosis.

    PubMed

    Zhu, Zhengbin; Zhu, Jinzhou; Zhao, Xiaoran; Yang, Ke; Lu, Lin; Zhang, Fengru; Shen, Weifeng; Zhang, Ruiyan

    2015-01-01

    Myocardial ischemia/reperfusion (I/R) injury interferes with the restoration of blood flow to ischemic myocardium. Oxidative stress-elicited apoptosis has been reported to contribute to I/R injury. All-trans retinoic acid (ATRA) has anti-apoptotic activity as previously reported. Here, we investigated the effects and the mechanism of action of ATRA on myocardial I/R injury both in vivo and in vitro. In vivo, ATRA reduced the size of the infarcted area (17.81±1.05% vs. 24.41±1.03%, P<0.05) and rescued cardiac function loss (ejection fraction 46.42±6.76% vs. 37.18±4.63%, P<0.05) after I/R injury. Flow-cytometric analysis and TUNEL assay demonstrated that the protective role of ATRA on myocardial I/R injury was related to its anti-apoptotic effects. The anti-apoptotic effects of ATRA were associated with partial inhibition of reactive oxygen species (ROS) production and significantly less phosphorylation of mitogen-activated protein kinases (MAPKs) including p38, JNK, and ERK. Western blot analysis also revealed that ATRA pre-treatment increased a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) expression (0.65 ± 0.20 vs. 0.41±0.02 in vivo) and reduced the level of receptor for advanced glycation end-products (RAGE) (0.38 ± 0.17 vs. 0.52 ± 0.11 in vivo). Concomitantly, the protective role of ATRA on I/R injury was not observed in RAGE-KO mice. The current results indicated that ATRA could prevent myocardial injury and reduced cardiomyocyte apoptosis after I/R effectively. One possible mechanism underlying these effects is that ATRA could increase ADAM10 expression and thus cleave RAGE, which is the main receptor up-stream of MAPKs in myocardial I/R injury, resulting in the down-regulation of MAPK signaling and protective role on myocardial I/R injury.

  6. Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells.

    PubMed

    Alvarez-Collazo, Julio; Alonso-Carbajo, Lucía; López-Medina, Ana I; Alpizar, Yeranddy A; Tajada, Sendoa; Nilius, Bernd; Voets, Thomas; López-López, José Ramón; Talavera, Karel; Pérez-García, María Teresa; Alvarez, Julio L

    2014-11-01

    Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

  7. Dioxin Exposure Disrupts the Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes

    PubMed Central

    Wang, Ying; Fan, Yunxia; Puga, Alvaro

    2010-01-01

    Experimental exposure of fish, birds, and rodents to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) causes multiple Ah receptor–mediated developmental abnormalities, an observation consistent with compelling evidence in human populations that TCDD exposure is responsible for a significant incidence of birth defects. To characterize molecular mechanisms that might explain the developmental effects of dioxin, we have studied the consequences of TCDD exposure on the differentiation of mouse embryonic stem (ES) cells in culture and on the expression of genes, including those coding for homeodomain containing transcription factors, with a role in progression of tissue differentiation and embryonic identity during development. We find that TCDD treatment causes expression changes in a number of homeobox genes concomitant with Ah receptor recruitment to the promoters of many of these genes, whether under naïve or dioxin-activated conditions. TCDD exposure also derails temporal expression trajectories of developmentally regulated genes in a wide diversity of differentiation pathways, including genes with functions in neural and cardiovascular development, self-renewal, hematopoiesis and mesenchymal lineage specification, and Notch and Wnt pathways. Among these, we find that TCDD represses the expression of the cardiac development–specific Nkx2.5 homeobox transcription factor, of cardiac troponin-T and of α- and β-myosin heavy chains, inhibiting the formation of beating cardiomyocytes, a characteristic phenotype of differentiating mouse ES cells in culture. These data identify potential pathways for dioxin to act as a developmental teratogen, possibly critical to cardiovascular development and disease, and provide molecular targets that may help us understand the molecular basis of Ah receptor–mediated developmental toxicity. PMID:20130022

  8. Dioxin exposure disrupts the differentiation of mouse embryonic stem cells into cardiomyocytes.

    PubMed

    Wang, Ying; Fan, Yunxia; Puga, Alvaro

    2010-05-01

    Experimental exposure of fish, birds, and rodents to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) causes multiple Ah receptor-mediated developmental abnormalities, an observation consistent with compelling evidence in human populations that TCDD exposure is responsible for a significant incidence of birth defects. To characterize molecular mechanisms that might explain the developmental effects of dioxin, we have studied the consequences of TCDD exposure on the differentiation of mouse embryonic stem (ES) cells in culture and on the expression of genes, including those coding for homeodomain containing transcription factors, with a role in progression of tissue differentiation and embryonic identity during development. We find that TCDD treatment causes expression changes in a number of homeobox genes concomitant with Ah receptor recruitment to the promoters of many of these genes, whether under naïve or dioxin-activated conditions. TCDD exposure also derails temporal expression trajectories of developmentally regulated genes in a wide diversity of differentiation pathways, including genes with functions in neural and cardiovascular development, self-renewal, hematopoiesis and mesenchymal lineage specification, and Notch and Wnt pathways. Among these, we find that TCDD represses the expression of the cardiac development-specific Nkx2.5 homeobox transcription factor, of cardiac troponin-T and of alpha- and beta-myosin heavy chains, inhibiting the formation of beating cardiomyocytes, a characteristic phenotype of differentiating mouse ES cells in culture. These data identify potential pathways for dioxin to act as a developmental teratogen, possibly critical to cardiovascular development and disease, and provide molecular targets that may help us understand the molecular basis of Ah receptor-mediated developmental toxicity.

  9. Subthreshold nitric oxide synthase inhibition improves synergistic effects of subthreshold MMP-2/MLCK-mediated cardiomyocyte protection from hypoxic injury.

    PubMed

    Bil-Lula, Iwona; Lin, Han-Bin; Biały, Dariusz; Wawrzyńska, Magdalena; Diebel, Lucas; Sawicka, Jolanta; Woźniak, Mieczyslaw; Sawicki, Grzegorz

    2016-06-01

    Injury of myocardium during ischaemia/reperfusion (I/R) is a complex and multifactorial process involving uncontrolled protein phosphorylation, nitration/nitrosylation by increased production of nitric oxide and accelerated contractile protein degradation by matrix metalloproteinase-2 (MMP-2). It has been shown that simultaneous inhibition of MMP-2 with doxycycline (Doxy) and myosin light chain kinase (MLCK) with ML-7 at subthreshold concentrations protects the heart from contractile dysfunction triggered by I/R in a synergistic manner. In this study, we showed that additional co-administration of nitric oxide synthase (NOS) inhibitor (1400W or L-NAME) in subthreshold concentrations improves this synergistic protection in the model of hypoxia-reoxygenation (H-R)-induced contractile dysfunction of cardiomyocytes. Isolated cardiomyocytes were subjected to 3 min. of hypoxia and 20 min. of reoxygenation in the presence or absence of the inhibitor cocktails. Contractility of cardiomyocytes was expressed as myocyte peak shortening. Inhibition of MMP-2 by Doxy (25-100 μM), MLCK by ML-7 (0.5-5 μM) and NOS by L-NAME (25-100 μM) or 1400W (25-100 μM) protected myocyte contractility after H-R in a concentration-dependent manner. Inhibition of these activities resulted in full recovery of cardiomyocyte contractility after H-R at the level of highest single-drug concentration. The combination of subthreshold concentrations of NOS, MMP-2 and MLCK inhibitors fully protected cardiomyocyte contractility and MLC1 from degradation by MMP-2. The observed protection with addition of L-NAME or 1400W was better than previously reported combination of ML-7 and Doxy. The results of this study suggest that addition of NOS inhibitor to the mixture of inhibitors is better strategy for protecting cardiomyocyte contractility.

  10. HDAC Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy

    PubMed Central

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

    2014-01-01

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

  11. 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.

  12. Conditional deletion of cardiomyocyte peroxisome proliferator-activated receptor γ enhances myocardial ischemia-reperfusion injury in mice.

    PubMed

    Hobson, Michael J; Hake, Paul W; O'Connor, Michael; Schulte, Christine; Moore, Victoria; James, Jeanne M; Piraino, Giovanna; Zingarelli, Basilia

    2014-01-01

    The nuclear transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of the inflammatory response to an array of biologic insults. We have previously demonstrated that PPARγ ligands reduce myocardial ischemia-reperfusion injury in rodents. In the current study, we directly determined the role of cardiomyocyte PPARγ in ischemia-reperfusion injury, using a model of conditional cardiomyocyte-specific deletion of PPARγ in vivo. In mice, α-myosin heavy chain-restricted Cre-mediated PPARγ deficiency was induced by tamoxifen treatment (30 mg/kg intraperitoneally) for 4 days (PPARγ mice), whereas controls included mice treated with the oil diluent vehicle (PPARγ mice). Western blot and histochemical analyses confirmed that expression of PPARγ protein was abolished in cardiomyocytes of mice treated with tamoxifen, but not with vehicle. After tamoxifen or vehicle treatment, animals were subjected to 30-min ligation of the left anterior descending coronary artery followed by 2-h reperfusion. In PPARγ mice, myocardial ischemia and reperfusion induced extensive myocardial damage, which was associated with elevated tissue activity of myeloperoxidase, indicating infiltration of neutrophils, and elevated plasma levels of troponin I when compared with PPARγ mice. Upon echocardiographic analysis, PPARγ mice also demonstrated ventricular dilatation and systolic dysfunction. Plasma levels of the proinflammatory cytokines interleukin 1β and interleukin 6 were higher in PPARγ mice when compared with PPARγ mice. These pathological events in PPARγ mice were associated with enhanced nuclear factor κB DNA binding in the infarcted hearts. Thus, our data suggest that cardiomyocyte PPARγ is a crucial protective receptor and may prevent reperfusion injury by modulating mechanisms of inflammation.

  13. Mitochondrial Toxicity of Perfluorooctane Sulfonate in Mouse Embryonic Stem Cell-Derived Cardiomyocytes.

    PubMed

    Tang, Lei-Lei; Wang, Jia-Dan; Xu, Ting-Ting; Zhao, Zhe; Zheng, Jia-Jie; Ge, Ren-Shan; Zhu, Dan-Yan

    2017-03-10

    Perfluorooctane sulfonate (PFOS) is a persistent organic contaminant that may cause cardiotoxicity in animals and humans. However, little is known about the underlying mechanism by which it affects the organelle toxicity in cardiomyocytes during the cardiogenesis. Our previous proteomic study showed that differences of protein expression mainly existed in mitochondria of cardiomyocytes differentiated from embryonic stem (ES) cells after exposure to PFOS. Here, we focused on mitochondrial toxicity of PFOS in ES cell-derived cardiomyocytes. The cardiomyogenesis from ES cells in vitro was inhibited, and the expression of L-type Ca(2+) channel (LTCC) was decreased to interrupt [Ca(2+)]c transient amplitude in cardiomyocytes after PFOS treatment. Transmission electron microscope revealed that swollen mitochondrion with vacuole in PFOS-treated cells. Meanwhile, mitochondrial transmembrane potential (ΔYm) was declined and ATP production was lowered. These changes were related to the increased EGFR phosphorylation, activated Rictor signaling, then mediated HK2 binding to mitochondrial membrane. Furthermore, PFOS reduced the interaction of IP3R-Grp75-VDAC and accumulated intracellular fatty acids by activating Rictor, thereby attenuating PGC-1a and Mfn2 expressions, then destroying mitochondria-associated endoplasmic reticulum membrane (MAM), which resulted in the decrease of [Ca(2+)]mito transient amplitude triggered by ATP. In conclusion, mitochondrial structure damages and abnormal Ca(2+) shuttle were the important aspects in PFOS-induced cardiomyocytes toxicity from ES cells by activating Rictor signaling pathway.

  14. The miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation of mouse embryonic cardiomyocytes.

    PubMed

    Xiang, Rui; Lei, Han; Chen, Mianzhi; Li, Qinwei; Sun, Huan; Ai, Jianzhong; Chen, Tielin; Wang, Honglian; Fang, Yin; Zhou, Qin

    2012-02-01

    MicroRNAs (miRNAs) have gradually been recognized as regulators of embryonic development; however, relatively few miRNAs have been identified that regulate cardiac development. A series of recent papers have established an essential role for the miRNA-17-92 (miR-17-92) cluster of miRNAs in the development of the heart. Previous research has shown that the Friend of Gata-2 (FOG-2) is critical for cardiac development. To investigate the possibility that the miR-17-92 cluster regulates FOG-2 expression and inhibits proliferation in mouse embryonic cardiomyocytes we initially used bioinformatics to analyze 3' untranslated regions (3'UTR) of FOG-2 to predict the potential of miR-17-92 to target it. We used luciferase assays to demonstrate that miR-17-5p and miR-20a of miR-17-92 interact with the predicted target sites in the 3'UTR of FOG-2. Furthermore, RT-PCR and Western blot were used to demonstrate the post-transcriptional regulation of FOG-2 by miR-17-92 in embryonic cardiomyocytes from E12.5-day pregnant C57BL/6J mice. Finally, EdU cell assays together with the FOG-2 rescue strategy were employed to evaluate the effect of proliferation on embryonic cardiomyocytes. We first found that the miR-17-5p and miR-20a of miR-17-92 directly target the 3'UTR of FOG-2 and post-transcriptionally repress the expression of FOG-2. Moreover, our findings demonstrated that over-expression of miR-17-92 may inhibit cell proliferation via post-transcriptional repression of FOG-2 in embryonic cardiomyocytes. These results indicate that the miR-17-92 cluster regulates the expression of FOG-2 protein and suggest that the miR-17-92 cluster might play an important role in heart development.

  15. Histamine deficiency exacerbates myocardial injury in acute myocardial infarction through impaired macrophage infiltration and increased cardiomyocyte apoptosis.

    PubMed

    Deng, Long; Hong, Tao; Lin, Jinyi; Ding, Suling; Huang, Zheyong; Chen, Jinmiao; Jia, Jianguo; Zou, Yunzeng; Wang, Timothy C; Yang, Xiangdong; Ge, Junbo

    2015-08-17

    Histamine is a biogenic amine that is widely distributed and has multiple functions, but the role it plays in acute myocardial infarction (AMI) remains unclear. In this study, we investigated the origin and contribution of endogenous histamine to AMI. Histidine decarboxylase (HDC) is the unique enzyme responsible for histamine generation. Using HDC-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the HDC promoter, we identified HDC expression primarily in CD11b(+)Gr-1(+) immature myeloid cells (IMCs) that markedly increase in the early stages of AMI. Deficiency of histamine in HDC knockout mice (HDC(-/-)) reduced cardiac function and exacerbated the injury of infarcted heart. Furthermore, administering either an H1 receptor antagonist (pyrilamine) or an H2 receptor antagonist (cimetidine) demonstrated a protective effect of histamine against myocardial injury. The results of in vivo and in vitro assays showed that histamine deficiency promotes the apoptosis of cardiomyocytes and inhibits macrophage infiltration. In conclusion, CD11b(+)Gr-1(+) IMCs are the predominant HDC-expressing sites in AMI, and histamine plays a protective role in the process of AMI through inhibition of cardiomyocyte apoptosis and facilitation of macrophage infiltration.

  16. MiR-146b protects cardiomyocytes injury in myocardial ischemia/reperfusion by targeting Smad4

    PubMed Central

    Di, Yun-Feng; Li, De-Cai; Shen, Yan-Qing; Wang, Chun-Lei; Zhang, Da-Yong; Shang, An-Quan; Hu, Teng

    2017-01-01

    MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, have been implicated as critical regulatory molecules in many cardiovascular diseases, including ischemia-/reperfusion-induced cardiac injury. In the present study, we report on the role of miR-146b in myocardial I/R injury and the underlying cardio-protective mechanism. Antagomir-146b was used to explore the effects of miR-146b on cardiac ischemia/reperfusion injury (30 min ischemia followed by 180 min reperfusion). As predicted, miR-146b overexpression significantly reduced the infarct size and cardiomyocytes apoptosis and release of creatine kinase and lactate dehydrogenase. In addition, miR-146b attenuated H9c2 cell apoptosis. Furthermore, Smad4 was predicted and verified as a potential miR-146b target using bioinformatics and luciferase assay. In summary, this study demonstrated that miR-146b plays a critical protective role in cardiac ischemic injury and may provide a new therapeutic approach for the treatment of myocardial I/R injury.

  17. Proliferation of mouse embryonic stem cell progeny and the spontaneous contractile activity of cardiomyocytes are affected by microtopography.

    PubMed

    Biehl, Jesse K; Yamanaka, Satoshi; Desai, Tejal A; Boheler, Kenneth R; Russell, Brenda

    2009-08-01

    The niche in which stem cells reside and differentiate is a complex physicochemical microenvironment that regulates cell function. The role played by three-dimensional physical contours was studied on cell progeny derived from mouse embryonic stem cells using microtopographies created on PDMS (poly-dimethyl-siloxane) membranes. While markers of differentiation were not affected, the proliferation of heterogeneous mouse embryonic stem cell-derived progeny was attenuated by 15 microm-, but not 5 microm-high microprojections. This reduction was reversed by Rho kinase and myosin light chain kinase inhibition, which diminishes the tension generating ability of stress fibers. Purified cardiomyocytes derived from embryonic stem cells also showed significant blunting of proliferation and increased beating rates compared with cells grown on flat substrates. Thus, proliferation of stem cell-derived progeny appears to be regulated by microtopography through tension-generation of contractility in the third-dimension. These results emphasize the importance of topographic cues in the modulation of stem cell progeny behavior.

  18. 1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells

    PubMed Central

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

    2016-01-01

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

  19. 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

  20. Generation of an inducible, cardiomyocyte-specific transgenic mouse model with PPAR β/δ overexpression.

    PubMed

    Kim, Teayoun; Zhelyabovska, Olga; Liu, Jian; Yang, Qinglin

    2013-01-01

    Peroxisome proliferator-activated receptors (PPARs) consist of three subtypes, each displaying distinctive tissue distribution. In general, the three PPAR subtypes exert overlapping function in transcriptional regulation of lipid metabolism. However, each PPAR subtype possesses distinctive functions in different tissues dependent on their expression abundance, endogenous ligands, and the PPAR coregulators in a specific tissue. Transgenesis is an invaluable technique in defining the in vivo function of a particular gene and its protein. Cre/LoxP-mediated gene targeting has been extensively used to explore the tissue-specific function of PPARs. While this tissue-specific loss-of-function approach is extremely useful in determining the essential role of a PPAR, the tissue-specific gain-of-function approach is another important technique used to understand the effects of PPAR activation in a particular tissue. Transgenic overexpression of PPAR in a specific tissue has been used. However, this conventional technique requires generating the transgenic models individually for each target tissue. In this chapter, we describe the methodology for a more efficient generation of transgenic mouse models with a constitutively active form of PPARβ/δ in different tissues.

  1. 9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes.

    PubMed

    Burt, Rees; Graves, Bridget M; Gao, Ming; Li, Chaunfu; Williams, David L; Fregoso, Santiago P; Hoover, Donald B; Li, Ying; Wright, Gary L; Wondergem, Robert

    2013-09-01

    It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca(2+)-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

  2. 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.

  3. Gypenoside Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of Mitogen-Activated Protein Kinase Mediated Nuclear Factor Kappa B Pathway In Vitro and In Vivo

    PubMed Central

    Yu, Haijie; Shi, Liye; Qi, Guoxian; Zhao, Shijie; Gao, Yuan; Li, Yuzhe

    2016-01-01

    Gypenoside (GP) is the major effective component of Gynostemma pentaphyllum and has been shown to encompass a variety of pharmacological activities. In this study, we investigated whether GP is able to protect cardiomyocytes against injury myocardial ischemia–reperfusion (I/R) injury by using in vitro oxygen-glucose deprivation–reoxygenation (OGD/R) H9c2 cell model and in vivo myocardial I/R rat model. We found that GP pre-treatment alleviated the impairments on the cardiac structure and function in I/R injured rats. Moreover, pre-treatment with GP significantly inhibited IκB-α phosphorylation and nuclear factor (NF)-κB p65 subunit translocation into nuclei. GP and the MAPK pathway inhibitors also reduced the phosphorylation of ERK, JNK, and p38 in vitro. Specific inhibition of ERK, JNK, and p38 increased the cell viability of OGD/R injured cells. Taken together, our data demonstrated that GP protects cardiomyocytes against I/R injury by inhibiting NF-κB p65 activation via the MAPK signaling pathway both in vitro and in vivo. These findings suggest that GP may be a promising agent for the prevention or treatment of myocardial I/R injury. PMID:27313532

  4. Effect of intracellular lipid droplets on cytosolic Ca2+ and cell death during ischaemia–reperfusion injury in cardiomyocytes

    PubMed Central

    Barba, Ignasi; Chavarria, Laia; Ruiz-Meana, Marisol; Mirabet, Maribel; Agulló, Esperanza; Garcia-Dorado, David

    2009-01-01

    Lipid droplets (LD) consist of accumulations of triacylglycerols and have been proposed to be markers of ischaemic but viable tissue. Previous studies have described the presence of LD in myocardium surviving an acute coronary occlusion. We investigated whether LD may be protective against cell death secondary to ischaemia–reperfusion injury. The addition of oleate–bovine serum albumin complex to freshly isolated adult rat cardiomyocytes or to HL-1 cells resulted in the accumulation of intracellular LD detectable by fluorescence microscopy, flow cytometry and 1H-nuclear magnetic resonance spectroscopy. Simulated ischaemia–reperfusion of HL-1 cells (respiratory inhibition at pH 6.4 followed by 30 min of reperfusion) resulted in significant cell death (29.7 ± 2.6% of total lactate dehydrogenase release). However, cell death was significantly attenuated in cells containing LD (40% reduction in LDH release compared with control cells, P= 0.02). The magnitude of LD accumulation was inversely correlated (r2= 0.68, P= 0.0003) with cell death. The protection associated with intracellular LD was not a direct effect of the fatty acids used to induce their formation, because oleate added 30 min before ischaemia, during ischaemia or during reperfusion did not form LD and did not protect against cell death. Increasing the concentration of free oleate during reperfusion progressively decreased the protection afforded by LD. HL-1 cells labelled with fluo-4, a Ca2+-sensitive fluorochrome, fluorescence within LD areas increased more throughout simulated ischaemia and reperfusion than in the cytosolic LD-free areas of the same cells. As a consequence, cells with LD showed less cytosolic Ca2+ overload than control cells. These results suggest that LD exert a protective effect during ischaemia–reperfusion by sequestering free fatty acids and Ca2+. PMID:19188253

  5. Cardiomyocyte­-specific expression of the nuclear matrix protein, CIZ1, stimulates production of mono-nucleated cells with an extended window of proliferation in the postnatal mouse heart

    PubMed Central

    Bageghni, Sumia A.; Frentzou, Georgia A.; Drinkhill, Mark J.; Mansfield, William; Coverley, Dawn

    2017-01-01

    ABSTRACT Myocardial injury in mammals leads to heart failure through pathological cardiac remodelling that includes hypertrophy, fibrosis and ventricular dilatation. Central to this is inability of the mammalian cardiomyocyte to self-renew due to entering a quiescent state after birth. Modulation of the cardiomyocyte cell-cycle after injury is therefore a target mechanism to limit damage and potentiate repair and regeneration. Here, we show that cardiomyocyte-specific over-expression of the nuclear-matrix­-associated DNA replication protein, CIZ1, extends their window of proliferation during cardiac development, delaying onset of terminal differentiation without compromising function. CIZ1-expressing hearts are enlarged, but the cardiomyocytes are smaller with an overall increase in number, correlating with increased DNA replication after birth and retention of an increased proportion of mono-nucleated cardiomyocytes into adulthood. Furthermore, these CIZ1 induced changes in the heart reduce the impact of myocardial injury, identifying CIZ1 as a putative therapeutic target for cardiac repair. PMID:27934662

  6. Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals During the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions.

    PubMed

    Wang, Qin; Kurita, Hisaka; Carreira, Vinicius; Ko, Chia-I; Fan, Yunxia; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Puga, Alvaro

    2016-02-01

    The AHR is a ligand-activated transcription factor that mediates gene-environment interactions. Genome-wide expression profiling during differentiation of mouse ES cells into cardiomyocytes showed that AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin; Dioxin (TCDD), its prototypical ligand, disrupted the expression of multiple homeobox transcription factors and inhibited cardiomyocyte contractility. Here we treated ES cells with TCDD at daily differentiation intervals to investigate whether TCDD-induced loss of contractility had a developmental window of sensitivity. Surprisingly, contractility was an AHR-dependent TCDD target solely between differentiation days 0 and 3 during the period of panmesoderm development, when TCDD also disrupted expression of genes in the TGFβ/BMP2/4 and wingless-type MMTV integration site (WNT)signaling pathways, suppressed the secretion of bone morphogenetic protein (BMP4), WNT3a, and WNT5a and elevated the secretion of Activin A, as determined by ELISA of the secreted proteins in the culture medium. Supplementing the culture medium with BMP4, WNT3a, or WNT5a during the first 3 days of differentiation successfully countered TCDD-induced impairment of contractility, while anti-WNT3a, or anti-WNT5a antibodies or continuous Noggin (a BMP4 antagonist) or Activin A treatment inhibited the contractile phenotype. In Ahr(+/+), but not in Ahr(-) (/) (-) ES cells, TCDD treatment significantly increased mitochondrial copy number, suggestive of mitochondrial stress and remodeling. Sustained AHR activation during ES cell differentiation appears to disrupt the expression of signals critical to the ontogeny of cardiac mesoderm and cause the loss of contractility in the resulting cardiomyocyte lineage.

  7. Ah Receptor Activation by Dioxin Disrupts Activin, BMP, and WNT Signals During the Early Differentiation of Mouse Embryonic Stem Cells and Inhibits Cardiomyocyte Functions

    PubMed Central

    Wang, Qin; Kurita, Hisaka; Carreira, Vinicius; Ko, Chia-I; Fan, Yunxia; Zhang, Xiang; Biesiada, Jacek; Medvedovic, Mario; Puga, Alvaro

    2016-01-01

    The AHR is a ligand-activated transcription factor that mediates gene-environment interactions. Genome-wide expression profiling during differentiation of mouse ES cells into cardiomyocytes showed that AHR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin; Dioxin (TCDD), its prototypical ligand, disrupted the expression of multiple homeobox transcription factors and inhibited cardiomyocyte contractility. Here we treated ES cells with TCDD at daily differentiation intervals to investigate whether TCDD-induced loss of contractility had a developmental window of sensitivity. Surprisingly, contractility was an AHR-dependent TCDD target solely between differentiation days 0 and 3 during the period of panmesoderm development, when TCDD also disrupted expression of genes in the TGFβ/BMP2/4 and wingless-type MMTV integration site (WNT)signaling pathways, suppressed the secretion of bone morphogenetic protein (BMP4), WNT3a, and WNT5a and elevated the secretion of Activin A, as determined by ELISA of the secreted proteins in the culture medium. Supplementing the culture medium with BMP4, WNT3a, or WNT5a during the first 3 days of differentiation successfully countered TCDD-induced impairment of contractility, while anti-WNT3a, or anti-WNT5a antibodies or continuous Noggin (a BMP4 antagonist) or Activin A treatment inhibited the contractile phenotype. In Ahr+/+, but not in Ahr−/− ES cells, TCDD treatment significantly increased mitochondrial copy number, suggestive of mitochondrial stress and remodeling. Sustained AHR activation during ES cell differentiation appears to disrupt the expression of signals critical to the ontogeny of cardiac mesoderm and cause the loss of contractility in the resulting cardiomyocyte lineage. PMID:26572662

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

    PubMed

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

    2014-01-01

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

  9. Syringaresinol protects against hypoxia/reoxygenation-induced cardiomyocytes injury and death by destabilization of HIF-1α in a FOXO3-dependent mechanism

    PubMed Central

    Cho, Siyoung; Cho, Miook; Kim, Juewon; Kaeberlein, Matt; Lee, Sang Jun; Suh, Yousin

    2015-01-01

    Hypoxia-inducible factor 1 (HIF-1) is a master regulator of hypoxic response and has been a prime therapeutic target for ischemia/reperfusion (I/R)-derived myocardial dysfunction and tissue damage. There is also increasing evidence that HIF-1 plays a central role in regulating aging, both through interactions with key longevity factors including Sirtuins and mTOR, as well as by directly promoting longevity in Caenorhabditis elegans. We investigated a novel function and the underlying mechanism of syringaresinol, a lignan compound, in modulation of HIF-1 and protection against cellular damage and death in a cardiomyocyte model of I/R injury. Syringaresinol caused destabilization of HIF-1α following H/R and then protected against hypoxia/reoxygenation (H/R)-induced cellular damage, apoptosis, and mitochondrial dysfunction in a dose-dependent manner. Knock-down of FOXO3 by specific siRNAs completely abolished the ability of syringaresinol to inhibit HIF-1 stabilization and apoptosis caused by H/R. Syringaresinol stimulated the nuclear localization and activity of FOXO3 leading to increased expression of antioxidant genes and decreased levels of reactive oxygen species (ROS) following H/R. Our results provide a new mechanistic insight into a functional role of syringaresinol against H/R-induced cardiomyocyte injury and death. The degradation of HIF-1α through activation of FOXO3 is a potential therapeutic strategy for ischemia-related diseases. PMID:25415049

  10. Astragalus polysaccharide restores autophagic flux and improves cardiomyocyte function in doxorubicin-induced cardiotoxicity

    PubMed Central

    Cao, Yuan; Shen, Tao; Huang, Xiuqing; Lin, Yajun; Chen, Beidong; Pang, Jing; Li, Guoping; Wang, Que; Zohrabian, Sylvia; Duan, Chao; Ruan, Yang; Man, Yong; Wang, Shu; Li, Jian

    2017-01-01

    Doxorubicin (adriamycin), an anthracycline antibiotic, is commonly used to treat many types of solid and hematological malignancies. Unfortunately, clinical usage of doxorubicin is limited due to the associated acute and chronic cardiotoxicity. Previous studies demonstrated that Astragalus polysaccharide (APS), the extracts of Astragalus membranaceus, had strong anti-tumor activities and anti-inflammatory effects. However, whether APS could mitigate chemotherapy-induced cardiotoxicity is unclear thus far. We used a doxorubicin-induced neonatal rat cardiomyocyte injury model and a mouse heart failure model to explore the function of APS. GFP-LC3 adenovirus-mediated autophagic vesicle assays, GFP and RFP tandemly tagged LC3 (tfLC3) assays and Western blot analyses were performed to analyze the cell function and cell signaling changes following APS treatment in cardiomyocytes. First, doxorubicin treatment led to C57BL/6J mouse heart failure and increased cardiomyocyte apoptosis, with a disturbed cell autophagic flux. Second, APS restored autophagy in doxorubicin-treated primary neonatal rat ventricular myocytes and in the doxorubicin-induced heart failure mouse model. Third, APS attenuated doxorubicin-induced heart injury by regulating the AMPK/mTOR pathway. The mTOR inhibitor rapamycin significantly abrogated the protective effect of APS. These results suggest that doxorubicin could induce heart failure by disturbing cardiomyocyte autophagic flux, which may cause excessive cell apoptosis. APS could restore normal autophagic flux, ameliorating doxorubicin-induced cardiotoxicity by regulating the AMPK/mTOR pathway. PMID:27902477

  11. Thioredoxin-2 protects against oxygen-glucose deprivation/reperfusion injury by inhibiting autophagy and apoptosis in H9c2 cardiomyocytes

    PubMed Central

    Li, Yan-Yan; Xiang, Yin; Zhang, Song; Wang, Yan; Yang, Jie; Liu, Wei; Xue, Feng-Tai

    2017-01-01

    The aim of this study is to examine the role of thioredoxin-2 (Trx2) in autophagy and apoptosis during myocardial ischemia-reperfusion (I/R) injury in vitro. We employed the oxygen-glucose deprivation and reperfusion (OGD/R) model of H9c2 cells and used lentiviral infection to overexpress Trx2. H9c2 cell viability and injury assays were conducted using a Cell Counting Kit-8 (CCK-8) and alactate dehydrogenase (LDH) kit. The effects of Trx2 on autophagy and apoptosis were measured by transmission electron microscopy (TEM), western blot, and flow cytometry. Our results showed that the expression of Trx2 was significantly decreased at reperfusion 6 h after OGD 12 h treatment. Trx2 overexpression inhibited autophagy in H9c2 cells subjected to OGD/R. As the underlying mechanisms, both Akt kinase/the mammalian target of rapamycin (Akt/mTOR) and AMP-activated protein kinase (AMPK)/mTOR signaling pathways were involved in the regulation of Trx2 during autophagy, which was also mediated by reactive oxygen species (ROS). 3-methyladenine (3-MA), an inhibitor of autophagy, not only suppressed OGD/R-induced autophagy but also decreased apoptosis. As a classical autophagy sensitizer, rapamycin (Rapa) augmented autophagy as well as apoptosis. Additionally, we further demonstrated that Trx2 could alleviate OGD/R-induced apoptosis via mitochondrion-mediated intrinsic apoptotic pathway. In summary, our data indicated that Trx2 protects cardiomyocytes under OGD/R by inhibiting autophagy and apoptosis. Trx2 may be a crucial regulatory protein during I/R-induced cardiomyocyte injury and death. PMID:28386372

  12. Traumatic Brain Injury Severity Affects Neurogenesis in Adult Mouse Hippocampus.

    PubMed

    Wang, Xiaoting; Gao, Xiang; Michalski, Stephanie; Zhao, Shu; Chen, Jinhui

    2016-04-15

    Traumatic brain injury (TBI) has been proven to enhance neural stem cell (NSC) proliferation in the hippocampal dentate gyrus. However, various groups have reported contradictory results on whether TBI increases neurogenesis, partially due to a wide range in the severities of injuries seen with different TBI models. To address whether the severity of TBI affects neurogenesis in the injured brain, we assessed neurogenesis in mouse brains receiving different severities of controlled cortical impact (CCI) with the same injury device. The mice were subjected to mild, moderate, or severe TBI by a CCI device. The effects of TBI severity on neurogenesis were evaluated at three stages: NSC proliferation, immature neurons, and newly-generated mature neurons. The results showed that mild TBI did not affect neurogenesis at any of the three stages. Moderate TBI promoted NSC proliferation without increasing neurogenesis. Severe TBI increased neurogenesis at all three stages. Our data suggest that the severity of injury affects adult neurogenesis in the hippocampus, and thus it may partially explain the inconsistent results of different groups regarding neurogenesis following TBI. Further understanding the mechanism of TBI-induced neurogenesis may provide a potential approach for using endogenous NSCs to protect against neuronal loss after trauma.

  13. Humid heat exposure induced oxidative stress and apoptosis in cardiomyocytes through the angiotensin II signaling pathway.

    PubMed

    Wang, Xiaowu; Yuan, Binbin; Dong, Wenpeng; Yang, Bo; Yang, Yongchao; Lin, Xi; Gong, Gu

    2015-05-01

    Exposure to humid heat stress leads to the initiation of serious physiological dysfunction that may result in heat-related diseases, including heat stroke, heat cramp, heat exhaustion, and even death. Increasing evidences have shown that the humid heat stress-induced dysfunction of the cardiovascular system was accompanied with severe cardiomyocyte injury; however, the precise mechanism of heat stress-induced injury of cardiomyocyte remains unknown. In the present study, we hypothesized that humid heat stress promoted oxidative stress through the activation of angiotensin II (Ang II) in cardiomyocytes. To test our hypothesis, we established mouse models of humid heat stress. Using the animal models, we found that Ang II levels in serum were significantly up-regulated and that the Ang II receptor AT1 was increased in cardiomyocytes. The antioxidant ability in plasma and heart tissues which was detected by the ferric reducing/antioxidant power assay was also decreased with the increased ROS production under humid heat stress, as was the expression of antioxidant genes (SOD2, HO-1, GPx). Furthermore, we demonstrated that the Ang II receptor antagonist, valsartan, effectively relieved oxidative stress, blocked Ang II signaling pathway and suppressed cardiomyocyte apoptosis induced by humid heat stress. In addition, overexpression of antioxidant genes reversed cardiomyocyte apoptosis induced by Ang II. Overall, these results implied that humid heat stress increased oxidative stress and caused apoptosis of cardiomyocytes through the Ang II signaling pathway. Thus, targeting the Ang II signaling pathway may provide a promising approach for the prevention and treatment of cardiovascular diseases caused by humid heat stress.

  14. Extracellular vesicles-mediated transfer of miR-208a/b exaggerate hypoxia/reoxygenation injury in cardiomyocytes by reducing QKI expression.

    PubMed

    Wang, Feng; Yuan, Yuxiang; Yang, Pirong; Li, Xia

    2017-03-10

    In this study, we tested the hypothesis that extracellular vesicles (EVs)-mediated transfer of miR-208a/b can exacerbate apoptosis of cardiomyocytes (CMs) induced by hypoxia/reoxygenation (H/R) injury by reducing the expression of the RNA-binding protein Quaking (QKI). EVs were isolated from culture medium of hypoxic H9c2 cells (EVs-H). In in vitro H9c2 cell model, the EVs-H could be taken up by normoxic CMs and exacerbated cell apoptosis induced by H/R injury. In addition, miR-208a and miR-208b were enriched in EVs-H. Suppression of miR-208a and miR-208b loading significantly suppressed the detrimental effect of EVs-H on H/R injury in H9c2 cells. Inhibition of endogenous miR-208a and miR-208b restored QKI5 and QKI6 after H/R treatment. Dual-luciferase assay confirmed direct bindings between miR-208a/b and QKI 3'UTR. Functionally, QKI5 overexpression significantly suppressed H/R-induced CM apoptosis and suppressed the enhancing effect of EVs-H on CM apoptosis. Therefore, we infer that EVs-mediated transfer of miR-208a/b can exaggerate H/R injury in CMs by reducing QKI expression. This represents a previously unrecognized pathway of H/R injury in CMs.

  15. A Neonatal Mouse Spinal Cord Compression Injury Model

    PubMed Central

    Züchner, Mark; Glover, Joel C.; Boulland, Jean-Luc

    2016-01-01

    Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1. PMID:27078037

  16. Sauchinone augments cardiomyocyte viability by enhancing autophagy proteins -PI3K, ERK(1/2), AMPK and Beclin-1 during early ischemia-reperfusion injury in vitro

    PubMed Central

    Thapalia, Bisharad Anil; Zhou, Zhen; Lin, Xianhe

    2016-01-01

    Background. Sauchinone has proved its anti-oxidant and anti-inflammatory properties in various animal tissues. This study sought to illustrate its regulatory nature on autophagy associated proteins (PI3K, ERK1/2, AMPK, and Beclin-1) during early cardiomyocyte ischemia and subsequent reperfusion. Methods. Cultured cardiomyocytes were subjected to simulated Ischemia/reperfusion with and without Sauchinone pretreatment and also in the presence of autophagy inhibitor (3-MA). Colorimetric analysis of CCK-8, LDH antibody assay as well as Western blot analysis were performed to observe the expressions of LC3B (II) and Beclin-1 protein (markers of autophagy), autophagy proteins (PI3K, ERK1/2 and AMPK) and apoptotic proteins (Bax and Bcl-2) and the results were quantified into their grey values and subjected to statistical analysis. Results. Sauchinone demonstrated cell survival enhancing properties with increase in CCK-8 (SD = 0.553±0.012) and decrease in LDH (SD = 0.183±0.054) expressions, both of which were best observed at test dose of 20 µmol/L. At this dose, there was increment in cellular autophagy as demonstrated by peaking of autophagy markers LC3B-II (p<0.05) and Beclin-1 (p<0.05) with strong correlations (r = 0.99). Similarly, the autophagy proteins, compared to control and I/R model, also showed a significant increased level with PI3K (p<0.0001), total p-ERK1/2 (p<0.0001) and p-AMPKα (p<0.0001). Simultaneously, a decrease in expressions of pro-apoptotic molecules Bax (r = 0.989, p<0.0001) with increment of in the anti-apoptotic protein Bcl-2 (r = 0.996, p<0.0001) was observed. The observed effects on cell density, viability and autophagy was abrogated in presence of 3-MA. Conclusions. Sauchinone enhances cell survival by promoting autophagy and inhibiting apoptosis in cardiomyocytes during early stages of Ischemia/reperfusion injury. PMID:27508047

  17. Cobalt protoporphyrin pretreatment protects human embryonic stem cell-derived cardiomyocytes from hypoxia/reoxygenation injury in vitro and increases graft size and vascularization in vivo.

    PubMed

    Luo, Jun; Weaver, Matthew S; Cao, Baohong; Dennis, James E; Van Biber, Benjamin; Laflamme, Michael A; Allen, Margaret D

    2014-06-01

    Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) can regenerate infarcted myocardium. However, when implanted into acutely infarcted hearts, few cells survive the first week postimplant. To improve early graft survival, hESC-CMs were pretreated with cobalt protoporphyrin (CoPP), a transcriptional activator of cytoprotective heme oxygenase-1 (HO-1). When hESC-CMs were challenged with an in vitro hypoxia/reoxygenation injury, mimicking cell transplantation into an ischemic site, survival was significantly greater among cells pretreated with CoPP versus phosphate-buffered saline (PBS)-pretreated controls. Compared with PBS-pretreated cells, CoPP-pretreated hESC-CM preparations exhibited higher levels of HO-1 expression, Akt phosphorylation, and vascular endothelial growth factor production, with reduced apoptosis, and a 30% decrease in intracellular reactive oxygen species. For in vivo translation, 1 × 10(7) hESC-CMs were pretreated ex vivo with CoPP or PBS and then injected intramyocardially into rat hearts immediately following acute infarction (permanent coronary ligation). At 1 week, hESC-CM content, assessed by quantitative polymerase chain reaction for human Alu sequences, was 17-fold higher in hearts receiving CoPP- than PBS-pretreated cells. On histomorphometry, cardiomyocyte graft size was 2.6-fold larger in hearts receiving CoPP- than PBS-pretreated cells, occupying up to 12% of the ventricular area. Vascular density of host-perfused human-derived capillaries was significantly greater in grafts composed of CoPP- than PBS-pretreated cells. Taken together, these experiments demonstrate that ex vivo pretreatment of hESC-CMs with a single dose of CoPP before intramyocardial implantation more than doubled resulting graft size and improved early graft vascularization in acutely infarcted hearts. These findings open the door for delivery of these, or other, stem cells during acute interventional therapy following myocardial infarction or ischemia.

  18. Contribution of mast cells to injury mechanisms in a mouse model of pediatric traumatic brain injury.

    PubMed

    Moretti, Raffaella; Chhor, Vibol; Bettati, Donatella; Banino, Elena; De Lucia, Silvana; Le Charpentier, Tifenn; Lebon, Sophie; Schwendimann, Leslie; Pansiot, Julien; Rasika, Sowmyalakshmi; Degos, Vincent; Titomanlio, Luigi; Gressens, Pierre; Fleiss, Bobbi

    2016-12-01

    The cognitive and behavioral deficits caused by traumatic brain injury (TBI) to the immature brain are more severe and persistent than injuries to the adult brain. Understanding this developmental sensitivity is critical because children under 4 years of age of sustain TBI more frequently than any other age group. One of the first events after TBI is the infiltration and degranulation of mast cells (MCs) in the brain, releasing a range of immunomodulatory substances; inhibition of these cells is neuroprotective in other types of neonatal brain injury. This study investigates for the first time the role of MCs in mediating injury in a P7 mouse model of pediatric contusion-induced TBI. We show that various neural cell types express histamine receptors and that histamine exacerbates excitotoxic cell death in primary cultured neurons. Cromoglycate, an inhibitor of MC degranulation, altered the inflammatory phenotype of microglia activated by TBI, reversing several changes but accentuating others, when administered before TBI. However, without regard to the time of cromoglycate administration, inhibiting MC degranulation did not affect cell loss, as evaluated by ventricular dilatation or cleaved caspase-3 labeling, or the density of activated microglia, neurons, or myelin. In double-heterozygous cKit mutant mice lacking MCs, this overall lack of effect was confirmed. These results suggest that the role of MCs in this model of pediatric TBI is restricted to subtle effects and that they are unlikely to be viable neurotherapeutic targets. © 2016 Wiley Periodicals, Inc.

  19. Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

    PubMed Central

    Richardson, Gavin D.

    2016-01-01

    Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4′,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types. PMID:27285379

  20. Intravenous Sphingosylphosphorylcholine Protects Ischemic and Postischemic Myocardial Tissue in a Mouse Model of Myocardial Ischemia/Reperfusion Injury

    PubMed Central

    Herzog, Christine; Schmitz, Martina; Levkau, Bodo; Herrgott, Ilka; Mersmann, Jan; Larmann, Jan; Johanning, Kai; Winterhalter, Michael; Chun, Jerold; Müller, Frank Ulrich; Echtermeyer, Frank; Hildebrand, Reinhard; Theilmeier, Gregor

    2010-01-01

    HDL, through sphingosine-1-phosphate (S1P), exerts direct cardioprotective effects on ischemic myocardium. It remains unclear whether other HDL-associated sphingophospholipids have similar effects. We therefore examined if HDL-associated sphingosylphosphorylcholine (SPC) reduces infarct size in a mouse model of transient myocardial ischemia/reperfusion. Intravenously administered SPC dose-dependently reduced infarct size after 30 minutes of myocardial ischemia and 24 hours reperfusion compared to controls. Infarct size was also reduced by postischemic, therapeutical administration of SPC. Immunohistochemistry revealed reduced polymorphonuclear neutrophil recruitment to the infarcted area after SPC treatment, and apoptosis was attenuated as measured by TUNEL. In vitro, SPC inhibited leukocyte adhesion to TNFα-activated endothelial cells and protected rat neonatal cardiomyocytes from apoptosis. S1P3 was identified as the lysophospholipid receptor mediating the cardioprotection by SPC, since its effect was completely absent in S1P3-deficient mice. We conclude that HDL-associated SPC directly protects against myocardial reperfusion injury in vivo via the S1P3 receptor. PMID:21274265

  1. Five new triterpenoidal saponins from the roots of Ilex cornuta and their protective effects against H₂O₂-induced cardiomyocytes injury.

    PubMed

    Wang, Wenlian; Zhao, Jianping; Li, Shanshan; Lu, Yuchen; Liu, Yanli; Xu, Qiongming; Li, Xiaoran; Khan, Ikhlas A; Yang, Shilin

    2014-12-01

    Five new ursane-type triterpenoidal saponins (1-5), together with five known ones (6-10), were isolated from the EtOH extract of the roots of Ilex cornuta. The structures of saponins 1-5 were elucidated as 19α-hydroxyurs-12-en-28-oic acid 3β-O-β-D-glucuronopyranoside (1), 19α-hydroxyurs-12-en-28-oic acid 3β-O-β-D-glucuronopyranoside-6-O-ethyl ester (2), 19α-hydroxyurs-12-en-28-oic acid 3β-O-α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranoside (3), 3β-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranosyl]-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (4) and 3β-O-[α-L-arabinopyranosyl-(1→2)-β-D-glucuronopyranoside-6-O-methyl ester]-19α-hydroxyurs-12-en-28-oic acid 28-O-β-D-glucopyranosyl ester (5), on the basis of spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR) and chemical reactions. Protective effects of compounds 1-10 against H₂O₂-induced H9c2 cardiomyocyte injury were tested. Compounds 1-5, 7, and 10 showed cell-protective effects. Among them compound 5 exhibited the highest activity. No significant DPPH radical scavenging activity was observed for compounds 1-10.

  2. Migration of cardiomyocytes is essential for heart regeneration in zebrafish.

    PubMed

    Itou, Junji; Oishi, Isao; Kawakami, Hiroko; Glass, Tiffany J; Richter, Jenna; Johnson, Austin; Lund, Troy C; Kawakami, Yasuhiko

    2012-11-01

    Adult zebrafish possess a significant ability to regenerate injured heart tissue through proliferation of pre-existing cardiomyocytes, which contrasts with the inability of mammals to do so after the immediate postnatal period. Zebrafish therefore provide a model system in which to study how an injured heart can be repaired. However, it remains unknown what important processes cardiomyocytes are involved in other than partial de-differentiation and proliferation. Here we show that migration of cardiomyocytes to the injury site is essential for heart regeneration. Ventricular amputation induced expression of cxcl12a and cxcr4b, genes encoding a chemokine ligand and its receptor. We found that cxcl12a was expressed in the epicardial tissue and that Cxcr4 was expressed in cardiomyocytes. We show that pharmacological blocking of Cxcr4 function as well as genetic loss of cxcr4b function causes failure to regenerate the heart after ventricular resection. Cardiomyocyte proliferation was not affected but a large portion of proliferating cardiomyocytes remained localized outside the injury site. A photoconvertible fluorescent reporter-based cardiomyocyte-tracing assay demonstrates that cardiomyocytes migrated into the injury site in control hearts but that migration was inhibited in the Cxcr4-blocked hearts. By contrast, the epicardial cells and vascular endothelial cells were not affected by blocking Cxcr4 function. Our data show that the migration of cardiomyocytes into the injury site is regulated independently of proliferation, and that coordination of both processes is necessary for heart regeneration.

  3. Fine oil combustion particle bioavailable constituents induce molecular profiles of oxidative stress, altered function, and cellular injury in cardiomyocytes.

    PubMed

    Knuckles, Travis L; Dreher, Kevin L

    2007-11-01

    Epidemiological studies have shown a positive association between exposure to air particulate matter (PM) pollution and adverse cardiovascular health effects in susceptible subpopulations such as those with pre-existing cardiovascular disease. The mechanism(s) through which pulmonary deposited PM, particularly fine PM2.5, PM with mass median aerodynamic diameter <2.5 microm, affects the cardiovascular system is currently not known and remains a major focus of investigation. In the present study, the transcriptosome and transcription factor proteome were examined in rat neonatal cardiomyocyte (RCM) cultures, following an acute exposure to bioavailable constituents of PM2.5 oil combustion particles designated residual oil fly ash leachate (ROFA-L). Out of 3924 genes examined, 38 genes were suppressed and 44 genes were induced following a 1-h exposure to 3.5 microg/ml of a particle-free leachate of ROFA (ROFA-L). Genomic alterations in pathways related to IGF-1, VEGF, IL-2, PI3/AKT, cardiovascular disease, and free radical scavenging, among others, were detected 1 h postexposure to ROFA-L. Global gene expression was altered in a manner consistent with cardiac myocyte electrophysiological remodeling, cellular oxidative stress, and apoptosis. ROFA-L altered the transcription factor proteome by suppressing activity of 24 and activating 40 transcription factors out of a total of 149. Genomic alterations were found to correlate with changes in transcription factor proteome. These acute changes indicate pathological molecular alterations, which may lead to possible chronic alterations to the cardiac myocyte. These data also potentially relate underlying cardiovascular effects from occupational exposure to ROFA and identify how particles from specific emission sources may mediate ambient PM cardiac effects.

  4. 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

  5. A proliferative burst during preadolescence establishes the final cardiomyocyte number.

    PubMed

    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

    2014-05-08

    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 that a thyroid hormone surge activates the IGF-1/IGF-1-R/Akt pathway on postnatal day 15 and initiates 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 day 15 is intermediate between that observed at postnatal days 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.

  6. Oxidant-induced cardiomyocyte injury: identification of the cytoprotective effect of a dopamine 1 receptor agonist using a cell-based high-throughput assay.

    PubMed

    Gerö, Domokos; Módis, Katalin; Nagy, Nóra; Szoleczky, Petra; Tóth, Zoltán Dóri; Dormán, György; Szabó, Csaba

    2007-11-01

    Myocyte injury due to myocardial reperfusion injury plays a crucial role in the pathogenesis of acute myocardial infarction even after successful coronary revascularization. Identification of compounds that reduce reperfusion-associated myocyte death is important. Therefore, we developed an in vitro model of myocardial reperfusion injury in H9c2 rat cardiomyocytes and applied a cell-based high-throughput approach to screen a standard library of pharmacologically active compounds (LOPAC) in order to identify drugs with cardioprotective effects. Oxidative stress was induced with hydrogen peroxide (H2O2) treatment, which resulted in approximately 50% reduction in cell viability. Test compounds were added at a 3-microM final concentration as a pretreatment or in a delayed fashion (30 min after the peroxide challenge in order to imitate pharmacological treatment following angioplasty). Cells were cultured for 3 or 24 h. Viability was quantitated with the methylthiazolyldiphenyl-tetrazolium bromide method. Cytotoxicity and cytoprotection were also evaluated by measuring the lactate dehydrogenase activity in the cell culture supernatant. The screening identified a number of compounds with cytoprotective action, including molecules that are known to interfere with components of DNA repair and cell cycle progression, e.g. poly(ADP-ribose) polymerase (PARP) inhibitors, topoisomerase inhibitors, and cyclin dependent kinase inhibitors, or reduce energy consumption by interfering with cardiac myofilament function. A number of dopamine D1 receptor agonists also provided significant cytoprotection at 3 h, but only three of them showed a similar effect at 24 h: chloro- and bromo-APB and chloro-PB hydrobromide. Chloro-APB hydrobromide significantly reduced peroxide-induced PARP activation in the myocytes independently of its action on dopamine D1 receptors, but lacked PARP inhibitor capacity in a cell-free PARP assay system. In conclusion, the pattern of cytoprotective drugs

  7. AMP-activated protein kinase-dependent autophagy mediated the protective effect of sonic hedgehog pathway on oxygen glucose deprivation-induced injury of cardiomyocytes.

    PubMed

    Xiao, Qing; Yang, Ya; Qin, Yuan; He, Yan-Hua; Chen, Kui-Xiang; Zhu, Jian-Wei; Zhang, Gui-Ping; Luo, Jian-Dong

    2015-02-13

    Sonic hedgehog (Shh) pathway has been reported to protect cardiomyocytes in myocardial infarction (MI), but the underlying mechanism is not clear. Here, we provide evidence that Shh pathway induces cardiomyocytes survival through AMP-activated protein kinase-dependent autophagy. Shh pathway agonist SAG increased the expression of LC3-II, and induced the formation of autophagosomes in cultured H9c2 cardiomyocytes under oxygen glucose deprivation (OGD) 1 h and 4 h. Moreover, SAG induced a profound AMP-activated protein kinase (AMPK) activation, and then directly phosphorylated and activated the downstream autophagy initiator Ulk1, independent of the autophagy suppressor mammalian target of rapamycin (mTOR) complex 1. Taken together, our results have shown that Shh activates AMPK-dependent autophagy in cardiomyocytes under OGD, suggesting a role of autophagy in Shh-induced cellular protection.

  8. Role of JAK-STAT pathway in reducing cardiomyocytes hypoxia/reoxygenation injury induced by S1P postconditioning.

    PubMed

    Wang, Yuqing; Wang, Dongfei; Zhang, Lizhi; Ye, Fangyu; Li, Mengmeng; Wen, Ke

    2016-08-05

    This experiment was designed to explore the protection of sphingosine1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation acting via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal pathway. The data showed that S1P could significantly increase cell viability, lower the rate of apoptosis, decrease the content of lactate dehydrogenase (LDH) and caspase3 activity in the culture medium, increase the activity of total superoxide dismutase (T-SOD) and manganese superoxide dismutase (Mn-SOD), reduce the loss of mitochondrial membrane potential and the fluorescence intensity of intracellular calcium, as well as increase the phosphorylation of JAK2 and STAT3 in comparison with the H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Our date shows that S1P protects H9c2 cells from hypoxia/reoxygenation injury and that the protection by S1P was inhibited by AG490 and stattic. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via the JAK-STAT pathway.

  9. GATA4 regulates Fgf16 to promote heart repair after injury.

    PubMed

    Yu, Wei; Huang, Xiuzhen; Tian, Xueying; Zhang, Hui; He, Lingjuan; Wang, Yue; Nie, Yu; Hu, Shengshou; Lin, Zhiqiang; Zhou, Bin; Pu, William; Lui, Kathy O; Zhou, Bin

    2016-03-15

    Although the mammalian heart can regenerate during the neonatal stage, this endogenous regenerative capacity is lost with age. Importantly, replication of cardiomyocytes has been found to be the key mechanism responsible for neonatal cardiac regeneration. Unraveling the transcriptional regulatory network for inducing cardiomyocyte replication will, therefore, be crucial for the development of novel therapies to drive cardiac repair after injury. Here, we investigated whether the key cardiac transcription factor GATA4 is required for neonatal mouse heart regeneration. Using the neonatal mouse heart cryoinjury and apical resection models with an inducible loss of GATA4 specifically in cardiomyocytes, we found severely depressed ventricular function in the Gata4-ablated mice (mutant) after injury. This was accompanied by reduced cardiomyocyte replication. In addition, the mutant hearts displayed impaired coronary angiogenesis and increased hypertrophy and fibrosis after injury. Mechanistically, we found that the paracrine factor FGF16 was significantly reduced in the mutant hearts after injury compared with littermate controls and was directly regulated by GATA4. Cardiac-specific overexpression of FGF16 via adeno-associated virus subtype 9 (AAV9) in the mutant hearts partially rescued the cryoinjury-induced cardiac hypertrophy, promoted cardiomyocyte replication and improved heart function after injury. Altogether, our data demonstrate that GATA4 is required for neonatal heart regeneration through regulation of Fgf16, suggesting that paracrine factors could be of potential use in promoting myocardial repair.

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

    PubMed Central

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

    2012-01-01

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

  11. Hierarchical organization of the hemostatic response to penetrating injuries in the mouse macrovasculature.

    PubMed

    Welsh, J D; Poventud-Fuentes, I; Sampietro, S; Diamond, S L; Stalker, T J; Brass, L F

    2017-03-01

    Essentials Methods were developed to image the hemostatic response in mouse femoral arteries in real time. Penetrating injuries produced thrombi consisting primarily of platelets. Similar to arterioles, a core-shell architecture of platelet activation occurs in the femoral artery. Differences from arterioles included slower platelet activation and reduced thrombin dependence.

  12. A mouse model of human repetitive mild traumatic brain injury

    PubMed Central

    Kane, Michael J.; Pérez, Mariana Angoa; Briggs, Denise I.; Viano, David C.; Kreipke, Christian W.; Kuhn, Donald M.

    2011-01-01

    A novel method for the study of repetitive mild traumatic brain injury (rmTBI) that models the most common form of head injury in humans is presented. Existing animal models of TBI impart focal, severe damage unlike that seen in repeated and mild concussive injuries, and few are configured for repetitive application. Our model is a modification of the Marmarou weight drop method and allows repeated head impacts to lightly anesthetized mice. A key facet of this method is the delivery of an impact to the cranium of an unrestrained subject allowing rapid acceleration of the free-moving head and torso, an essential characteristic known to be important for concussive injury in humans, and a factor that is missing from existing animal models of TBI. Our method does not require scalp incision, emplacement of protective skull helmets or surgery and the procedure can be completed in 1-2 minutes. Mice spontaneously recover the righting reflex and show no evidence of seizures, paralysis or impaired behavior. Skull fractures and intracranial bleeding are very rare. Minor deficits in motor coordination and locomotor hyperactivity recover over time. Histological analyses reveal mild astrocytic reactivity (increased expression of GFAP) and increased phospho-tau but a lack of blood-brain-barrier disruption, edema and microglial activation. This new animal model is simple and cost-effective and will facilitate characterization of the neurobiological and behavioral consequences of rmTBI. It is also ideal for high throughput screening of potential new therapies for mild concussive injuries as experienced by athletes and military personnel. PMID:21930157

  13. A mouse model of human repetitive mild traumatic brain injury.

    PubMed

    Kane, Michael J; Angoa-Pérez, Mariana; Briggs, Denise I; Viano, David C; Kreipke, Christian W; Kuhn, Donald M

    2012-01-15

    A novel method for the study of repetitive mild traumatic brain injury (rmTBI) that models the most common form of head injury in humans is presented. Existing animal models of TBI impart focal, severe damage unlike that seen in repeated and mild concussive injuries, and few are configured for repetitive application. Our model is a modification of the Marmarou weight drop method and allows repeated head impacts to lightly anesthetized mice. A key facet of this method is the delivery of an impact to the cranium of an unrestrained subject allowing rapid acceleration of the free-moving head and torso, an essential characteristic known to be important for concussive injury in humans, and a factor that is missing from existing animal models of TBI. Our method does not require scalp incision, emplacement of protective skull helmets or surgery and the procedure can be completed in 1-2 min. Mice spontaneously recover the righting reflex and show no evidence of seizures, paralysis or impaired behavior. Skull fractures and intracranial bleeding are very rare. Minor deficits in motor coordination and locomotor hyperactivity recover over time. Histological analyses reveal mild astrocytic reactivity (increased expression of GFAP) and increased phospho-tau but a lack of blood-brain-barrier disruption, edema and microglial activation. This new animal model is simple and cost-effective and will facilitate characterization of the neurobiological and behavioral consequences of rmTBI. It is also ideal for high throughput screening of potential new therapies for mild concussive injuries as experienced by athletes and military personnel.

  14. A Novel Mouse Model of Penetrating Brain Injury

    PubMed Central

    Cernak, Ibolja; Wing, Ian D.; Davidsson, Johan; Plantman, Stefan

    2014-01-01

    Penetrating traumatic brain injury (pTBI) has been difficult to model in small laboratory animals, such as rats or mice. Previously, we have established a non-fatal, rat model for pTBI using a modified air-rifle that accelerates a pellet, which hits a small probe that then penetrates the experimental animal’s brain. Knockout and transgenic strains of mice offer attractive tools to study biological reactions induced by TBI. Hence, in the present study, we adapted and modified our model to be used with mice. The technical characterization of the impact device included depth and speed of impact, as well as dimensions of the temporary cavity formed in a brain surrogate material after impact. Biologically, we have focused on three distinct levels of severity (mild, moderate, and severe), and characterized the acute phase response to injury in terms of tissue destruction, neural degeneration, and gliosis. Functional outcome was assessed by measuring bodyweight and motor performance on rotarod. The results showed that this model is capable of reproducing major morphological and neurological changes of pTBI; as such, we recommend its utilization in research studies aiming to unravel the biological events underlying injury and regeneration after pTBI. PMID:25374559

  15. NF-kB activation as a biomarker of light injury using a transgenic mouse model

    NASA Astrophysics Data System (ADS)

    Pocock, Ginger M.; Boretsky, Adam; Wang, Heuy-Ching; Golden, Dallas; Gupta, Praveena; Vargas, Gracie; Oliver, Jeffrey W.; Motamedi, Massoud

    2012-03-01

    The spatial and temporal activation of NF-kB (p65) was monitored in the retina of a transgenic mouse model (cis-NFkB-EGFP) in vivo after receiving varying grades of laser induced thermal injury in one eye. Baseline images of the retinas from 26 mice were collected prior to injury and up to five months post-exposure using a Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope (cSLO) with a spectral domain optical coherence tomographer (SDOCT). Injured and control eyes were enucleated at discrete time points following laser exposure for cryosectioning to determine localization of NF-kB dependent enhanced green fluorescent protein (EGFP) reporter gene expression within the retina using fluorescence microscopy. In addition, EGFP basal expression in brain and retinal tissue from the cis-NFkB-EGFP was characterized using two-photon imaging. Regions of the retina exposed to threshold and supra-threshold laser damage evaluated using fluorescence cSLO showed increased EGFP fluorescence localized to the exposed region for a duration that was dependent upon the degree of injury. Fluorescence microscopy of threshold damage revealed EGFP localized to the outer nuclear region and retinal pigment epithelial layer. Basal expression of EGFP imaged using two-photon microscopy was heterogeneously distributed throughout brain tissue and confined to the inner retina. Results show cis-NF-kB-EGFP reporter mouse can be used for in vivo studies of light induced injury to the retina and possibly brain injury.

  16. Pluripotent Stem Cell Derived Cardiomyocytes for Cardiac Repair

    PubMed Central

    Lundy, Scott D.; Gantz, Jay A.; Pagan, Chelsea M.; Filice, Dominic; Laflamme, Michael A.

    2014-01-01

    Opinion Statement The adult mammalian heart has limited capacity for generation, so a major injury such as a myocardial infarction results in the permanent loss of up to one billion cardiomyocytes. The field of cardiac cell therapy aims to replace these lost contractile units with de novo cardiomyocytes to restore lost systolic function and prevent progression to heart failure. Arguably the ideal cell for this application is the human cardiomyocyte itself, which can electromechanically couple with host myocardium and contribute active systolic force. Pluripotent stem cells from both human embryonic or induced pluripotent lineages are attractive sources for cardiomyocytes, and preclinical investigation of these cells is in progress. Recent work has focused on efficient generation and purification of cardiomyocytes, tissue engineering efforts, and examining the consequences of cell transplantation from mechanical, vascular, and electrical standpoints. Here we discuss historical and contemporary aspects of pluripotent stem cell-based cardiac cell therapy, with an emphasis on recent preclinical studies with translational goals. PMID:24838687

  17. Epiplakin attenuates experimental mouse liver injury by chaperoning keratin reorganization

    PubMed Central

    Szabo, Sandra; Wögenstein, Karl L.; Österreicher, Christoph H.; Guldiken, Nurdan; Chen, Yu; Doler, Carina; Wiche, Gerhard; Boor, Peter; Haybaeck, Johannes; Strnad, Pavel; Fuchs, Peter

    2015-01-01

    Background & Aims Epiplakin is a member of the plakin protein family and exclusively expressed in epithelial tissues where it binds to keratins. Epiplakin-deficient (Eppk1−/−) mice displayed no obvious spontaneous phenotype, but their keratinocytes showed a faster keratin network breakdown in response to stress. The role of epiplakin in the stressed liver remained to be elucidated. Methods Wild-type (WT) and Eppk1−/− mice were subjected to common bile duct ligation (CBDL) or fed with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet. The importance of epiplakin during keratin reorganization was assessed in primary hepatocytes. Results Our experiments revealed that epiplakin is expressed in hepatocytes and cholangiocytes, and binds to keratin 8 (K8) and K18 via multiple domains. In several liver stress models epiplakin and K8 genes displayed identical expression patterns and transgenic K8 overexpression resulted in elevated hepatic epiplakin levels. After CBDL and DDC treatment, Eppk1−/− mice developed a more pronounced liver injury and their livers contained larger amounts of hepatocellular keratin granules, indicating impaired disease-induced keratin network reorganization. In line with these findings, primary Eppk1−/− hepatocytes showed increased formation of keratin aggregates after treatment with the phosphatase inhibitor okadaic acid, a phenotype which was rescued by the chemical chaperone trimethylamine N-oxide (TMAO). Finally, transfection experiments revealed that Eppk1−/− primary hepatocytes were less able to tolerate forced K8 overexpression and that TMAO treatment rescued this phenotype. Conclusion Our data indicate that epiplakin plays a protective role during experimental liver injuries by chaperoning disease-induced keratin reorganization. PMID:25617501

  18. A 2 week routine stretching programme did not prevent contraction-induced injury in mouse muscle.

    PubMed

    Black, Jonathon D J; Freeman, Marcus; Stevens, E Don

    2002-10-01

    Most athletes stretch as part of their training regimen and it is commonly believed that this practice prevents muscle injury. We tested this belief using an animal model, in situ mouse extensor digitorum longus (EDL) muscle. One lower hindlimb was slowly stretched for 1 min on alternate days for 12 days; the other leg served as a control. The mouse was lightly anaesthetized during the stretching protocol (isofluorane). Both legs were tested in situ by measuring maximum isometric force and maximum work before and after an eccentric contraction that was designed to cause a contraction-induced injury. The difference between a contraction before and after (i.e. the deficit) was used as a measure of damage caused by the eccentric contraction. There was a threshold for force deficit at a peak to peak eccentric excursion amplitude of 19.5 % (i.e. L(o) +/- 9.75 %, where L(o) is muscle length at peak isometric force). There was a significant increase in force deficit, work deficit, and curve shift with an increase in eccentric excursion amplitude above the threshold. There was no statistical difference in the force deficit, work deficit, or curve shift between the stretched leg and the control leg (P > 0.05). A routine stretching programme, at least at the intensities employed in this experiment, did not prevent contraction-induced injury in the in situ mouse EDL muscle.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  20. 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

  1. Building and re-building the heart by cardiomyocyte proliferation

    PubMed Central

    Foglia, Matthew J.; Poss, Kenneth D.

    2016-01-01

    The adult human heart does not regenerate significant amounts of lost tissue after injury. Rather than making new, functional muscle, human hearts are prone to scarring and hypertrophy, which can often lead to fatal arrhythmias and heart failure. The most-cited basis of this ineffective cardiac regeneration in mammals is the low proliferative capacity of adult cardiomyocytes. However, mammalian cardiomyocytes can avidly proliferate during fetal and neonatal development, and both adult zebrafish and neonatal mice can regenerate cardiac muscle after injury, suggesting that latent regenerative potential exists. Dissecting the cellular and molecular mechanisms that promote cardiomyocyte proliferation throughout life, deciphering why proliferative capacity normally dissipates in adult mammals, and deriving means to boost this capacity are primary goals in cardiovascular research. Here, we review our current understanding of how cardiomyocyte proliferation is regulated during heart development and regeneration. PMID:26932668

  2. A mouse model of weight-drop closed head injury: emphasis on cognitive and neurological deficiency

    PubMed Central

    Khalin, Igor; Jamari, Nor Laili Azua; Razak, Nadiawati Bt Abdul; Hasain, Zubaidah Bt; Nor, Mohd Asri bin Mohd; Zainudin, Mohd Hakimi bin Ahmad; Omar, Ainsah Bt; Alyautdin, Renad

    2016-01-01

    Traumatic brain injury (TBI) is a leading cause of death and disability in individuals worldwide. Producing a clinically relevant TBI model in small-sized animals remains fairly challenging. For good screening of potential therapeutics, which are effective in the treatment of TBI, animal models of TBI should be established and standardized. In this study, we established mouse models of closed head injury using the Shohami weight-drop method with some modifications concerning cognitive deficiency assessment and provided a detailed description of the severe TBI animal model. We found that 250 g falling weight from 2 cm height produced severe closed head injury in C57BL/6 male mice. Cognitive disorders in mice with severe closed head injury could be detected using passive avoidance test on day 7 after injury. Findings from this study indicate that weight-drop injury animal models are suitable for further screening of brain neuroprotectants and potentially are similar to those seen in human TBI. PMID:27212925

  3. Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

    PubMed Central

    Barazzone, C; Belin, D; Piguet, P F; Vassalli, J D; Sappino, A P

    1996-01-01

    Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury. PMID:8981909

  4. Cardiac RNA induces inflammatory responses in cardiomyocytes and immune cells via Toll-like receptor 7 signaling.

    PubMed

    Feng, Yan; Chen, Hongliang; Cai, Jiayan; Zou, Lin; Yan, Dan; Xu, Ganqiong; Li, Dan; Chao, Wei

    2015-10-30

    We have recently reported that extracellular RNA (exRNA) released from necrotic cells induces cytokine production in cardiomyocytes and immune cells and contributes to myocardial ischemia/reperfusion injury. However, the signaling mechanism by which exRNA exhibits its pro-inflammatory effect is unknown. Here we hypothesize that exRNA directly induces inflammation through specific Toll-like receptors (TLRs). To test the hypothesis, we treated rat neonatal cardiomyocytes, mouse bone marrow-derived macrophages (BMDM), or mouse neutrophils with RNA (2.5-10 μg/ml) isolated from rat cardiomyocytes or the hearts from mouse, rat, and human. We found that cellular RNA induced production of several cytokines such as macrophage inflammatory protein-2 (MIP-2), ILs, TNFα, and the effect was completely diminished by RNase, but not DNase. The RNA-induced cytokine production was partially inhibited in cells treated with TLR7 antagonist or genetically deficient in TLR7. Deletion of myeloid differentiation primary response protein 88 (MyD88), a downstream adapter of TLRs including TLR7, abolished the RNA-induced MIP-2 production. Surprisingly, genetic deletion of TLR3 had no impact on the RNA-induced MIP-2 response. Importantly, extracellular RNA released from damaged cardiomyocytes also induced cytokine production. Finally, mice treated with 50 μg of RNA intraperitoneal injection exhibited acute peritonitis as evidenced by marked neutrophil and monocyte migration into the peritoneal space. Together, these data demonstrate that exRNA of cardiac origin exhibits a potent pro-inflammatory property in vitro and in vivo and that exRNA induces cytokine production through TLR7-MyD88 signaling.

  5. PTGER1 deletion attenuates renal injury in diabetic mouse models.

    PubMed

    Thibodeau, Jean-François; Nasrallah, Rania; Carter, Anthony; He, Ying; Touyz, Rhian; Hébert, Richard L; Kennedy, Christopher R J

    2013-12-01

    We hypothesized that the EP1 receptor promotes renal damage in diabetic nephropathy. We rendered EP1 (PTGER1, official symbol) knockout mice (EP1(-/-)) diabetic using the streptozotocin and OVE26 models. Albuminuria, mesangial matrix expansion, and glomerular hypertrophy were each blunted in EP1(-/-) streptozotocin and OVE26 cohorts compared with wild-type counterparts. Although diabetes-associated podocyte depletion was unaffected by EP1 deletion, EP1 antagonism with ONO-8711 in cultured podocytes decreased angiotensin II-mediated superoxide generation, suggesting that EP1-associated injury of remaining podocytes in vivo could contribute to filtration barrier dysfunction. Accordingly, EP1 deletion in OVE26 mice prevented nephrin mRNA expression down-regulation and ameliorated glomerular basement membrane thickening and foot process effacement. Moreover, EP1 deletion reduced diabetes-induced expression of fibrotic markers fibronectin and α-actin, whereas EP1 antagonism decreased fibronectin in cultured proximal tubule cells. Similarly, proximal tubule megalin expression was reduced by diabetes but was preserved in EP1(-/-) mice. Finally, the diabetes-associated increase in angiotensin II-mediated constriction of isolated mesenteric arteries was blunted in OVE26EP1(-/-) mice, demonstrating a role for EP1 receptors in the diabetic vasculature. These data suggest that EP1 activation contributes to diabetic nephropathy progression at several locations, including podocytes, proximal tubule, and the vasculature. The EP1 receptor facilitates the actions of angiotensin II, thereby suggesting that targeting of both the renin-angiotensin system and the EP1 receptor could be beneficial in diabetic nephropathy.

  6. Recombinant erythropoietin is neuroprotective in a novel mouse oxidative injury model.

    PubMed

    Juul, Sandra E; McPherson, Ronald J; Bammler, Theodor K; Wilkerson, Jasmine; Beyer, Richard P; Farin, Federico M

    2008-01-01

    To identify neuroprotective changes in gene expression, we developed a neonatal mouse model of moderate to severe oxidative brain injury and hypothesized that recombinant erythropoietin (rEpo) would decrease the expression of proapoptotic and proinflammatory genes 24 and 48 h, respectively, after injury and increase the expression of neurogenic and angiogenic genes 168 h after injury. Postnatal day 10 BALB-c mice underwent sham surgery or right common carotid artery occlusion followed by alternating hypoxia and hyperoxia and were then treated with rEpo (5,000 U/kg s.c.) or saline (vehicle) daily for up to three doses. At death, gross brain injury was assessed, then hippocampus, cortex, and thalamus were isolated for RNA or protein extraction. Microarray analysis, real-time polymerase chain reaction, and Bio-Plex suspension array system validation were performed. rEpo decreased both incidence and severity of brain injury (median injury score 3 vs. 0, p < 0.0001) and reduced the injury-induced increases in interleukin-1alpha and interleukin-6 gene expression (p < 0.001), with corresponding effects on protein translation. Similarly, the expression of caspase-1, caspase-4, and caspase-6 and of p53 was increased by brain injury at 24 h, but mitigated by rEpo (p < 0.01). The interleukin-10 expression was higher in the rEpo-treated animals. Apoptotic and proinflammatory gene expression persisted for 168 h. There was no increase in angiogenic gene expression at the time points studied.

  7. Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes.

    PubMed

    Trivedi, Purvi C; Bartlett, Jordan J; Perez, Lester J; Brunt, Keith R; Legare, Jean Francois; Hassan, Ansar; Kienesberger, Petra C; Pulinilkunnil, Thomas

    2016-12-01

    Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.

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

    PubMed Central

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

    2016-01-01

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

  9. Liver transplantation in the mouse: Insights into liver immunobiology, tissue injury, and allograft tolerance.

    PubMed

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

    2016-04-01

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

  10. Regional susceptibility to stress-induced intestinal injury in the mouse.

    PubMed

    Novosad, Veronica L; Richards, Jennifer L; Phillips, Neil A; King, Michelle A; Clanton, Thomas L

    2013-09-15

    Injury to the intestinal mucosa is a life-threatening problem in a variety of clinical disorders, including hemorrhagic shock, trauma, burn, pancreatitis, and heat stroke. The susceptibility to injury of different regions of intestine in these disorders is not well understood. We compared histological injury across the small intestine in two in vivo mouse models of injury, hemorrhagic shock (30% loss of blood volume) and heat stroke (peak core temperature 42.4°C). In both injury models, areas near the duodenum showed significantly greater mucosal injury and reductions in villus height. To determine if these effects were dependent on circulating factors, experiments were performed on isolated intestinal segments to test for permeability to 4-kDa FITC-dextran. The segments were exposed to hyperthermia (42°C for 90 min), moderate simulated ischemia (Po2 ∼30 Torr, Pco2 ∼60 Torr, pH 7.1), severe ischemia (Po2 ∼20 Torr, Pco2 ∼80 Torr, pH 6.9), or severe hypoxia (Po2 ∼0 Torr, Pco2 ∼35 Torr) for 90 min, and each group was compared with sham controls. All treatments resulted in marked elevations in permeability within segments near the duodenum. In severe hypoxia or hyperthermia, permeability was also moderately elevated in the jejunum and ileum; in moderate or severe ischemia, permeability was unaffected in these regions. The results demonstrate increased susceptibility of proximal regions of the small intestine to acute stress-induced damage, irrespective of circulating factors. The predominant injury in the duodenum may impact the pattern of acute inflammatory responses arising from breach of the intestinal barrier, and such knowledge may be useful for designing therapeutic strategies.

  11. Role of microglia in a mouse model of paediatric traumatic brain injury.

    PubMed

    Chhor, Vibol; Moretti, Raffaella; Le Charpentier, Tifenn; Sigaut, Stephanie; Lebon, Sophie; Schwendimann, Leslie; Oré, Marie-Virginie; Zuiani, Chiara; Milan, Valentina; Josserand, Julien; Vontell, Regina; Pansiot, Julien; Degos, Vincent; Ikonomidou, Chrysanthy; Titomanlio, Luigi; Hagberg, Henrik; Gressens, Pierre; Fleiss, Bobbi

    2016-11-04

    The cognitive and behavioural deficits caused by traumatic brain injury (TBI) to the immature brain are more severe and persistent than TBI in the mature brain. Understanding this developmental sensitivity is critical as children under four years of age sustain TBI more frequently than any other age group. Microglia (MG), resident immune cells of the brain that mediate neuroinflammation, are activated following TBI in the immature brain. However, the type and temporal profile of this activation and the consequences of altering it are still largely unknown. In a mouse model of closed head weight drop paediatric brain trauma, we characterized i) the temporal course of total cortical neuroinflammation and the phenotype of ex vivo isolated CD11B-positive microglia/macrophage (MG/MΦ) using a battery of 32 markers, and ii) neuropathological outcome 1 and 5days post-injury. We also assessed the effects of targeting MG/MΦ activation directly, using minocycline a prototypical microglial activation antagonist, on these processes and outcome. TBI induced a moderate increase in both pro- and anti-inflammatory cytokines/chemokines in the ipsilateral hemisphere. Isolated cortical MG/MΦ expressed increased levels of markers of endogenous reparatory/regenerative and immunomodulatory phenotypes compared with shams. Blocking MG/MΦ activation with minocycline at the time of injury and 1 and 2days post-injury had only transient protective effects, reducing ventricular dilatation and cell death 1day post-injury but having no effect on injury severity at 5days. This study demonstrates that, unlike in adults, the role of MG/MΦ in injury mechanisms following TBI in the immature brain may not be negative. An improved understanding of MG/MΦ function in paediatric TBI could support translational efforts to design therapeutic interventions.

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

    SciTech Connect

    Desai, Varsha G.; Kwekel, Joshua C.; Vijay, Vikrant; Moland, Carrie L.; Herman, Eugene H.; Lee, Taewon; Han, Tao; Lewis, Sherry M.; Davis, Kelly J.; Muskhelishvili, Levan; Kerr, Susan; Fuscoe, James C.

    2014-12-01

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

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

    PubMed Central

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

    2011-01-01

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

  14. A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

    PubMed

    Miyakawa, Kazuhisa; Albee, Ryan; Letzig, Lynda G; Lehner, Andreas F; Scott, Michael A; Buchweitz, John P; James, Laura P; Ganey, Patricia E; Roth, Robert A

    2015-08-01

    Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies.

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

    PubMed

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

    2015-09-22

    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.

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

    PubMed

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

    2017-03-01

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

  17. Hepcidin protects against lipopolysaccharide-induced liver injury in a mouse model of obstructive jaundice.

    PubMed

    Huang, Ying-Hsien; Yang, Ya-Ling; Tiao, Mao-Meng; Kuo, Ho-Chang; Huang, Li-Tung; Chuang, Jiin-Haur

    2012-06-01

    Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.

  18. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.

    PubMed

    Basso, D Michele; Fisher, Lesley C; Anderson, Aileen J; Jakeman, Lyn B; McTigue, Dana M; Popovich, Phillip G

    2006-05-01

    Genetically engineered mice are used extensively to examine molecular responses to spinal cord injury (SCI). Inherent strain differences may confound behavioral outcomes; therefore, behavioral characterization of several strains after SCI is warranted. The Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) for rats has been widely used for SCI mice, but may not accurately reflect their unique recovery pattern. This study's purpose was to develop a valid locomotor rating scale for mice and to identify strain differences in locomotor recovery after SCI. We examined C57BL/6, C57BL/10, B10.PL, BALB/c, and C57BL/6x129S6 F1 strains for 42 days after mild, moderate, and severe contusive SCI or transection of the mid thoracic spinal cord. Contusions were created using the Ohio State University electromagnetic SCI device which is a displacement-driven model, and the Infinite Horizon device, which is a force-driven model. Attributes and rankings for the Basso Mouse Scale for Locomotion (BMS) were determined from frequency analyses of seven locomotor categories. Mouse recovery differed from rats for coordination, paw position and trunk instability. Disagreement occurred across six expert raters using BBB (p < 0.05) but not BMS to assess the same mice. BMS detected significant differences in locomotor outcomes between severe contusion and transection (p < 0.05) and SCI severity gradations resulting from displacement variations of only 0.1 mm (p < 0.05). BMS demonstrated significant face, predictive and concurrent validity. Novice BMS raters with training scored within 0.5 points of experts and demonstrated high reliability (0.92-0.99). The BMS is a sensitive, valid and reliable locomotor measure in SCI mice. BMS revealed significantly higher recovery in C57BL/10, B10.PL and F1 than the C57BL/6 and BALB/c strains after moderate SCI (p < 0.05). The differing behavioral response to SCI suggests inherent genetic factors significantly impact locomotor recovery and must be

  19. Microwave & Magnetic (M2) Proteomics of a Mouse Model of Mild Traumatic Brain Injury

    PubMed Central

    Evans, Teresa M.; Van Remmen, Holly; Purkar, Anjali; Mahesula, Swetha; Gelfond, J AL; Sabia, Marian; Qi, Wenbo; Lin, Ai-Ling; Jaramillo, Carlos A.; Haskins, William E.

    2014-01-01

    Short-term increases in oxidative stress and decreases in motor function, including debilitating effects on balance and motor control, can occur following primary mild traumatic brain injuries (mTBI). However, the long-term effects on motor unit impairment and integrity as well as the molecular mechanisms underlying secondary injuries are poorly understood. We hypothesized that changes in central nervous system-specific protein (CSP) expression might correlate to these long-term effects. To test our hypothesis, we longitudinally assessed a closed-skull mTBI mouse model, vs. sham control, at 1, 7, 30, and 120 days post-injury. Motor impairment was determined by rotarod and grip strength performance measures, while motor unit integrity was determined using electromyography. Relative protein expression was determined by microwave & magnetic (M2) proteomics of ipsilateral brain tissue, as previously described. Isoprostane measurements were performed to confirm a primary oxidative stress response. Decoding the relative expression of 476 ± 56 top-ranked proteins for each specimen revealed statistically significant changes in the expression of two well-known CSPs at 1, 7 and 30 days post-injury: P < 0.001 for myelin basic protein (MBP) and P < 0.05 for myelin associated glycoprotein (MAG). This was confirmed by Western blot. Moreover, MAG, αII-spectrin (SPNA2) and neurofilament light (NEFL) expression at 30 days post-injury were directly related to grip strength (P < 0.05). While higher-powered studies of larger cohorts merit further investigation, this study supports the proof-of-concept that M2 proteomics is a rapid method to quantify putative protein biomarkers and therapeutic targets of mTBI and suggests the feasibility of CSP expression correlations to long-term effects on motor impairment. PMID:26157646

  20. Altered Neuroinflammation and Behavior after Traumatic Brain Injury in a Mouse Model of Alzheimer's Disease.

    PubMed

    Kokiko-Cochran, Olga; Ransohoff, Lena; Veenstra, Mike; Lee, Sungho; Saber, Maha; Sikora, Matt; Teknipp, Ryan; Xu, Guixiang; Bemiller, Shane; Wilson, Gina; Crish, Samuel; Bhaskar, Kiran; Lee, Yu-Shang; Ransohoff, Richard M; Lamb, Bruce T

    2016-04-01

    Traumatic brain injury (TBI) has acute and chronic sequelae, including an increased risk for the development of Alzheimer's disease (AD). TBI-associated neuroinflammation is characterized by activation of brain-resident microglia and infiltration of monocytes; however, recent studies have implicated beta-amyloid as a major manipulator of the inflammatory response. To examine neuroinflammation after TBI and development of AD-like features, these studies examined the effects of TBI in the presence and absence of beta-amyloid. The R1.40 mouse model of cerebral amyloidosis was used, with a focus on time points well before robust AD pathologies. Unexpectedly, in R1.40 mice, the acute neuroinflammatory response to TBI was strikingly muted, with reduced numbers of CNS myeloid cells acquiring a macrophage phenotype and decreased expression of inflammatory cytokines. At chronic time points, macrophage activation substantially declined in non-Tg TBI mice; however, it was relatively unchanged in R1.40 TBI mice. The persistent inflammatory response coincided with significant tissue loss between 3 and 120 days post-injury in R1.40 TBI mice, which was not observed in non-Tg TBI mice. Surprisingly, inflammatory cytokine expression was enhanced in R1.40 mice compared with non-Tg mice, regardless of injury group. Although R1.40 TBI mice demonstrated task-specific deficits in cognition, overall functional recovery was similar to non-Tg TBI mice. These findings suggest that accumulating beta-amyloid leads to an altered post-injury macrophage response at acute and chronic time points. Together, these studies emphasize the role of post-injury neuroinflammation in regulating long-term sequelae after TBI and also support recent studies implicating beta-amyloid as an immunomodulator.

  1. Regulatory T cells exhibit neuroprotective effect in a mouse model of traumatic brain injury

    PubMed Central

    Yu, Yunhu; Cao, Fang; Ran, Qishan; Sun, Xiaochuan

    2016-01-01

    Traumatic brain injury (TBI) is a major health and socioeconomic problem as it is associated with high rates of mortality and morbidity worldwide. Regulatory T cells (Tregs) have been reported to reduce inflammatory response in several diseases, including myasthenia gravis, viral myocarditis and cerebral infarction. The present study investigated the role of Tregs in mediating neuro-protective effects in a mouse model of TBI. Initially, Treg levels were determined, and compared between the controlled cortical impact (CCI) model for moderate TBI and the sham group, by using flow cytometry and ELISA. Afterwards, the number of Tregs was upregulated (by injection) and downregulated (by depletion), respectively, to elucidate the effect of Tregs in the presence of an inflammatory reaction and a deficient neurological function and consequently, in the prognosis of TBI in the mouse. The expression of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6)] and anti-inflammatory cytokines [IL-10, transforming growth factor (TGF)-β] in blood and brain tissues was also measured in the five groups: Μice receiving a saline injection, mice experiencing Treg depletion, small-dose (SD Tregs, 1.25×105), and mice receiving different doses of Tregs: Moderate-dose (MD Tregs, 2.5×105) and large-dose (LD Tregs, 5×105), using ELISA and PCR. Co-cultures of Tregs and microglia were performed to evaluate the expression of pro-inflammatory cytokines and observe the interaction between the two types of cells. The regulation patterns in JNK-NF-κB pathway by Tregs were also evaluated by western blot analysis. Treg levels were significantly reduced in TBI mouse group on the 3rd day after TBI (P<0.05). In the mouse model of TBI, the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) was enhanced, while the expression of anti-inflammatory cytokines (IL-10, TGF-β) was reduced (P<0.05). Tregs exhibited a suppressive effect on inflammatory reactions

  2. Regulatory T cells exhibit neuroprotective effect in a mouse model of traumatic brain injury.

    PubMed

    Yu, Yunhu; Cao, Fang; Ran, Qishan; Sun, Xiaochuan

    2016-12-01

    Traumatic brain injury (TBI) is a major health and socioeconomic problem as it is associated with high rates of mortality and morbidity worldwide. Regulatory T cells (Tregs) have been reported to reduce inflammatory response in several diseases, including myasthenia gravis, viral myocarditis and cerebral infarction. The present study investigated the role of Tregs in mediating neuro‑protective effects in a mouse model of TBI. Initially, Treg levels were determined, and compared between the controlled cortical impact (CCI) model for moderate TBI and the sham group, by using flow cytometry and ELISA. Afterwards, the number of Tregs was upregulated (by injection) and downregulated (by depletion), respectively, to elucidate the effect of Tregs in the presence of an inflammatory reaction and a deficient neurological function and consequently, in the prognosis of TBI in the mouse. The expression of pro‑inflammatory cytokines [tumor necrosis factor (TNF)‑α, interleukin (IL)‑1β, IL‑6)] and anti‑inflammatory cytokines [IL‑10, transforming growth factor (TGF)‑β] in blood and brain tissues was also measured in the five groups: Μice receiving a saline injection, mice experiencing Treg depletion, small‑dose (SD Tregs, 1.25x105), and mice receiving different doses of Tregs: Moderate‑dose (MD Tregs, 2.5x105) and large‑dose (LD Tregs, 5x105), using ELISA and PCR. Co‑cultures of Tregs and microglia were performed to evaluate the expression of pro‑inflammatory cytokines and observe the interaction between the two types of cells. The regulation patterns in JNK‑NF‑κB pathway by Tregs were also evaluated by western blot analysis. Treg levels were significantly reduced in TBI mouse group on the 3rd day after TBI (P<0.05). In the mouse model of TBI, the expression of pro‑inflammatory cytokines (TNF‑α, IL‑1β, IL‑6) was enhanced, while the expression of anti‑inflammatory cytokines (IL‑10, TGF‑β) was reduced (P<0.05). Tregs exhibited a

  3. Repetitive mild traumatic brain injury in a mouse model produces learning and memory deficits accompanied by histological changes.

    PubMed

    Mouzon, Benoit; Chaytow, Helena; Crynen, Gogce; Bachmeier, Corbin; Stewart, Janice; Mullan, Michael; Stewart, William; Crawford, Fiona

    2012-12-10

    Concussion or mild traumatic brain injury (mTBI) represents the most common type of brain injury. However, in contrast with moderate or severe injury, there are currently few non-invasive experimental studies that investigate the cumulative effects of repetitive mTBI using rodent models. Here we describe and compare the behavioral and pathological consequences in a mouse model of single (s-mTBI) or repetitive injury (r-mTBI, five injuries given at 48 h intervals) administered by an electromagnetic controlled impactor. Our results reveal that a single mTBI is associated with transient motor and cognitive deficits as demonstrated by rotarod and the Barnes Maze respectively, whereas r-mTBI results in more significant deficits in both paradigms. Histology revealed no overt cell loss in the hippocampus, although a reactive gliosis did emerge in hippocampal sector CA1 and in the deeper cortical layers beneath the injury site in repetitively injured animals, where evidence of focal injury also was observed in the brainstem and cerebellum. Axonal injury, manifest as amyloid precursor protein immunoreactive axonal profiles, was present in the corpus callosum of both injury groups, though more evident in the r-mTBI animals. Our data demonstrate that this mouse model of mTBI is reproducible, simple, and noninvasive, with behavioral impairment after a single injury and increasing deficits after multiple injuries accompanied by increased focal and diffuse pathology. As such, this model may serve as a suitable platform with which to explore repetitive mTBI relevant to human brain injury.

  4. Hepatocyte Tissue Factor Contributes to the Hypercoagulable State in a Mouse Model of Chronic Liver Injury

    PubMed Central

    Rautou, Pierre-Emmanuel; Tatsumi, Kohei; Antoniak, Silvio; Owens, A. Phillip; Sparkenbaugh, Erica; Holle, Lori A.; Wolberg, Alisa S.; Kopec, Anna K.; Pawlinski, Rafal; Luyendyk, James P.; Mackman, Nigel

    2015-01-01

    Summary Background & Aims Patients with chronic liver disease and cirrhosis have a dysregulated coagulation system and are prone to thrombosis. The basis for this hypercoagulable state is not completely understood. Tissue factor (TF) is the primary initiator of coagulation in vivo. Patients with cirrhosis have increased TF activity in white blood cells and circulating microparticles. The aim of our study was to determine the contribution of TF to the hypercoagulable state in a mouse model of chronic liver injury. Methods We measured levels of TF activity in the liver, white blood cells and circulating microparticles, and a marker of activation of coagulation [thrombinantithrombin complexes (TATc)] in the plasma of mice subjected to bile duct ligation for 12 days. We used wild-type mice, mice with a global TF deficiency (low TF mice), and mice deficient for TF in either myeloid cells (TFflox/flox, LysMCre mice) or in hepatocytes (TFflox/flox, AlbCre). Results Wild-type mice with liver injury had increased levels of white blood cell, microparticle TF activity and TATc compared to sham mice. Low TF mice and mice lacking TF in hepatocytes had reduced levels of TF in the liver and in microparticles and exhibited reduced activation of coagulation without a change in liver fibrosis. In contrast, mice lacking TF in myeloid cells had reduced white blood cell TF but no change in microparticle TF activity or TATc. Conclusions Hepatocyte TF activates coagulation in a mouse model of chronic liver injury. TF may contribute to the hypercoagulable state associated with chronic liver diseases in patients. PMID:26325534

  5. A Mouse Model for Fetal Maternal Stem Cell Transfer During Ischemic Cardiac Injury

    PubMed Central

    Kara, Rina J.; Bolli, Paola; Matsunaga, Iwao; Tanweer, Omar; Altman, Perry; Chaudhry, Hina W.

    2012-01-01

    Fetal cells enter the maternal circulation during pregnancies and can persist in blood and tissues for decades, creating a state of physiologic microchimerism. Microchimerism refers to acquisition of cells from another individual and can be due to bi-directional cell traffic between mother and fetus during pregnancy. Peripartum cardiomyopathy, a rare cardiac disorder associated with high mortality rates has the highest recovery rate amongst all etiologies of heart failure although the reason is unknown. Collectively, these observations led us to hypothesize that fetal cells enter the maternal circulation and may be recruited to the sites of myocardial disease or injury. The ability to genetically modify mice makes them an ideal system for studying the phenomenon of microchimerism in cardiac disease. Described here is a mouse model for ischemic cardiac injury during pregnancy designed to study microchimerism. Wild-type virgin female mice mated with eGFP male mice underwent ligation of the left anterior descending artery to induce a myocardial infarction at gestation day 12. We demonstrate the selective homing of eGFP cells to the site of cardiac injury without such homing to nonfinjured tissues suggesting the presence of precise signals sensed by fetal cells enabling them to target diseased myocardium specifically. PMID:22883609

  6. MMI-0100 inhibits cardiac fibrosis in myocardial infarction by direct actions on cardiomyocytes and fibroblasts via MK2 inhibition

    PubMed Central

    Xu, Lei; Yates, Cecelia C.; Lockyer, Pamela; Xie, Liang; Bevilacqua, Ariana; He, Jun; Lander, Cynthia; Patterson, Cam; Willis, Monte

    2014-01-01

    The cell-permeant peptide inhibitor of MAPKAP kinase 2 (MK2), MMI-0100, inhibits MK2 and downstream fibrosis and inflammation. Recent studies have demonstrated that MMI-0100 reduces intimal hyperplasia in a mouse vein graft model, pulmonary fibrosis in a murine bleomycin-induced model and development of adhesions in conjunction with abdominal surgery. MK2 is critical to the pathogenesis of ischemic heart injury as MK2 −/− mice are resistant to ischemic remodeling. Therefore, we tested the hypothesis that inhibiting MK2 with MMI-0100 would protect the heart after acute myocardial infarction (AMI) in vivo. AMI was induced by placing a permanent LAD coronary ligation. When MMI-0100 peptide was given 30 minutes after permanent LAD coronary artery ligation, the resulting fibrosis was reduced/prevented ~50% at a 2 week time point, with a corresponding improvement in cardiac function and decrease in left ventricular dilation. In cultured cardiomyocytes and fibroblasts, MMI-0100 inhibited MK2 to reduce cardiomyocyte caspase 3/7 activity, while enhancing primary cardiac fibroblast caspase 3/7 activity, which may explain MMI-0100’s salvage of cardiac function and anti-fibrotic effects in vivo. These findings suggest that therapeutic inhibition of MK2 after acute MI, using rationally-designed cell-permeant peptides, inhibits cardiac fibrosis and maintains cardiac function by mechanisms that involve inhibiting cardiomyocyte apoptosis, while enhancing primary cardiac fibroblast cell death. PMID:25257914

  7. Baicalin Attenuates IL-17-Mediated Acetaminophen-Induced Liver Injury in a Mouse Model

    PubMed Central

    Liao, Chia-Chih; Day, Yuan-Ji; Lee, Hung-Chen; Liou, Jiin-Tarng; Chou, An-Hsun; Liu, Fu-Chao

    2016-01-01

    Background IL-17 has been shown to be involved in liver inflammatory disorders in both mice and humans. Baicalin (BA), a major compound extracted from traditional herb medicine (Scutellariae radix), has potent hepatoprotective properties. Previous study showed that BA inhibits IL-17-mediated lymphocyte adhesion and downregulates joint inflammation. The aim of this study is to investigate the role of IL-17 in the hepatoprotective effects of BA in an acetaminophen (APAP)-induced liver injury mouse model. Methods Eight weeks male C57BL/6 (B6) mice were used for this study. Mice received intraperitoneal hepatotoxic injection of APAP (300 mg/kg) and after 30 min of injection, the mice were treated with BA at a concentration of 30 mg/kg. After 16 h of treatment, mice were killed. Blood samples and liver tissues were harvested for analysis of liver injury parameters. Results APAP overdose significantly increased the serum alanine transferase (ALT) levels, hepatic activities of myeloperoxidase (MPO), expression of cytokines (TNF-α, IL-6, and IL-17), and malondialdehyde (MDA) activity when compared with the control animals. BA treatment after APAP administration significantly attenuated the elevation of these parameters in APAP-induced liver injury mice. Furthermore, BA treatment could also decrease hepatic IL-17-producing γδT cells recruitment, which was induced after APAP overdose. Conclusion Our data suggested that baicalin treatment could effectively decrease APAP-induced liver injury in part through attenuation of hepatic IL-17 expression. These results indicate that baicalin is a potential hepatoprotective agent. PMID:27855209

  8. Cathepsin B-dependent motor neuron death after nerve injury in the adult mouse

    SciTech Connect

    Sun, Li; Wu, Zhou; Baba, Masashi; Peters, Christoph; Uchiyama, Yasuo; Nakanishi, Hiroshi

    2010-08-27

    Research highlights: {yields} Cathepsin B (CB), a lysosomal cysteine protease, is expressed in neuron and glia. {yields} CB increased in hypogrossal nucleus neurons after nerve injury in adult mice. {yields} CB-deficiency significantly increased the mean survival ratio of injured neurons. {yields} Thus, CB plays a critical role in axotomy-induced neuronal death in adult mice. -- Abstract: There are significant differences in the rate of neuronal death after peripheral nerve injury between species. The rate of neuronal death of motor neurons after nerve injury in the adult rats is very low, whereas that in adult mice is relatively high. However, the understanding of the mechanism underlying axotomy-induced motor neuron death in adult mice is limited. Cathepsin B (CB), a typical cysteine lysosomal protease, has been implicated in three major morphologically distinct pathways of cell death; apoptosis, necrosis and autophagic cell death. The possible involvement of CB in the neuronal death of hypogrossal nucleus (HGN) neurons after nerve injury in adult mice was thus examined. Quantitative analyses showed the mean survival ratio of HGN neurons in CB-deficient (CB-/-) adult mice after nerve injury was significantly greater than that in the wild-type mice. At the same time, proliferation of microglia in the injured side of the HGN of CB-/- adult mice was markedly reduced compared with that in the wild-type mice. On the injured side of the HGN in the wild-type adult mice, both pro- and mature forms of CB markedly increased in accordance with the increase in the membrane-bound form of LC3 (LC3-II), a marker protein of autophagy. Furthermore, the increase in CB preceded an increase in the expression of Noxa, a major executor for axotomy-induced motor neuron death in the adult mouse. Conversely, expression of neither Noxa or LC3-II was observed in the HGN of adult CB-/- mice after nerve injury. These observations strongly suggest that CB plays a critical role in axotomy

  9. Magnetic resonance microscopy of spinal cord injury in mouse using a miniaturized implantable RF coil.

    PubMed

    Bilgen, Mehmet

    2007-01-15

    A magnetic resonance neuroimaging method is described for high-resolution imaging of spinal cord injury in live mouse. The method is based on a specially designed radio frequency coil system formed by a combination of an implantable coil and an external volume coil. The implantable coil is a 5 mm x 10 mm rectangular design with a 9.1 pF capacitor and 22 gauge copper wire and optimal for surgical implantation over the cervical or thoracic spine. The external volume coil is a standard birdcage resonator. The coils are inductively overcoupled for imaging the spinal cord at 9.4 T magnetic field strength. The inductive overcoupling provides flexibility in tuning the resonant frequency and matching the impedance of the implanted coil remotely using the tuning and matching capabilities of the volume coil. After describing the implementation of the imaging setup, in vivo data are gathered to demonstrate the imaging performance of the coil system and the feasibility of performing MR microscopy on injured mouse spinal cord.

  10. Rapamycin attenuates the development of posttraumatic epilepsy in a mouse model of traumatic brain injury.

    PubMed

    Guo, Dongjun; Zeng, Linghui; Brody, David L; Wong, Michael

    2013-01-01

    Posttraumatic epilepsy is a major source of disability following traumatic brain injury (TBI) and a common cause of medically-intractable epilepsy. Previous attempts to prevent the development of posttraumatic epilepsy with treatments administered immediately following TBI have failed. Recently, the mammalian target of rapamycin complex 1 (mTORC1) pathway has been implicated in mechanisms of epileptogenesis and the mTORC1 inhibitor, rapamycin, has been proposed to have antiepileptogenic effects in preventing some types of epilepsy. In this study, we have tested the hypothesis that rapamycin has antiepileptogenic actions in preventing the development of posttraumatic epilepsy in an animal model of TBI. A detailed characterization of posttraumatic epilepsy in the mouse controlled cortical impact model was first performed using continuous video-EEG monitoring for 16 weeks following TBI. Controlled cortical impact injury caused immediate hyperactivation of the mTORC1 pathway lasting at least one week, which was reversed by rapamycin treatment. Rapamycin decreased neuronal degeneration and mossy fiber sprouting, although the effect on mossy fiber sprouting was reversible after stopping rapamycin and did not directly correlate with inhibition of epileptogenesis. Most posttraumatic seizures occurred greater than 10 weeks after TBI, and rapamycin treatment for one month after TBI decreased the seizure frequency and rate of developing posttraumatic epilepsy during the entire 16 week monitoring session. These results suggest that rapamycin may represent a rational treatment for preventing posttraumatic epilepsy in patients with TBI.

  11. Troxerutin protects the mouse liver against oxidative stress-mediated injury induced by D-galactose.

    PubMed

    Zhang, Zi-feng; Fan, Shao-hua; Zheng, Yuan-lin; Lu, Jun; Wu, Dong-mei; Shan, Qun; Hu, Bin

    2009-09-09

    Troxerutin, a trihydroxyethylated derivative of rutin, has been well-demonstrated to exert hepatoprotective properties. In the present study, we attempted to explore whether the antioxidant and anti-inflammatory mechanisms were involved in troxerutin-mediated protection from D-gal-induced liver injury. The effects of troxerutin on liver lipid peroxidation, antioxidant enzymatic activities, and the expression of inflammatory mediator were investigated in D-gal-treated mice. The results showed that troxerutin largely attenuated the D-gal-induced TBARS content increase and also markedly renewed the activities of Cu, Zn-SOD, CAT, and GPx in the livers of D-gal-treated mice. Furthermore, troxerutin inhibited the upregulation of the expression of NF-kappaB p65, iNOS, and COX-2 induced by D-gal. D-Gal-induced tissue architecture changes and serum ALT and AST increases were effectively suppressed by troxerutin. In conclusion, these results suggested that troxerutin could protect the mouse liver from D-gal-induced injury by attenuating lipid peroxidation, renewing the activities of antioxidant enzymes and suppressing inflammatory response. This study provided novel insights into the mechanisms of troxerutin in the protection of the liver.

  12. Signaling through hepatocyte vasopressin receptor 1 protects mouse liver from ischemia-reperfusion injury

    PubMed Central

    Jiang, Jingbo; Ma, Tonghui; Lin, Xiaozhu; Jiang, Liping; Cheng, Jilin; Tao, Ran

    2016-01-01

    Terlipressin has been used extensively in the management of certain complications associated with end-stage liver diseases (ESLDs). In our pilot study, terlipressin treatment showed beneficial effects on liver function in patients with decompensated cirrhosis, however whether it plays a role in liver ischemia-reperfusion injury (IRI) remains unknown. Using a mouse nonlethal hepatic IR model, we found terlipressin administration significantly ameliorated IR-induced liver apoptosis, necrosis and inflammation. Furthermore, despite its known effect on visceral vasoconstriction, hemodynamic evaluation of murine hepatic tissue after IR revealed no change of overall hepatic blood flow after terlipressin treatment. Further studies identified the upregulation of vasopressin receptor 1 (V1R) expression on hepatocytes upon IR. In isolated hepatocyte hypoxia/reoxygenation model, the active component of terlipressin, lysine vasopressin, conferred hepatocytes resistant to oxidative stress-induced apoptosis. Mechanistic studies revealed the V1R engagement activated the Wnt/β-catenin/FoxO3a/AKT pathway, which subsequently circumvented the proapoptotic events, thus ameliorated hepatocyte apoptosis. Furthermore, genetic knockdown of V1R expression in hepatocyte cell lines or blockade of this signaling pathway abrogated such protective effect. Conclusion: These data highlight the functional importance of the hepatocyte V1R/Wnt/β-catenin/FoxO3a/AKT pathway in protecting liver from oxidative stress-induced injury. PMID:27713143

  13. Systemic Administration of Induced Neural Stem Cells Regulates Complement Activation in Mouse Closed Head Injury Models

    PubMed Central

    Gao, Mou; Dong, Qin; Yao, Hui; Lu, Yingzhou; Ji, Xinchao; Zou, Mingming; Yang, Zhijun; Xu, Minhui; Xu, Ruxiang

    2017-01-01

    Complement activation plays important roles in the pathogenesis of central nervous system (CNS) diseases. Patients face neurological disorders due to the development of complement activation, which contributes to cell apoptosis, brain edema, blood-brain barrier dysfunction and inflammatory infiltration. We previously reported that induced neural stem cells (iNSCs) can promote neurological functional recovery in closed head injury (CHI) animals. Remarkably, we discovered that local iNSC grafts have the potential to modulate CNS inflammation post-CHI. In this study, we aimed to explore the role of systemically delivered iNSCs in complement activation following CNS injury. Our data showed that iNSC grafts decreased the levels of sera C3a and C5a and down-regulated the expression of C3d, C9, active Caspase-3 and Bax in the brain, kidney and lung tissues of CHI mice. Furthermore, iNSC grafts decreased the levels of C3d+/NeuN+, C5b-9+/NeuN+, C3d+/Map2+ and C5b-9+/Map2+ neurons in the injured cortices of CHI mice. Subsequently, we explored the mechanisms underlying these effects. With flow cytometry analysis, we observed a dramatic increase in complement receptor type 1-related protein y (Crry) expression in iNSCs after CHI mouse serum treatment. Moreover, both in vitro and in vivo loss-of-function studies revealed that iNSCs could modulate complement activation via Crry expression. PMID:28383046

  14. Generation of a tamoxifen inducible Tnnt2MerCreMer knock-in mouse model for cardiac studies.

    PubMed

    Yan, Jianyun; Sultana, Nishat; Zhang, Lu; Park, David S; Shekhar, Akshay; Hu, Jun; Bu, Lei; Cai, Chen-Leng

    2015-06-01

    Tnnt2, encoding thin-filament sarcomeric protein cardiac troponin T, plays critical roles in heart development and function in mammals. To develop an inducible genetic deletion strategy in myocardial cells, we generated a new Tnnt2:MerCreMer (Tnnt2(MerCreMer/+)) knock-in mouse. Rosa26 reporter lines were used to examine the specificity and efficiency of the inducible Cre recombinase. We found that Cre was specifically and robustly expressed in the cardiomyocytes at embryonic and adult stages following tamoxifen induction. The knock-in allele on Tnnt2 locus does not impact cardiac function. These results suggest that this new Tnnt2(MerCreMer/+) mouse could be applied towards the temporal genetic deletion of genes of interests in cardiomyocytes with Cre-LoxP technology. The Tnnt2(MerCreMer/+) mouse model also provides a useful tool to trace myocardial lineage during development and repair after cardiac injury.

  15. Drinking citrus fruit juice inhibits vascular remodeling in cuff-induced vascular injury mouse model.

    PubMed

    Ohnishi, Arika; Asayama, Rie; Mogi, Masaki; Nakaoka, Hirotomo; Kan-No, Harumi; Tsukuda, Kana; Chisaka, Toshiyuki; Wang, Xiao-Li; Bai, Hui-Yu; Shan, Bao-Shuai; Kukida, Masayoshi; Iwanami, Jun; Horiuchi, Masatsugu

    2015-01-01

    Citrus fruits are thought to have inhibitory effects on oxidative stress, thereby attenuating the onset and progression of cancer and cardiovascular disease; however, there are few reports assessing their effect on vascular remodeling. Here, we investigated the effect of drinking the juice of two different citrus fruits on vascular neointima formation using a cuff-induced vascular injury mouse model. Male C57BL6 mice were divided into five groups as follows: 1) Control (water) (C), 2) 10% Citrus unshiu (CU) juice (CU10), 3) 40% CU juice (CU40), 4) 10% Citrus iyo (CI) juice (CI10), and 5) 40% CI juice (CI40). After drinking them for 2 weeks from 8 weeks of age, cuff injury was induced by polyethylene cuff placement around the femoral artery. Neointima formation was significantly attenuated in CU40, CI10 and CI40 compared with C; however, no remarkable preventive effect was observed in CU10. The increases in levels of various inflammatory markers including cytokines such as monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α in response to vascular injury did not differ significantly between C, CU10 and CI10. The increases in cell proliferation and superoxide anion production were markedly attenuated in CI10, but not in CU10 compared with C. The increase in phosphorylated ERK expression was markedly attenuated both in CU10 and CI10 without significant difference between CU10 and CI10. Accumulation of immune cells did not differ between CU10 and CI10. These results indicate that drinking citrus fruit juice attenuates vascular remodeling partly via a reduction of oxidative stress. Interestingly, the preventive efficacy on neointima formation was stronger in CI than in CU at least in part due to more prominent inhibitory effects on oxidative stress by CI.

  16. NG2 cell response in the CNP-EGFP mouse after contusive spinal cord injury

    PubMed Central

    Lytle, Judith M.; Chittajallu, Ramesh; Wrathall, Jean R.; Gallo, Vittorio

    2009-01-01

    NG2+ cells in the adult CNS are a heterogeneous population. The extent to which the subpopulation of NG2+ cells that function as oligodendrocyte progenitor cells (OPCs) respond to spinal cord injury (SCI) and recapitulate their normal developmental progression remains unclear. We used the CNP-EGFP mouse, in which oligodendrocyte lineage cells express EGFP, to study NG2+ cells in the normal and injured spinal cord. In white matter of uninjured mice, bipolar EGFP+NG2+ cells and multipolar EGFPnegNG2+ cells were identified. After SCI, EGFP+NG2+ cell proliferation in residual white matter peaked at 3 days post injury (DPI) rostral to the epicenter, while EGFPnegNG2+ cell proliferation peaked at 7 DPI at the epicenter. The expression of transcription factors Olig2, Sox10 and Sox17, and the basic electrophysiological membrane parameters and potassium current phenotype of the EGFP+NG2+ population after injury were consistent with those of proliferative OPCs during development. EGFPnegNG2+ cells did not express transcription factors involved in oligodendrogenesis. EGFP+CC1+ oligodendrocytes at 6 weeks included cells that incorporated BrdU during the peak of EGFP+NG2+ cell proliferation. EGFPnegCC1+ oligodendrocytes were never observed. Treatment with glial growth factor 2 and fibroblast growth factor 2 enhanced oligodendrogenesis and increased the number of EGFPnegNG2+ cells. Therefore, based on EGFP and transcription factor expression, spatio-temporal proliferation patterns, and response to growth factors, two populations of NG2+ cells can be identified that react to SCI. The EGFP+NG2+ cells undergo cellular and physiological changes in response to SCI that are similar to those that occur in early postnatal NG2+ cells during developmental oligodendrogenesis. PMID:18756526

  17. Macrophage recruitment and epithelial repair following hair cell injury in the mouse utricle.

    PubMed

    Kaur, Tejbeer; Hirose, Keiko; Rubel, Edwin W; Warchol, Mark E

    2015-01-01

    The sensory organs of the inner ear possess resident populations of macrophages, but the function of those cells is poorly understood. In many tissues, macrophages participate in the removal of cellular debris after injury and can also promote tissue repair. The present study examined injury-evoked macrophage activity in the mouse utricle. Experiments used transgenic mice in which the gene for the human diphtheria toxin receptor (huDTR) was inserted under regulation of the Pou4f3 promoter. Hair cells in such mice can be selectively lesioned by systemic treatment with diphtheria toxin (DT). In order to visualize macrophages, Pou4f3-huDTR mice were crossed with a second transgenic line, in which one or both copies of the gene for the fractalkine receptor CX3CR1 were replaced with a gene for GFP. Such mice expressed GFP in all macrophages, and mice that were CX3CR1(GFP/GFP) lacked the necessary receptor for fractalkine signaling. Treatment with DT resulted in the death of ∼70% of utricular hair cells within 7 days, which was accompanied by increased numbers of macrophages within the utricular sensory epithelium. Many of these macrophages appeared to be actively engulfing hair cell debris, indicating that macrophages participate in the process of 'corpse removal' in the mammalian vestibular organs. However, we observed no apparent differences in injury-evoked macrophage numbers in the utricles of CX3CR1(+/GFP) mice vs. CX3CR1(GFP/GFP) mice, suggesting that fractalkine signaling is not necessary for macrophage recruitment in these sensory organs. Finally, we found that repair of sensory epithelia at short times after DT-induced hair cell lesions was mediated by relatively thin cables of F-actin. After 56 days recovery, however, all cell-cell junctions were characterized by very thick actin cables.

  18. Inhibition of ischemic cardiomyocyte apoptosis through targeted ablation of Bnip3 restrains postinfarction remodeling in mice

    PubMed Central

    Diwan, Abhinav; Krenz, Maike; Syed, Faisal M.; Wansapura, Janaka; Ren, Xiaoping; Koesters, Andrew G.; Li, Hairong; Kirshenbaum, Lorrie A.; Hahn, Harvey S.; Robbins, Jeffrey; Jones, W. Keith; Dorn, Gerald W.

    2007-01-01

    Following myocardial infarction, nonischemic myocyte death results in infarct expansion, myocardial loss, and ventricular dysfunction. Here, we demonstrate that a specific proapoptotic gene, Bnip3, minimizes ventricular remodeling in the mouse, despite having no effect on early or late infarct size. We evaluated the effects of ablating Bnip3 on cardiomyocyte death, infarct size, and ventricular remodeling after surgical ischemia/reperfusion (IR) injury in mice. Immediately following IR, no significant differences were observed between Bnip3–/– and WT mice. However, at 2 days after IR, apoptosis was diminished in Bnip3–/– periinfarct and remote myocardium, and at 3 weeks after IR, Bnip3–/– mice exhibited preserved LV systolic performance, diminished LV dilation, and decreased ventricular sphericalization. These results suggest myocardial salvage by inhibition of apoptosis. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice, causing progressive LV dilation and diminished systolic function. Conditional Bnip3 overexpression prior to coronary ligation increased apoptosis and infarct size. These studies identify postischemic apoptosis by myocardial Bnip3 as a major determinant of ventricular remodeling in the infarcted heart, suggesting that Bnip3 may be an attractive therapeutic target. PMID:17909626

  19. Full tensor diffusion imaging is not required to assess the white-matter integrity in mouse contusion spinal cord injury.

    PubMed

    Tu, Tsang-Wei; Kim, Joong H; Wang, Jian; Song, Sheng-Kwei

    2010-01-01

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

  20. Thermomineral water promotes axonal sprouting but does not reduce glial scar formation in a mouse model of spinal cord injury

    PubMed Central

    Aleksić, Dubravka; Aksić, Milan; Divac, Nevena; Radonjić, Vidosava; Filipović, Branislav; Jakovčevski, Igor

    2014-01-01

    Thermomineral water from the Atomic Spa Gornja Trepča has been used for a century in the treatment of neurologic disease. The thermomineral water contains microelements, including lithium and magnesium, which show neural regeneration-promoting effects after central nervous system injury. In this study, we investigated the effects of oral intake of thermomineral water from the Atomic Spa Gornja Trepča on nerve regeneration in a 3-month-old mouse model of spinal cord injury. The mice receiving oral intake of thermomineral water showed better locomotor recovery than those without administration of thermomineral water at 8 and 12 weeks after lower thoracic spinal cord compression. At 12 weeks after injury, sprouting of catecholaminergic axons was better in mice that drank thermomineral water than in those without administration of thermomineral water, but there was no difference in glial reaction to injury between mice with and without administration of thermomineral water. These findings suggest that thermomineral water can promote the nerve regeneration but cannot reduce glial scar formation in a mouse model of spinal cord injury. PMID:25657739

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

    PubMed Central

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

    2016-01-01

    Abstract Background Kidney injury molecule‐1 (Kim‐1) has been validated as a urinary biomarker for acute and chronic renal damage. The expression of Kim‐1 mRNA is also activated by acute kidney injury induced by cisplatin in rodents and humans. To date, the measurement of Kim‐1 expression has not fully allowed the detection of in vitro cisplatin nephrotoxicity in immortalized culture cells, such as human kidney‐2 cells and immortalized proximal tubular epithelial cells. Methods We measured the augmentation of Kim‐1 mRNA expression after the addition of cisplatin using immortalized S3 cells established from the kidneys of transgenic mice harboring temperature‐sensitive large T antigen from Simian virus 40. Results A mouse Kim‐1 gene luciferase reporter in conjunction with an Hprt gene reporter detected cisplatin‐induced nephrotoxicity in S3 cells. These two reporter genes were contained in a mouse artificial chromosome, and two luciferases that emitted different wavelengths were used to monitor the respective gene expression. However, the Kim‐1 reporter gene failed to respond to cisplatin in A9 fibroblast cells that contained the same reporter mouse artificial chromosome, suggesting cell type‐specificity for activation of the reporter. Conclusions We report the feasibility of measuring in vitro cisplatin nephrotoxicity using a Kim‐1 reporter gene in S3 cells. PMID:27591740

  2. Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy

    PubMed Central

    Kooij, Viola; Viswanathan, Meera C.; Lee, Dong I.; Rainer, Peter P.; Schmidt, William; Kronert, William A.; Harding, Sian E.; Kass, David A.; Bernstein, Sanford I.; Van Eyk, Jennifer E.; Cammarato, Anthony

    2016-01-01

    Aims Heart failure is often preceded by cardiac hypertrophy, which is characterized by increased cell size, altered protein abundance, and actin cytoskeletal reorganization. Profilin is a well-conserved, ubiquitously expressed, multifunctional actin-binding protein, and its role in cardiomyocytes is largely unknown. Given its involvement in vascular hypertrophy, we aimed to test the hypothesis that profilin-1 is a key mediator of cardiomyocyte-specific hypertrophic remodelling. Methods and results Profilin-1 was elevated in multiple mouse models of hypertrophy, and a cardiomyocyte-specific increase of profilin in Drosophila resulted in significantly larger heart tube dimensions. Moreover, adenovirus-mediated overexpression of profilin-1 in neonatal rat ventricular myocytes (NRVMs) induced a hypertrophic response, measured by increased myocyte size and gene expression. Profilin-1 silencing suppressed the response in NRVMs stimulated with phenylephrine or endothelin-1. Mechanistically, we found that profilin-1 regulates hypertrophy, in part, through activation of the ERK1/2 signalling cascade. Confocal microscopy showed that profilin localized to the Z-line of Drosophila myofibrils under normal conditions and accumulated near the M-line when overexpressed. Elevated profilin levels resulted in elongated sarcomeres, myofibrillar disorganization, and sarcomeric disarray, which correlated with impaired muscle function. Conclusion Our results identify novel roles for profilin as an important mediator of cardiomyocyte hypertrophy. We show that overexpression of profilin is sufficient to induce cardiomyocyte hypertrophy and sarcomeric remodelling, and silencing of profilin attenuates the hypertrophic response. PMID:26956799

  3. Protective Effect of Quercetin on the Development of Preimplantation Mouse Embryos against Hydrogen Peroxide-Induced Oxidative Injury

    PubMed Central

    Zhang, Qin-hua; Yan, Zhi-guang; Liang, Hong-xing; Chai, Wei-ran; Yan, Zheng; Kuang, Yan-ping; Qi, Cong

    2014-01-01

    Quercetin, a plant-derived flavonoid in Chinese herbs, fruits and wine, displays antioxidant properties in many pathological processes associated with oxidative stress. However, the effect of quercetin on the development of preimplantation embryos under oxidative stress is unclear. The present study sought to determine the protective effect and underlying mechanism of action of quercetin against hydrogen peroxide (H2O2)-induced oxidative injury in mouse zygotes. H2O2 treatment impaired the development of mouse zygotes in vitro, decreasing the rates of blastocyst formation and hatched, and increasing the fragmentation, apoptosis and retardation in blastocysts. Quercetin strongly protected zygotes from H2O2-induced oxidative injury by decreasing the reactive oxygen species level, maintaining mitochondrial function and modulating total antioxidant capability, the activity of the enzymatic antioxidants, including glutathione peroxidase and catalase activity to keep the cellular redox environment. Additionally, quercetin had no effect on the level of glutathione, the main non-enzymatic antioxidant in embryos. PMID:24586844

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

    PubMed Central

    Yajima, Toshitaka

    2011-01-01

    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. PMID:21585262

  5. Cold stress aggravates inflammatory responses in an LPS-induced mouse model of acute lung injury

    NASA Astrophysics Data System (ADS)

    Joo, Su-Yeon; Park, Mi-Ju; Kim, Kyun-Ha; Choi, Hee-Jung; Chung, Tae-Wook; Kim, Yong Jin; Kim, Joung Hee; Kim, Keuk-Jun; Joo, Myungsoo; Ha, Ki-Tae

    2016-08-01

    Although the relationship between environmental cold temperature and susceptibility to respiratory infection is generally accepted, the effect of ambient cold temperature on host reactivity in lung inflammation has not been fully studied. To examine the function of ambient cold temperature on lung inflammation, mice were exposed to 4 °C for 8 h each day for 14 days. In the lungs of mice exposed to cold stress, inflammatory cells in bronchoalveolar lavage (BAL) fluid and lung tissues were slightly increased by about twofold. However, the structures of pulmonary epithelial cells were kept within normal limits. Next, we examined the effect of cold stress on the inflammatory responses in a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The infiltration of neutrophils and inflammation of lung tissue determined by histology were significantly increased by exposure to ambient cold temperature. In addition, the production of pro-inflammatory cytokines including interleukin (IL)-12, IL-17, and monokine induced by gamma interferon (MIG) was elevated by exposure to cold stress. Therefore, we suggest that cold stress is a factor that exacerbates lung inflammation including ALI. To our knowledge, this is the first report on the relationship between cold stress and severity of lung inflammation.

  6. Lipopolysaccharide-induced inflammation aggravates irradiation-induced injury to the young mouse brain.

    PubMed

    Roughton, Karolina; Andreasson, Ulf; Blomgren, Klas; Kalm, Marie

    2013-01-01

    Radiotherapy is an effective treatment strategy in the treatment of brain tumors, but it is also a major cause of long-term complications, especially in survivors of pediatric brain tumors. Cognitive decline caused by cranial radiotherapy is thought, at least partly, to depend on injury to stem and progenitor cells in the dentate gyrus of the hippocampus. This study investigated the effects of lipopolysaccharide (LPS)-induced inflammation at the time of irradiation (IR) in the growing mouse brain. A single injection of LPS (0.3 mg/kg) was administered 24 h prior to cranial IR of 14-day-old male mice. LPS pretreatment increased the levels of the chemokine CCL2 and the cytokine IL-1β in the brain by 440 and 560%, respectively, compared to IR alone. IR disrupted hippocampal neurogenesis and the growth of the dentate gyrus, and the mice pretreated with LPS displayed an even more pronounced lack of growth than the vehicle-treated group 2 months after IR. The density of microglia was not affected, but LPS-pretreated mice displayed 48% fewer bromodeoxyuridine-positive cells and 43% fewer doublecortin-positive cells in the granule cell layer 2 months after IR compared with the vehicle-treated group. In conclusion, an ongoing inflammation in the brain at the time of IR further enhanced the IR-induced loss of neurogenesis, and may aggravate future cognitive deficits in patients treated with cranial radiotherapy.

  7. Chitooligosaccharide Inhibits Scar Formation and Enhances Functional Recovery in a Mouse Model of Sciatic Nerve Injury.

    PubMed

    Hou, Hongping; Zhang, Lihai; Ye, Zuguang; Li, Jianrong; Lian, Zijian; Chen, Chao; He, Rong; Peng, Bo; Xu, Qihua; Zhang, Guangping; Gan, Wenbiao; Tang, Peifu

    2016-05-01

    Chitooligosaccharide (COS) has been shown to induce fibroblast apoptosis, indicating that it could be used as a material to inhibit scar formation. In the present study, we used a mouse model of sciatic nerve injury (SNI) to determine the role of COS in scar inhibition and functional recovery. The animals were divided into three groups: SNI, SNI + vehicle, and SNI + COS group. We performed a series of functional and histological examinations at ctrl, 0 min, 14 days, and 42 days, including behavioral recovery, percentage of regenerating axons, degree of scar formation, vascular changes, type I and type III collagen ratio, and percentage of demyelinated axons. The SNI + COS group exhibited better recovery of sensory and motor function and less scar formation. Two-photon microscopy showed that the percentage of regenerating axons was highest in the SNI + COS group at 14 and 42 days. Our results suggested that COS can inhibit scar formation and enhance functional recovery by inducing fibroblast death, altering the proportion of different vascular diameters, changing the ratio of type I/type III collagen, and reducing the percentage of demyelinated axons. COS might be a useful drug in the treatment of SNI to reduce scar formation, but additional research is required to clarify the relevant molecular pathways.

  8. Cardiomyocyte Hypertrophy in Arrhythmogenic Cardiomyopathy.

    PubMed

    Gerçek, Mustafa; Gerçek, Muhammed; Kant, Sebastian; Simsekyilmaz, Sakine; Kassner, Astrid; Milting, Hendrik; Liehn, Elisa A; Leube, Rudolf E; Krusche, Claudia A

    2017-04-01

    Arrhythmogenic cardiomyopathy (AC) is a hereditary disease leading to sudden cardiac death or heart failure. AC pathology is characterized by cardiomyocyte loss and replacement fibrosis. Our goal was to determine whether cardiomyocytes respond to AC progression by pathological hypertrophy. To this end, we examined tissue samples from AC patients with end-stage heart failure and tissue samples that were collected at different disease stages from desmoglein 2-mutant mice, a well characterized AC model. We find that cardiomyocyte diameters are significantly increased in right ventricles of AC patients. Increased mRNA expression of the cardiac stress marker natriuretic peptide B is also observed in the right ventricle of AC patients. Elevated myosin heavy chain 7 mRNA expression is detected in left ventricles. In desmoglein 2-mutant mice, cardiomyocyte diameters are normal during the concealed disease phase but increase significantly after acute disease onset on cardiomyocyte death and fibrotic myocardial remodeling. Hypertrophy progresses further during the chronic disease stage. In parallel, mRNA expression of myosin heavy chain 7 and natriuretic peptide B is up-regulated in both ventricles with right ventricular preference. Calcineurin/nuclear factor of activated T cells (Nfat) signaling, which is linked to pathological hypertrophy, is observed during AC progression, as evidenced by Nfatc2 and Nfatc3 mRNA in cardiomyocytes and increased mRNA of the Nfat target regulator of calcineurin 1. Taken together, we demonstrate that pathological hypertrophy occurs in AC and is secondary to cardiomyocyte loss and cardiac remodeling.

  9. A novel acute lethal liver injury mouse model with visualization of NF-κB activity for treatment of severe acute liver injury

    PubMed Central

    Liao, Huanjin; Cai, Jun; Zhang, Lifang; Peng, Yanxia; Wu, Ping; Xie, Tong; Pan, Qingjun

    2017-01-01

    Acute lethal inflammation, especially that related to liver injury, is an important clinical issue. To date, however, there is no model that can be used to assess this serious condition. This study was designed to establish a novel lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute lethal liver injury model in nuclear factor-κB (NF-κB) transgenic mice. The results show that a high dose of LPS (500 μg/kg) plus D-GalN (800 mg/kg) successfully established a novel mouse model of acute lethal liver injury with a lifespan of 8-10 h. Significantly increased NF-κB activity, detected with an in vivo imaging system (IVIS), peaked at approximately 4 h post-LPS/D-GalN challenge in NF-κB transgenic mice. Moreover, the serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1 were significantly increased and peaked at approximately 4 h post-i.p. injection of LPS/D-GalN. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) also sharply increased. Correlation analyses showed that NF-κB activity was significantly correlated with serum levels of ALT and AST. The mouse model livers showed marked congestion and hemorrhage, and hematoxylin and eosin (H&E) staining confirmed the destruction of the lobular structure and severe hepatocyte necrosis and hemorrhage. None of these changes were observed in the control mice. In summary, a novel LPS/D-GalN-induced acute lethal liver injury model with visualization of NF-κB activity was established in NF-κB transgenic mice. This model will provide the technology for developing new therapeutic strategies for treatment of severe acute liver injury complicated by endotoxemia or septicemia. PMID:28386325

  10. Microarray analysis of high-dose recombinant erythropoietin treatment of unilateral brain injury in neonatal mouse hippocampus.

    PubMed

    Juul, Sandra E; Beyer, Richard P; Bammler, Theo K; McPherson, Ronald J; Wilkerson, Jasmine; Farin, Federico M

    2009-05-01

    Recombinant human erythropoietin (rEpo) is neuroprotective in neonatal models of brain injury. Proposed mechanisms of neuroprotection include activation of gene pathways that decrease oxidative injury, inflammation, and apoptosis, while increasing vasculogenesis and neurogenesis. To determine the effects of rEpo on gene expression in 10-d-old BALB-c mice with unilateral brain injury, we compared microarrays from the hippocampi of brain-injured pups treated with saline or rEpo to similarly treated sham animals. Total RNA was extracted 24 h after brain injury and analyzed using Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. We identified sex-specific differences in hippocampal gene expression after brain injury and after high-dose rEpo treatment using single-gene and gene set analysis. Although high-dose rEpo had minimal effects on hippocampal gene expression in shams, at 24-h post brain injury, high-dose rEpo treatment significantly decreased the proinflammatory and antiapoptotic response noted in saline-treated brain-injured comparison animals.

  11. Burn Injury Leads to Increased Long-Term Susceptibility to Respiratory Infection in both Mouse Models and Population Studies

    PubMed Central

    Fear, Vanessa S.; Boyd, James H.; Rea, Suzanne; Wood, Fiona M.

    2017-01-01

    Background Burn injury initiates an acute inflammatory response that subsequently drives wound repair. However, acute disruption to the immune response is also common, leading to susceptibility to sepsis and increased morbidity and mortality. Despite increased understanding of the impact of burn injury on the immune system in the acute phase, little is known about long-term consequences of burn injury on immune function. This study was established to determine whether burn injury has long-term clinical impacts on patients’ immune responses. Methods Using a population-based retrospective longitudinal study and linked hospital morbidity and death data from Western Australia, comparative rates of hospitalisation for respiratory infections in burn patients and a non-injured comparator cohort were assessed. In addition, a mouse model of non-severe burn injury was also used in which viral respiratory infection was induced at 4 weeks post-injury using a mouse modified version of the Influenza A virus (H3NN; A/mem/71-a). Results and conclusions The burn injured cohort contained 14893 adult patients from 1980–2012 after removal of those patients with evidence of smoke inhalation or injury to the respiratory tract. During the study follow-up study a total of 2,884 and 2,625 respiratory infection hospital admissions for the burn and uninjured cohorts, respectively, were identified. After adjusting for covariates, the burn cohort experienced significantly elevated admission rates for influenza and viral pneumonia (IRR, 95%CI: 1.73, 1.27–2.36), bacterial pneumonia (IRR, 95%CI: 2.05, 1.85–2.27) and for other types of upper and lower respiratory infections (IRR, 95% CI: 2.38, 2.09–2.71). In the mouse study an increased viral titre was observed after burn injury, accompanied by a reduced CD8 response and increased NK and NKT cells in the draining lymph nodes. This data suggests burn patients are at long-term increased risk of infection due to sustained modulation of the

  12. Cardiomyocyte proliferation in cardiac development and regeneration: a guide to methodologies and interpretations.

    PubMed

    Leone, Marina; Magadum, Ajit; Engel, Felix B

    2015-10-01

    The newt and the zebrafish have the ability to regenerate many of their tissues and organs including the heart. Thus, a major goal in experimental medicine is to elucidate the molecular mechanisms underlying the regenerative capacity of these species. A wide variety of experiments have demonstrated that naturally occurring heart regeneration relies on cardiomyocyte proliferation. Thus, major efforts have been invested to induce proliferation of mammalian cardiomyocytes in order to improve cardiac function after injury or to protect the heart from further functional deterioration. In this review, we describe and analyze methods currently used to evaluate cardiomyocyte proliferation. In addition, we summarize the literature on naturally occurring heart regeneration. Our analysis highlights that newt and zebrafish heart regeneration relies on factors that are also utilized in cardiomyocyte proliferation during mammalian fetal development. Most of these factors have, however, failed to induce adult mammalian cardiomyocyte proliferation. Finally, our analysis of mammalian neonatal heart regeneration indicates experiments that could resolve conflicting results in the literature, such as binucleation assays and clonal analysis. Collectively, cardiac regeneration based on cardiomyocyte proliferation is a promising approach for improving adult human cardiac function after injury, but it is important to elucidate the mechanisms arresting mammalian cardiomyocyte proliferation after birth and to utilize better assays to determine formation of new muscle mass.

  13. Comparative transcriptome profiling of the injured zebrafish and mouse hearts identifies miRNA-dependent repair pathways

    PubMed Central

    Crippa, Stefania; Nemir, Mohamed; Ounzain, Samir; Ibberson, Mark; Berthonneche, Corinne; Sarre, Alexandre; Boisset, Gaëlle; Maison, Damien; Harshman, Keith; Xenarios, Ioannis; Diviani, Dario; Schorderet, Daniel; Pedrazzini, Thierry

    2016-01-01

    Aims The adult mammalian heart has poor regenerative capacity. In contrast, the zebrafish heart retains a robust capacity for regeneration into adulthood. These distinct responses are consequences of a differential utilization of evolutionary-conserved gene regulatory networks in the damaged heart. To systematically identify miRNA-dependent networks controlling cardiac repair following injury, we performed comparative gene and miRNA profiling of the cardiac transcriptome in adult mice and zebrafish. Methods and results Using an integrated approach, we show that 45 miRNA-dependent networks, involved in critical biological pathways, are differentially modulated in the injured zebrafish vs. mouse hearts. We study, more particularly, the miR-26a-dependent response. Therefore, miR-26a is down-regulated in the fish heart after injury, whereas its expression remains constant in the mouse heart. Targets of miR-26a involve activators of the cell cycle and Ezh2, a component of the polycomb repressive complex 2 (PRC2). Importantly, PRC2 exerts repressive functions on negative regulators of the cell cycle. In cultured neonatal cardiomyocytes, inhibition of miR-26a stimulates, therefore, cardiomyocyte proliferation. Accordingly, miR-26a knockdown prolongs the proliferative window of cardiomyocytes in the post-natal mouse heart. Conclusions This novel strategy identifies a series of miRNAs and associated pathways, in particular miR-26a, which represent attractive therapeutic targets for inducing repair in the injured heart. PMID:26857418

  14. Alterations of lung microbiota in a mouse model of LPS-induced lung injury

    PubMed Central

    Meng, Fanyong; Meliton, Angelo; Afonyushkin, Taras; Ulanov, Alexander; Semenyuk, Ekaterina; Latif, Omar; Tesic, Vera; Birukova, Anna A.; Birukov, Konstantin G.

    2015-01-01

    Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3–V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents. PMID:25957290

  15. Alterations of lung microbiota in a mouse model of LPS-induced lung injury.

    PubMed

    Poroyko, Valeriy; Meng, Fanyong; Meliton, Angelo; Afonyushkin, Taras; Ulanov, Alexander; Semenyuk, Ekaterina; Latif, Omar; Tesic, Vera; Birukova, Anna A; Birukov, Konstantin G

    2015-07-01

    Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents.

  16. Intra-articular injection of synovial mesenchymal stem cells improves cartilage repair in a mouse injury model

    PubMed Central

    Mak, J.; Jablonski, C. L.; Leonard, C. A.; Dunn, J. F.; Raharjo, E.; Matyas, J. R.; Biernaskie, J.; Krawetz, R. J.

    2016-01-01

    Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ “super-healer” strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not. PMID:26983696

  17. Experimental traumatic brain injury induces rapid aggregation and oligomerization of amyloid-beta in an Alzheimer's disease mouse model.

    PubMed

    Washington, Patricia M; Morffy, Nicholas; Parsadanian, Maia; Zapple, David N; Burns, Mark P

    2014-01-01

    Soluble amyloid-beta (Aβ) oligomers are hypothesized to be the pathogenic species in Alzheimer's disease (AD), and increased levels of oligomers in the brain subsequent to traumatic brain injury (TBI) may exacerbate secondary injury pathways and underlie increased risk of developing AD in later life. To determine whether TBI causes Aβ aggregation and oligomerization in the brain, we exposed triple transgenic AD model mice to controlled cortical impact injury and measured levels of soluble, insoluble, and oligomeric Aβ by enzyme-linked immunosorbent assay (ELISA) at 1, 3, and 7 days postinjury. TBI rapidly increased levels of both soluble and insoluble Aβ40 and Aβ42 in the injured cortex at 1 day postinjury. We confirmed previous findings that identified damaged axons as a major site of Aβ accumulation using both immunohistochemistry and biochemistry. We also report that soluble Aβ oligomers were significantly increased in the injured cortex, as demonstrated by both ELISA and Western blot. Interestingly, the mouse brain is able to rapidly clear trauma-induced Aβ, with both soluble and insoluble Aβ species returning to sham levels by 7 days postinjury. In conclusion, we demonstrate that TBI causes acute accumulation and aggregation of Aβ in the brain, including the formation of low- and high-molecular-weight Aβ oligomers. The formation and aggregation of Aβ into toxic species acutely after injury may play a role in secondary injury cascades after trauma and, chronically, may contribute to increased risk of developing AD in later life.

  18. Intra-articular injection of synovial mesenchymal stem cells improves cartilage repair in a mouse injury model.

    PubMed

    Mak, J; Jablonski, C L; Leonard, C A; Dunn, J F; Raharjo, E; Matyas, J R; Biernaskie, J; Krawetz, R J

    2016-03-17

    Controversy remains whether articular cartilage has an endogenous stem/progenitor cell population, since its poor healing capacity after injury can lead to diseases such as osteoarthritis. In the joint environment there are mesenchymal stem/progenitor cells (MSCs) in the synovial membrane and synovial fluid that can differentiate into cartilage, but it is still under debate if these cells contribute to cartilage repair in vivo. In this study, we isolated a Sca-1 positive, chondrogenesis capable population of mouse synovial MSCs from C57BL6 and MRL/MpJ "super-healer" strains. Intra-articular injection of Sca-1 + GFP + synovial cells from C57BL6 or MRL/MpJ into C57BL6 mice following cartilage injury led to increased cartilage repair by 4 weeks after injury. GFP expression was detected in the injury site at 2 weeks, but not 4 weeks after injury. These results suggest that synovial stem/progenitor cells, regardless of strain background, have beneficial effects when injected into an injured joint. MSCs derived from MRL/MpJ mice did not promote an increased repair capacity compared to MSCs derived from non-healing C57BL6 controls; however, MRL/MpJ MSCs were observed within the defect area at the time points examined, while C57BL6 MSCs were not.

  19. Mouse model of liver ischemia and reperfusion injury: method for studying reactive oxygen and nitrogen metabolites in vivo.

    PubMed

    Abe, Yuta; Hines, Ian N; Zibari, Gazi; Pavlick, Kevin; Gray, Laura; Kitagawa, Yuko; Grisham, Matthew B

    2009-01-01

    The mouse model of liver ischemia and reperfusion injury has proven to be valuable for our understanding of the role that reactive oxygen and nitrogen metabolites play in postischemic tissue injury. This methods paper provides a detailed protocol for inducing partial liver ischemia followed by reperfusion. Liver ischemia is induced in anesthetized mice by cross-clamping the hepatic artery and portal vein for varying lengths of time, resulting in deprivation of blood flow to approximately 70% of the liver. Restoration of blood flow to the ischemic lobes enhances superoxide production concomitant with a rapid and marked decrease in the bioavailability of nitric oxide, resulting in alterations in the redox state of the liver in favor of a more oxidative environment. This hepatocellular oxidative stress induces the activation of oxidant-sensitive transcription factors followed by the upregulation of proinflammatory cytokines and mediators that ultimately lead to liver injury. This model can be induced in any strain or sex of mouse and requires 1-2 months of practice to become proficient in the surgery and animal manipulation. The roles of various reactive metabolites of oxygen and nitrogen may be evaluated using genetically engineered mice as well as selective molecular, cellular, and/or pharmacological agents.

  20. 17β-Estradiol Promotes Schwann Cell Proliferation and Differentiation, Accelerating Early Remyelination in a Mouse Peripheral Nerve Injury Model

    PubMed Central

    Chen, Yan; Guo, Wenjie; Li, Wenjuan; Cheng, Meng; Hu, Ying; Xu, Wenming

    2016-01-01

    Estrogen induces oligodendrocyte remyelination in response to demyelination in the central nervous system. Our objective was to determine the effects of 17β-estradiol (E2) on Schwann cell function and peripheral nerve remyelination after injury. Adult male C57BL/6J mice were used to prepare the sciatic nerve transection injury model and were randomly categorized into control and E2 groups. To study myelination in vitro, dorsal root ganglion (DRG) explant culture was prepared using 13.5-day-old mouse embryos. Primary Schwann cells were isolated from the sciatic nerves of 1- to 3-day-old Sprague–Dawley rats. Immunostaining for myelin basic protein (MBP) expression and toluidine blue staining for myelin sheaths demonstrated that E2 treatment accelerates early remyelination in the “nerve bridge” region between the proximal and distal stumps of the transection injury site in the mouse sciatic nerve. The 5-bromo-2′-deoxyuridine incorporation assay revealed that E2 promotes Schwann cell proliferation in the bridge region and in the primary culture, which is blocked using AKT inhibitor MK2206. The in vitro myelination in the DRG explant culture determined showed that the MBP expression in the E2-treated group is higher than that in the control group. These results show that E2 promotes Schwann cell proliferation and myelination depending on AKT activation. PMID:27872858

  1. Lasting retinal injury in a mouse model of blast-induced trauma

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Traumatic brain injury (TBI) due to blast exposure is currently the most prevalent of war injuries. While secondary ocular blast injuries due to flying debris are more common, primary ocular blast exposure has been reported among survivors of explosions, but with limited understanding of the resulti...

  2. Berberine Inhibits Doxorubicin-Triggered Cardiomyocyte Apoptosis via Attenuating Mitochondrial Dysfunction and Increasing Bcl-2 Expression

    PubMed Central

    Lv, Xiuxiu; Yu, Xiaohui; Wang, Yiyang; Wang, Faqiang; Li, Hongmei; Wang, Yanping; Lu, Daxiang; Qi, Renbin; Wang, Huadong

    2012-01-01

    Cardiomyocyte apoptosis is an important event in doxorubicin (DOX)-induced cardiac injury. The aim of the present study was to investigate the protection of berberine (Ber) against DOX- triggered cardiomyocyte apoptosis in neonatal rat cardiomyocytes and rats. In neonatal rat cardiomyocytes, Ber attenuated DOX-induced cellular injury and apoptosis in a dose-dependent manner. However, Ber has no significant effect on viability of MCF-7 breast cancer cells treated with DOX. Ber reduced caspase-3 and caspase-9, but not caspase-8 activity in DOX-treated cardiomyocytes. Furthermore, Ber decreased adenosine monophosphate-activated protein kinase α (AMPKα) and p53 phosphorylation at 2 h, cytosolic cytochrome c and mitochondrial Bax levels and increased Bcl-2 level at 6 h in DOX-stimulated cardiomyocytes. Pretreatment with compound C, an AMPK inhibitor, also suppressed p53 phosphorylation and apoptosis in DOX-treated cardiomyocytes. DOX stimulation for 30 min led to a loss of mitochondrial membrane potential and a rise in the AMP/ATP ratio. Ber markedly reduced DOX-induced mitochondrial membrane potential loss and an increase in the AMP/ATP ratio at 1 h and 2 h post DOX exposure. In in vivo experiments, Ber significantly improved survival, increased stroke volume and attenuated myocardial injury in DOX-challenged rats. TUNEL and Western blot assays showed that Ber not only decreased myocardial apoptosis, caspase-3 activation, AMPKα and p53 phosphorylation, but also increased Bcl-2 expression in myocardium of rats exposed to DOX for 84 h. These findings indicate that Ber attenuates DOX-induced cardiomyocyte apoptosis via protecting mitochondria, inhibiting an increase in the AMP/ATP ratio and AMPKα phosphorylation as well as elevating Bcl-2 expression, which offer a novel mechanism responsible for protection of Ber against DOX-induced cardiomyopathy. PMID:23077597

  3. Local Ca2+ releases enable rapid heart rates in developing cardiomyocytes

    PubMed Central

    Korhonen, Topi; Rapila, Risto; Ronkainen, Veli-Pekka; Koivumäki, Jussi T; Tavi, Pasi

    2010-01-01

    The ability to generate homogeneous intracellular Ca2+ oscillations at high frequency is the basis of the rhythmic contractions of mammalian cardiac myocytes. While the specific mechanisms and structures enabling homogeneous high-frequency Ca2+ signals in adult cardiomyocytes are well characterized, it is not known how these kind of Ca2+ signals are produced in developing cardiomyocytes. Here we investigated the mechanisms reducing spatial and temporal heterogeneity of cytosolic Ca2+ signals in mouse embryonic ventricular cardiomyocytes. We show that in developing cardiomyocytes the propagating Ca2+ signals are amplified in cytosol by local Ca2+ releases. Local releases are based on regular 3-D sarcoplasmic reticulum (SR) structures containing SR Ca2+ uptake ATPases (SERCA) and Ca2+ release channels (ryanodine receptors, RyRs) at regular intervals throughout the cytosol. By evoking [Ca2+]i-induced Ca2+ sparks, the local release sites promote a 3-fold increase in the cytosolic Ca2+ propagation speed. We further demonstrate by mathematical modelling that without these local release sites the developing cardiomyocytes lose their ability to generate homogeneous global Ca2+ signals at a sufficiently high frequency. The mechanism described here is robust and indispensable for normal mammalian cardiomyocyte function from the first heartbeats during the early embryonic phase till terminal differentiation after birth. These results suggest that local cytosolic Ca2+ releases are indispensable for normal cardiomyocyte development and function of developing heart. PMID:20211983

  4. Local Ca2+ releases enable rapid heart rates in developing cardiomyocytes.

    PubMed

    Korhonen, Topi; Rapila, Risto; Ronkainen, Veli-Pekka; Koivumäki, Jussi T; Tavi, Pasi

    2010-05-01

    The ability to generate homogeneous intracellular Ca(2+) oscillations at high frequency is the basis of the rhythmic contractions of mammalian cardiac myocytes. While the specific mechanisms and structures enabling homogeneous high-frequency Ca(2+) signals in adult cardiomyocytes are well characterized, it is not known how these kind of Ca(2+) signals are produced in developing cardiomyocytes. Here we investigated the mechanisms reducing spatial and temporal heterogeneity of cytosolic Ca(2+) signals in mouse embryonic ventricular cardiomyocytes. We show that in developing cardiomyocytes the propagating Ca(2+) signals are amplified in cytosol by local Ca(2+) releases. Local releases are based on regular 3-D sarcoplasmic reticulum (SR) structures containing SR Ca(2+) uptake ATPases (SERCA) and Ca(2+) release channels (ryanodine receptors, RyRs) at regular intervals throughout the cytosol. By evoking [Ca(2+)](i)-induced Ca(2+) sparks, the local release sites promote a 3-fold increase in the cytosolic Ca(2+) propagation speed. We further demonstrate by mathematical modelling that without these local release sites the developing cardiomyocytes lose their ability to generate homogeneous global Ca(2+) signals at a sufficiently high frequency. The mechanism described here is robust and indispensable for normal mammalian cardiomyocyte function from the first heartbeats during the early embryonic phase till terminal differentiation after birth. These results suggest that local cytosolic Ca(2+) releases are indispensable for normal cardiomyocyte development and function of developing heart.

  5. Comparison Analysis of Dysregulated LncRNA Profile in Mouse Plasma and Liver after Hepatic Ischemia/Reperfusion Injury.

    PubMed

    Chen, Zhenzhen; Luo, Yanjin; Yang, Weili; Ding, Liwei; Wang, Junpei; Tu, Jian; Geng, Bin; Cui, Qinghua; Yang, Jichun

    2015-01-01

    Long noncoding RNAs (LncRNAs) have been believed to be the major transcripts in various tissues and organs, and may play important roles in regulation of many biological processes. The current study determined the LncRNA profile in mouse plasma after liver ischemia/reperfusion injury (IRI) using microarray technology. Microarray assays revealed that 64 LncRNAs were upregulated, and 244 LncRNAs were downregulated in the plasma of liver IRI mouse. Among these dysregulated plasma LncRNAs, 59-61% were intergenic, 22-25% were antisense overlap, 8-12% were sense overlap and 6-7% were bidirectional. Ten dysregulated plasma LncRNAs were validated by quantitative PCR assays, confirming the accuracy of microarray analysis result. Comparison analysis between dysregulated plasma and liver LncRNA profile after liver IRI revealed that among the 308 dysregulated plasma LncRNAs, 245 LncRNAs were present in the liver, but remained unchanged. In contrast, among the 98 dysregulated liver LncRNAs after IRI, only 19 were present in the plasma, but remained unchanged. LncRNA AK139328 had been previously reported to be upregulated in the liver after IRI, and silencing of hepatic AK139328 ameliorated liver IRI. Both microarray and RT-PCR analyses failed to detect the presence of AK139328 in mouse plasma. In summary, the current study compared the difference between dysregulated LncRNA profile in mouse plasma and liver after liver IRI, and suggested that a group of dysregulated plasma LncRNAs have the potential of becoming novel biomarkers for evaluation of ischemic liver injury.

  6. Unfractionated heparin attenuates intestinal injury in mouse model of sepsis by inhibiting heparanase

    PubMed Central

    Chen, Song; Zhang, Xiaojuan; Sun, Yini; Hu, Ziwei; Lu, Siyu; Ma, Xiaochun

    2015-01-01

    Intestinal injury is a key feature in sepsis. Heparanase, a heparin sulfate-specific glucuronidase, mediates the onset of organ injury during early sepsis. Heparin has the function to attenuate inflammation and injury induced by multiple factors; however, whether unfractionated heparin (UFH) can attenuate the intestinal injury induced by sepsis as well as the underlying mechanism is still unknown. In the present study, the function of UFH in intestinal injury induced by sepsis was explored. Results of our study showed that after CLP operation, the inflammatory response and expression of heparanase were increased and NF-κB and MAPK P38 signaling pathways were activated. However, pretreatment with UFH will inhibit the expression and activation of heparanase, and reverse the activation of NF-κB and MAPK P38 signaling pathways, thus attenuating inflammatory responses induced by sepsis. These results suggest that UFH may be a promising therapeutic drug for intestinal injury caused by sepsis. PMID:26191183

  7. The antifibrinolytic drug tranexamic acid reduces liver injury and fibrosis in a mouse model of chronic bile duct injury.

    PubMed

    Joshi, Nikita; Kopec, Anna K; Towery, Keara; Williams, Kurt J; Luyendyk, James P

    2014-06-01

    Hepatic fibrin deposition has been shown to inhibit hepatocellular injury in mice exposed to the bile duct toxicant α-naphthylisothiocyanate (ANIT). Degradation of fibrin clots by fibrinolysis controls the duration and extent of tissue fibrin deposition. Thus, we sought to determine the effect of treatment with the antifibrinolytic drug tranexamic acid (TA) and plasminogen activator inhibitor-1 (PAI-1) deficiency on ANIT-induced liver injury and fibrosis in mice. Plasmin-dependent lysis of fibrin clots was impaired in plasma from mice treated with TA (1200 mg/kg i.p., administered twice daily). Prophylactic TA administration reduced hepatic inflammation and hepatocellular necrosis in mice fed a diet containing 0.025% ANIT for 2 weeks. Hepatic type 1 collagen mRNA expression and deposition increased markedly in livers of mice fed ANIT diet for 4 weeks. To determine whether TA treatment could inhibit this progression of liver fibrosis, mice were fed ANIT diet for 4 weeks and treated with TA for the last 2 weeks. Interestingly, TA treatment largely prevented increased deposition of type 1 collagen in livers of mice fed ANIT diet for 4 weeks. In contrast, biliary hyperplasia/inflammation and liver fibrosis were significantly increased in PAI-1(-/-) mice fed ANIT diet for 4 weeks. Overall, the results indicate that fibrinolytic activity contributes to ANIT diet-induced liver injury and fibrosis in mice. In addition, these proof-of-principle studies suggest the possibility that therapeutic intervention with an antifibrinolytic drug could form a novel strategy to prevent or reduce liver injury and fibrosis in patients with liver disease.

  8. 64CuCl2 PET/CT imaging of mouse muscular injury induced by electroporation

    PubMed Central

    Xie, Fang; Cai, Huawei; Peng, Fangyu

    2017-01-01

    Skeletal muscle injury is common in body injuries suffered in sports and car accidents. Development of new tracers is significant for assessing muscular injury with positron emission tomography/computed tomography (PET/CT) and monitoring repair of muscle injury in response to treatment. Copper is required for wound healing and increased copper ions were detected in the soft tissue of wound in rodents and human. Based on the recent finding of increased 64Cu uptake in the traumatic brain injury, this study aimed to explore use of 64CuCl2 as a radiotracer for molecular imaging of muscular injury using PET/CT. Focally increased 64Cu uptake by the injured muscular tissue (5.4 ± 1.2% ID/g) was detected in the C57BL/6 mice with electroporation-induced skeletal muscle injury by PET/CT after intravenous injection of 64CuCl2 as a tracer, compared to low 64Cu uptake associated with muscular inflammation induced by intramuscular injection of lipopolysaccharides (0.82 ± 0.26% ID/g, P < 0.01) or physiological 64Cu uptake of the non-injured muscular tissues (0.78 ± 0.20% ID/g, P < 0.01). The findings support further investigation of 64CuCl2 as a new radiotracer for molecular imaging of skeletal muscle injury using PET/CT. PMID:28123866

  9. Lens injury stimulates adult mouse retinal ganglion cell axon regeneration via both macrophage- and lens-derived factors.

    PubMed

    Lorber, Barbara; Berry, Martin; Logan, Ann

    2005-04-01

    In the present study the effects of lens injury on retinal ganglion cell axon/neurite re-growth were investigated in adult mice. In vivo, lens injury promoted successful regeneration of retinal ganglion cell axons past the optic nerve lesion site, concomitant with the invasion of macrophages into the eye and the presence of activated retinal astrocytes/Muller cells. In vitro, retinal ganglion cells from lens-lesioned mice grew significantly longer neurites than those from intact mice, which correlated with the presence of enhanced numbers of activated retinal astrocytes/Muller cells. Co-culture of retinal ganglion cells from intact mice with macrophage-rich lesioned lens/vitreous body led to increased neurite lengths compared with co-culture with macrophage-free intact lens/vitreous body, pointing to a neurotrophic effect of macrophages. Furthermore, retinal ganglion cells from mice that had no lens injury but had received intravitreal Zymosan injections to stimulate macrophage invasion into the eye grew significantly longer neurites compared with controls, as did retinal ganglion cells from intact mice co-cultured with macrophage-rich vitreous body from Zymosan-treated mice. The intact lens, but not the intact vitreous body, exerted a neurotrophic effect on retinal ganglion cell neurite outgrowth, suggesting that lens-derived neurotrophic factor(s) conspire with those derived from macrophages in lens injury-stimulated axon regeneration. Together, these results show that lens injury promotes retinal ganglion cell axon regeneration/neurite outgrowth in adult mice, an observation with important implications for axon regeneration studies in transgenic mouse models.

  10. Heme oxygenase-1 deficiency promotes the development of necrotizing enterocolitis-like intestinal injury in a newborn mouse model.

    PubMed

    Schulz, Stephanie; Wong, Ronald J; Jang, Kyu Yun; Kalish, Flora; Chisholm, Karen M; Zhao, Hui; Vreman, Hendrik J; Sylvester, Karl G; Stevenson, David K

    2013-06-01

    Necrotizing enterocolitis (NEC) is typified by mucosal destruction, which subsequently can lead to intestinal necrosis. Prematurity, enteral feeding, and bacterial colonization are the main risk factors and, combined with other stressors, can cause increased intestinal permeability, injury, and an exaggerated inflammatory response. Heme oxygenase-1 (HO-1) mediates intestinal protection due to anti-inflammatory, antioxidative, and antiapoptotic effects of its products carbon monoxide, biliverdin, and bilirubin. This study investigates a possible role of HO-1 in the pathogenesis of NEC using a newborn mouse model. We induced NEC-like intestinal injury in 7-day-old HO-1 heterozygous (HO-1 Het, Hmox1(+/-)) and wild-type (Wt, Hmox1(+/+)) mice by gavage feeding and hypoxic exposures. Control (Con) pups of both genotypes were dam-fed. Intestines of HO-1 Het Con pups appeared predisposed to injury, with higher histological damage scores, more TUNEL-positive cells, and a significant reduction in muscularis externa thickness compared with Wt Con pups. The increase in HO activity after HO-1 induction by the substrate heme or by hypoxic stress was significantly impaired in HO-1 Het pups. After induction of intestinal injury, HO-1 Het pups displayed significantly higher NEC incidence (78 vs. 43%), mortality (83 vs. 54%), and median scores (2.5 vs. 1.5) than Wt NEC pups. PCR array analyses revealed increased expressions of IL-1β, P-selectin, matrix metallopeptidase 2, collagen type XVIII-α1, serpine 1, and others in NEC-induced HO-1 Het ileal and jejunal tissues. We conclude that a partial HO-1 deficiency promotes experimental NEC-like intestinal injury, possibly mediated by exaggerated inflammation and disruption in tissue repair.

  11. Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

    PubMed Central

    Evans, Teresa A.; Barkauskas, Deborah S.; Myers, Jay T.; Huang, Alex Y.

    2014-01-01

    Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury. PMID:25489963

  12. The in-vivo monitoring method for traumatic brain injury of mouse based on near-infrared light intensity

    NASA Astrophysics Data System (ADS)

    Li, Weitao; Wang, Xuena; Qian, Zhiyu; Xie, Jieru; Liu, Xing

    2012-02-01

    A system based on near-infrared light intensity was used to monitor mouse model of traumatic brain injury (TBI) noninvasively. The measurement system was controlled by microcontroller. Light from a 760/850nm dual-wavelength light emitting diode was coupled to a 0.6-mm-diameter optical fiber. The collection fibers were coupled to optoelectronic detectors, which were placed in the different distance from the source fiber. The system consisted of a constant current bias, a circuit lock-in amplifier (including band pass filter, lock-in amplifier, and low pass filter), a PCI 6240 data acquisition card and a multi-core-processor computer. The modified Lambert Beer law was used to calculate the concentration of ΔHbO2 and ΔHb. The sensitivity matrix was defined to evaluate the region of effective detection of optical probe. Five groups of TBI mouse models were built by Feeney's free-falling method. The data measured by system show after TBI the concentration of ΔHbO2 decreased and that of ΔHb increased. It can be concluded that the system can be used to monitor the changes of TBI of mouse non-invasively.

  13. Injury effects of ginkgolide B on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo.

    PubMed

    Shiao, Nion-Heng; Chan, Wen-Hsiung

    2009-07-10

    Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1-6 microM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3-6 microM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.

  14. The myelopoietic effects of a Serratia marcescens-derived biologic response modifier in a mouse model of thermal injury.

    PubMed

    Peterson, V M; Rundus, C H; Reinoehl, P J; Schroeter, S R; McCall, C A; Bartle, E J

    1992-04-01

    The proliferative defects observed in phagocytic stem cells after major thermal injuries may be caused by an inadequate production of colony-stimulating factors (CSFs), a family of hemopoietic cytokines necessary for the production and function of granulocytes and monocytes. In this study a biologic response modifier (S-BRM) consisting of sized vesicles derived from the cell membrane and ribosomes of Serratia marcescens was investigated in a mouse model of thermal injury to determine its ability to augment postburn myelopoiesis. Treatment of burned mice with S-BRM was well tolerated and was associated with statistically significant increases in absolute numbers of circulating granulocytes and monocytes compared with burned mice receiving saline solution. In addition, the size of the splenic myeloid stem cell compartment, as measured by granulocyte-macrophage stem cell colony formation in soft agar, was markedly expanded. Finally, plasma levels of CSF were increased significantly in burned mice receiving S-BRM but were not elevated in burned littermates treated with saline solution. These data suggest that production of CSF is suboptimal after thermal injury and S-BRM is capable of up-regulating postburn myelopoiesis by causing the release of CSF into the systemic circulation.

  15. Inhibitory effects of prior low-dose x-irradiation on ischemia-reperfusion injury in mouse paw.

    PubMed

    Kataoka, Takahiro; Mizuguchi, Yuko; Yoshimoto, Masaaki; Taguchi, Takehito; Yamaoka, Kiyonori

    2007-11-01

    We have reported that low-dose, unlike high-dose, irradiation enhanced antioxidation function and reduced oxidative damage. On the other hand, ischemia-reperfusion injury is induced by reactive oxygen species. In this study, we examined the inhibitory effects of prior low-dose X-irradiation on ischemia-reperfusion injury in mouse paw. BALB/c mice were irradiated by sham or 0.5 Gy of X-ray. At 4 hrs after irradiation, the left hind leg was bound 10 times with a rubber ring for 0.5, 1, or 2 hrs and the paw thickness was measured. Results show that the paw swelling thickness by ischemia for 0.5 hr was lower than that for 2 hrs. At 1 hr after reperfusion from ischemia for 1 hr, superoxide dismutase activity in serum was increased in those mice which received 0.5 Gy irradiation and in the case of the ischemia for 0.5 or 1 hr, the paw swelling thicknesses were inhibited by 0.5 Gy irradiation. In addition, interstitial edema in those mice which received 0.5 Gy irradiation was less than that in the mice which underwent by sham irradiation. These findings suggest that the ischemia-reperfusion injury is inhibited by the enhancement of antioxidation function by 0.5 Gy irradiation.

  16. PD-L1 Blockade Attenuated Sepsis-Induced Liver Injury in a Mouse Cecal Ligation and Puncture Model

    PubMed Central

    Bao, Rui; Zhu, Jiali; Wang, Jiafeng; Li, Jinbao

    2013-01-01

    Liver plays a major role in hypermetabolism and produces acute phase proteins during systemic inflammatory response syndrome and it is of vital importance in host defense and bacteria clearance. Our previous studies indicated that programmed death-1 (PD-1) and its ligand programmed death ligand-1 (PD-L1) are crucial modulators of host immune responses during sepsis. Our current study was designed to investigate the role of PD-L1 in sepsis-induced liver injury by a mouse cecal ligation and puncture (CLP) model. Our results indicated that there was a significant increase of PD-L1 expression in liver after CLP challenge compared to sham-operated controls, in terms of levels of mRNA transcription and immunohistochemistry. Anti-PD-L1 antibody significantly alleviated the morphology of liver injury in CLP mice. Anti-PD-L1 antibody administration decreased ALT and AST release in CLP mice, decreased the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 mRNA in liver after sepsis challenge. Thus, anti-PD-L1 antibody might have a therapeutic potential in attenuating liver injury in sepsis. PMID:24324295

  17. In vivo photoacoustic tomography of mouse cerebral edema induced by cold injury

    NASA Astrophysics Data System (ADS)

    Xu, Zhun; Zhu, Quing; Wang, Lihong V.

    2011-06-01

    For the first time, we have implemented photoacoustic tomography (PAT) to image the water content of an edema in vivo. We produced and imaged a cold-induced cerebral edema transcranially, then obtained blood vessel and water accumulation images at 610 and 975 nm, respectively. We tracked the changes at 12, 24, and 36 h after the cold injury. The blood volume decreased after the cold injury, and the maximum area of edema was observed 24 h after the cold injury. We validated PAT of the water content of the edema through magnetic Resonance Imaging and the water spectrum from the spectrophotometric measurement.

  18. A New Transgenic Mouse Model for Studying the Neurotoxicity of Spermine Oxidase Dosage in the Response to Excitotoxic Injury

    PubMed Central

    Cervelli, Manuela; Bellavia, Gabriella; D'Amelio, Marcello; Cavallucci, Virve; Moreno, Sandra; Berger, Joachim; Nardacci, Roberta; Marcoli, Manuela; Maura, Guido; Piacentini, Mauro; Amendola, Roberto; Cecconi, Francesco; Mariottini, Paolo

    2013-01-01

    Spermine oxidase is a FAD-containing enzyme involved in polyamines catabolism, selectively oxidizing spermine to produce H2O2, spermidine, and 3-aminopropanal. Spermine oxidase is highly expressed in the mouse brain and plays a key role in regulating the levels of spermine, which is involved in protein synthesis, cell division and cell growth. Spermine is normally released by neurons at synaptic sites where it exerts a neuromodulatory function, by specifically interacting with different types of ion channels, and with ionotropic glutamate receptors. In order to get an insight into the neurobiological roles of spermine oxidase and spermine, we have deregulated spermine oxidase gene expression producing and characterizing the transgenic mouse model JoSMOrec, conditionally overexpressing the enzyme in the neocortex. We have investigated the effects of spermine oxidase overexpression in the mouse neocortex by transcript accumulation, immunohistochemical analysis, enzymatic assays and polyamine content in young and aged animals. Transgenic JoSMOrec mice showed in the neocortex a higher H2O2 production in respect to Wild-Type controls, indicating an increase of oxidative stress due to SMO overexpression. Moreover, the response of transgenic mice to excitotoxic brain injury, induced by kainic acid injection, was evaluated by analysing the behavioural phenotype, the immunodistribution of neural cell populations, and the ultrastructural features of neocortical neurons. Spermine oxidase overexpression and the consequently altered polyamine levels in the neocortex affects the cytoarchitecture in the adult and aging brain, as well as after neurotoxic insult. It resulted that the transgenic JoSMOrec mouse line is more sensitive to KA than Wild-Type mice, indicating an important role of spermine oxidase during excitotoxicity. These results provide novel evidences of the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain. PMID

  19. Cardiomyocyte-specific ablation of CD36 improves post-ischemic functional recovery.

    PubMed

    Nagendran, Jeevan; Pulinilkunnil, Thomas; Kienesberger, Petra C; Sung, Miranda M; Fung, David; Febbraio, Maria; Dyck, Jason R B

    2013-10-01

    Although pre-clinical evidence has suggested that partial inhibition of myocardial fatty acid oxidation (FAO) and subsequent switch to greater glucose oxidation for ATP production can prevent ischemia/reperfusion injury, controversy about this approach persists. For example, mice with germline deletion of the FA transporter CD36, exhibited either impaired or unchanged post-ischemic functional recovery despite a 40-60% reduction in FAO rates. Because there are limitations to cardiac studies utilizing whole body CD36 knockout (totalCD36KO) mice, we have now generated an inducible and cardiomyocyte-specific CD36 KO (icCD36KO) mouse to better address the role of cardiomyocyte CD36 and its regulation of FAO and post-ischemic functional recovery. Four to six weeks following CD36 ablation, hearts from icCD36KO mice had significantly decreased FA uptake compared to controls, which was paralleled by significant reductions in intramyocardial triacylglycerol content. Analysis of cardiac energy metabolism using ex vivo working heart perfusions showed that reduced FAO rates were compensated by enhanced glucose oxidation in the hearts from icCD36KO mice. In contrast to the totalCD36KO mice, hearts from icCD36KO mice exhibited significantly improved functional recovery following ischemia/reperfusion (18min of global no-flow ischemia followed by 40min of aerobic reperfusion). This improved recovery was associated with lower calculated proton production prior to and following ischemia compared to controls. Moreover, the amount of ATP generated relative to cardiac work was significantly lower in the hearts from icCD36KO mice compared to controls, indicating significantly increased cardiac efficiency in the hearts from icCD36KO mice. These data provide genetic evidence that reduced FAO as a result of diminished CD36-mediated FA uptake improves post-ischemic cardiac efficiency and functional recovery. As such, targeting cardiomyocyte FA uptake and FAO via inhibition of CD36 in the

  20. MK801 attenuates secondary injury in a mouse experimental compression model of spinal cord trauma

    PubMed Central

    2011-01-01

    Background Glutamergic excitotoxicity has been shown to play a deleterious role in the pathophysiology of spinal cord injury (SCI). The aim of this study was to investigate the neuroprotective effect of dizocilpine maleate, MK801 (2 mg/Kg, 30 min and 6 hours after injury) in a mice model of SCI. The spinal cord trauma was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. Results Spinal cord injury in mice resulted in severe trauma characterized by edema, neutrophil infiltration and apoptosis. In this study we clearly demonstrated that administration of MK801 attenuated all inflammatory parameters. In fact 24 hours after injury, the degree of spinal cord inflammation and tissue injury (evaluated as histological score), infiltration of neutrophils, NF-κB activation, iNOS, cytokines levels (TNF-α and IL-1β), neurotrophin expression were markedly reduced by MK801 treatment. Moreover, in a separate set of experiments, we have demonstrated that MK801 treatment significantly improved the recovery of locomotory function. Conclusions Blockade of NMDA by MK801 lends support to the potential importance of NMDA antagonists as therapeutic agents in the treatment of acute spinal cord injury. PMID:21492450

  1. Long-Term Cognitive Impairments and Pathological Alterations in a Mouse Model of Repetitive Mild Traumatic Brain Injury

    PubMed Central

    Luo, Jian; Nguyen, Andy; Villeda, Saul; Zhang, Hui; Ding, Zhaoqing; Lindsey, Derek; Bieri, Gregor; Castellano, Joseph M.; Beaupre, Gary S.; Wyss-Coray, Tony

    2014-01-01

    Mild traumatic brain injury (mTBI, also referred to as concussion) accounts for the majority of all traumatic brain injuries. The consequences of repetitive mTBI have become of particular concern for individuals engaged in certain sports or in military operations. Many mTBI patients suffer long-lasting neurobehavioral impairments. In order to expedite pre-clinical research and therapy development, there is a need for animal models that reflect the long-term cognitive and pathological features seen in patients. In the present study, we developed and characterized a mouse model of repetitive mTBI, induced onto the closed head over the left frontal hemisphere with an electromagnetic stereotaxic impact device. Using GFAP-luciferase bioluminescence reporter mice that provide a readout of astrocyte activation, we observed an increase in bioluminescence relative to the force delivered by the impactor after single impact and cumulative effects of repetitive mTBI. Using the injury parameters established in the reporter mice, we induced a repetitive mTBI in wild-type C57BL/6J mice and characterized the long-term outcome. Animals received repetitive mTBI showed a significant impairment in spatial learning and memory when tested at 2 and 6 months after injury. A robust astrogliosis and increased p-Tau immunoreactivity were observed upon post-mortem pathological examinations. These findings are consistent with the deficits and pathology associated with mTBI in humans and support the use of this model to evaluate potential therapeutic approaches. PMID:24550885

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Functional MRI for characterization of renal perfusion impairment and edema formation due to acute kidney injury in different mouse strains

    PubMed Central

    Chen, Rongjun; Gutberlet, Marcel; Jang, Mi-Sun; Meier, Martin; Mengel, Michael; Hartung, Dagmar; Wacker, Frank; Rong, Song; Hueper, Katja

    2017-01-01

    Purpose The purpose was to characterize acute kidney injury (AKI) in C57BL/6 (B6)- and 129/Sv (Sv)-mice by noninvasive measurement of renal perfusion and tissue edema using functional MRI. Methods Different severities of AKI were induced in B6- and Sv-mice by renal ischemia reperfusion injury (IRI). Unilateral clamping of the renal pedicle for 35 min (moderate AKI) or 45 min (severe AKI) was done. MRI (7-Tesla) was performed 1, 7 and 28 days after surgery using a flow alternating inversion recovery (FAIR) arterial spin labeling (ASL) sequence. Maps of perfusion and T1-relaxation time were calculated. Relative MRI-parameters of the IRI kidney compared to the contralateral not-clipped kidney were compared between AKI severities and between mouse strains using unpaired t-tests. In addition, fibrosis was assessed by Masson Trichrome and collagen IV staining. Results After moderate AKI relative perfusion impairment was significantly higher in B6- than in Sv-mice at d7 (55±7% vs. 82±8%, p<0.05) and d28 (76±7% vs. 102±3%, p<0.01). T1-values increased in the early phase after AKI in both mouse strains. T1-increase was more severe after prolonged ischemia times of 45 min compared to 35 min in both mouse strains, measured in the renal cortex and outer stripe of outer medulla. Kidney volume loss (compared to the contralateral kidney) occurred already after 7 days but proceeded markedly towards 4 weeks in severe AKI. Early renal perfusion impairment was predictive for later kidney volume loss. The progression to chronic kidney disease (CKD) in the severe AKI model was similar in both mouse strains as revealed by histology. Conclusion Quantification of renal perfusion and tissue edema by functional MRI allows characterization of strain differences upon AKI. Renal perfusion impairment was stronger in B6- compared to Sv-animals following moderate AKI. Prolonged ischemia times were associated with more severe perfusion impairment and edema formation in the early phase and

  4. Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections

    NASA Astrophysics Data System (ADS)

    Bensley, Jonathan Guy; de Matteo, Robert; Harding, Richard; Black, Mary Jane

    2016-04-01

    Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell). Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. Thick (40 μm) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4‧,6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. Previous methods have been restricted to thin sections (2–10 μm) and offer an incomplete picture of cardiomyocytes. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections.

  5. 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

  6. 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.

  7. Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections

    PubMed Central

    Bensley, Jonathan Guy; De Matteo, Robert; Harding, Richard; Black, Mary Jane

    2016-01-01

    Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell). Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. Thick (40 μm) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4′,6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. Previous methods have been restricted to thin sections (2–10 μm) and offer an incomplete picture of cardiomyocytes. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections. PMID:27048757

  8. Cardiomyocyte-specific ablation of CD36 accelerates the progression from compensated cardiac hypertrophy to heart failure.

    PubMed

    Sung, Miranda M; Byrne, Nikole J; Kim, Ty T; Levasseur, Jody; Masson, Grant; Boisvenue, Jamie J; Febbraio, Maria; Dyck, Jason R B

    2017-03-01

    Previous studies have shown that loss of CD36 protects the heart from dysfunction induced by pressure overload in the presence of diet-induced insulin resistance and/or obesity. The beneficial effects of CD36 ablation in this context are mediated by preventing excessive cardiac fatty acid (FA) entry and reducing lipotoxic injury. However, whether or not the loss of CD36 can prevent pressure overload-induced cardiac dysfunction in the absence of chronic exposure to high circulating FAs is presently unknown. To address this, we utilized a tamoxifen-inducible cardiomyocyte-specific CD36 knockout (icCD36KO) mouse and genetically deleted CD36 in adulthood. Control mice (CD36 floxed/floxed mice) and icCD36KO mice were treated with tamoxifen and subsequently subjected to transverse aortic constriction (TAC) surgery to generate pressure overload-induced cardiac hypertrophy. Consistent with CD36 mediating a significant proportion of FA entry into the cardiomyocyte and subsequent FA utilization for ATP production, hearts from icCD36KO mice were metabolically inefficient and displayed signs of energetic stress, including activation of the energetic stress kinase, AMPK. In addition, impaired energetics in icCD36KO mice contributed to a rapid progression from compensated hypertrophy to heart failure. However, icCD36KO mice fed a medium-chain FA diet, whereby medium-chain FAs can enter into the cardiomyocyte independent from CD36, were protected from TAC-induced heart failure. Together these data suggest that limiting FA uptake and partial inhibition of FA oxidation in the heart via CD36 ablation may be detrimental for the compensated hypertrophic heart in the absence of sufficiently elevated circulating FAs to provide an adequate energy source.NEW & NOTEWORTHY Limiting CD36-mediated fatty acid uptake in the setting of obesity and/or insulin resistance protects the heart from cardiac hypertrophy and dysfunction. However, cardiomyocyte-specific CD36 ablation in the absence of

  9. p62/SQSTM1 Plays a Protective Role in Oxidative Injury of Steatotic Liver in a Mouse Hepatectomy Model

    PubMed Central

    Haga, Sanae; Ozawa, Takeaki; Yamada, Yuma; Morita, Naoki; Nagashima, Izuru; Inoue, Hiroshi; Inaba, Yuka; Noda, Natsumi; Abe, Riichiro; Umezawa, Kazuo

    2014-01-01

    Abstract Aims: Liver injury and regeneration involve complicated processes and are affected by various physio-pathological factors. We investigated the mechanisms of steatosis-associated liver injury and delayed regeneration in a mouse model of partial hepatectomy. Results: Initial regeneration of the steatotic liver was significantly delayed after hepatectomy. Although hepatocyte proliferation was not significantly suppressed, severe liver injury with oxidative stress (OS) occurred immediately after hepatectomy in the steatotic liver. Fas-ligand (FasL)/Fas expression was upregulated in the steatotic liver, whereas the expression of antioxidant and anti-apoptotic molecules (catalase/MnSOD/Ref-1 and Bcl-2/Bcl-xL/FLIP, respectively) and p62/SQSTM1, a steatosis-associated protein, was downregulated. Interestingly, pro-survival Akt was not activated in response to hepatectomy, although it was sufficiently expressed even before hepatectomy. Suppression of p62/SQSTM1 increased FasL/Fas expression and reduced nuclear factor erythroid 2-related factor-2 (Nrf-2)-dependent antioxidant response elements activity and antioxidant responses in steatotic and nonsteatotic hepatocytes. Exogenously added FasL induced severe cellular OS and necrosis/apoptosis in steatotic hepatocytes, with only the necrosis being inhibited by pretreatment with antioxidants, suggesting that FasL/Fas-induced OS mainly leads to necrosis. Furthermore, p62/SQSTM1 re-expression in the steatotic liver markedly reduced liver injury and improved liver regeneration. Innovation: This study is the first which demonstrates that reduced expression of p62/SQSTM1 plays a crucial role in posthepatectomy acute injury and delayed regeneration of steatotic liver, mainly via redox-dependent mechanisms. Conclusion: In the steatotic liver, reduced expression of p62/SQSTM1 induced FasL/Fas overexpression and suppressed antioxidant genes, mainly through Nrf-2 inactivation, which, along with the hypo-responsiveness of Akt

  10. Dysferlin Deficiency and the Development of Cardiomyopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2B

    PubMed Central

    Chase, Thomas H.; Cox, Gregory A.; Burzenski, Lisa; Foreman, Oded; Shultz, Leonard D.

    2009-01-01

    Limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages. PMID:19875504

  11. A Possible Role of Neuroglobin in the Retina After Optic Nerve Injury: A Comparative Study of Zebrafish and Mouse Retina.

    PubMed

    Sugitani, Kayo; Koriyama, Yoshiki; Ogai, Kazuhiro; Wakasugi, Keisuke; Kato, Satoru

    2016-01-01

    Neuroglobin (Ngb) is a new member of the family of heme proteins and is specifically expressed in neurons of the central and peripheral nervous systems in all vertebrates. In particular, the retina has a 100-fold higher concentration of Ngb than do other nervous tissues. The role of Ngb in the retina is yet to be clarified. Therefore, to understand the functional role of Ngb in the retina after optic nerve injury (ONI), we used two types of retina, from zebrafish and mice, which have permissible and non-permissible capacity for nerve regeneration after ONI, respectively. After ONI, the Ngb protein in zebrafish was upregulated in the amacrine cells within 3 days, whereas in the mouse retina, Ngb was downregulated in the retinal ganglion cells (RGCs) within 3 days. Zebrafish Ngb (z-Ngb) significantly enhanced neurite outgrowth in retinal explant culture. According to these results, we designed an overexpression experiment with the mouse Ngb (m-Ngb) gene in RGC-5 cells (retinal precursor cells). The excess of m-Ngb actually rescued RGC-5 cells under hypoxic conditions and significantly enhanced neurite outgrowth in cell culture. These data suggest that mammalian Ngb has positive neuroprotective and neuritogenic effects that induce nerve regeneration after ONI.

  12. Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes.

    PubMed

    Sharma, Alok; Nguyen, Hieu; Geng, Cuiyu; Hinman, Melissa N; Luo, Guangbin; Lou, Hua

    2014-11-18

    In cardiomyocytes, calcium is known to control gene expression at the level of transcription, whereas its role in regulating alternative splicing has not been explored. Here we report that, in mouse primary or embryonic stem cell-derived cardiomyocytes, increased calcium levels induce robust and reversible skipping of several alternative exons from endogenously expressed genes. Interestingly, we demonstrate a calcium-mediated splicing regulatory mechanism that depends on changes of histone modifications. Specifically, the regulation occurs through changes in calcium-responsive kinase activities that lead to alterations in histone modifications and subsequent changes in the transcriptional elongation rate and exon skipping. We demonstrate that increased intracellular calcium levels lead to histone hyperacetylation along the body of the genes containing calcium-responsive alternative exons by disrupting the histone deacetylase-to-histone acetyltransferase balance in the nucleus. Consequently, the RNA polymerase II elongation rate increases significantly on those genes, resulting in skipping of the alternative exons. These studies reveal a mechanism by which calcium-level changes in cardiomyocytes impact on the output of gene expression through altering alternative pre-mRNA splicing patterns.

  13. Enhanced Expression of TREK-1 Is Related with Chronic Constriction Injury of Neuropathic Pain Mouse Model in Dorsal Root Ganglion

    PubMed Central

    Han, Hyo Jo; Lee, Seung Wook; Kim, Gyu-Tae; Kim, Eun-Jin; Kwon, Byeonghun; Kang, Dawon; Kim, Hyun Jeong; Seo, Kwang-Suk

    2016-01-01

    Neuropathic pain is a complex state showing increased pain response with dysfunctional inhibitory neurotransmission. The TREK family, one of the two pore domain K+ (K2P) channel subgroups were focused among various mechanisms of neuropathic pain. These channels influence neuronal excitability and are thought to be related in mechano/thermosensation. However, only a little is known about the expression and role of TREK-1 and TREK-2, in neuropathic pain. It is performed to know whether TREK-1 and/or 2 are positively related in dorsal root ganglion (DRG) of a mouse neuropathic pain model, the chronic constriction injury (CCI) model. Following this purpose, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot analyses were performed using mouse DRG of CCI model and compared to the sham surgery group. Immunofluorescence staining of isolectin-B4 (IB4) and TREK were performed. Electrophysiological recordings of single channel currents were analyzed to obtain the information about the channel. Interactions with known TREK activators were tested to confirm the expression. While both TREK-1 and TREK-2 mRNA were significantly overexpressed in DRG of CCI mice, only TREK-1 showed significant increase (∼9 fold) in western blot analysis. The TREK-1-like channel recorded in DRG neurons of the CCI mouse showed similar current-voltage relationship and conductance to TREK-1. It was easily activated by low pH solution (pH 6.3), negative pressure, and riluzole. Immunofluorescence images showed the expression of TREK-1 was stronger compared to TREK-2 on IB4 positive neurons. These results suggest that modulation of the TREK-1 channel may have beneficial analgesic effects in neuropathic pain patients. PMID:27133259

  14. Enhanced Expression of TREK-1 Is Related with Chronic Constriction Injury of Neuropathic Pain Mouse Model in Dorsal Root Ganglion.

    PubMed

    Han, Hyo Jo; Lee, Seung Wook; Kim, Gyu-Tae; Kim, Eun-Jin; Kwon, Byeonghun; Kang, Dawon; Kim, Hyun Jeong; Seo, Kwang-Suk

    2016-05-01

    Neuropathic pain is a complex state showing increased pain response with dysfunctional inhibitory neurotransmission. The TREK family, one of the two pore domain K⁺ (K2P) channel subgroups were focused among various mechanisms of neuropathic pain. These channels influence neuronal excitability and are thought to be related in mechano/thermosensation. However, only a little is known about the expression and role of TREK-1 and TREK-2, in neuropathic pain. It is performed to know whether TREK-1 and/ or 2 are positively related in dorsal root ganglion (DRG) of a mouse neuropathic pain model, the chronic constriction injury (CCI) model. Following this purpose, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and western blot analyses were performed using mouse DRG of CCI model and compared to the sham surgery group. Immunofluorescence staining of isolectin- B4 (IB4) and TREK were performed. Electrophysiological recordings of single channel currents were analyzed to obtain the information about the channel. Interactions with known TREK activators were tested to confirm the expression. While both TREK-1 and TREK-2 mRNA were significantly overexpressed in DRG of CCI mice, only TREK-1 showed significant increase (~9 fold) in western blot analysis. The TREK-1-like channel recorded in DRG neurons of the CCI mouse showed similar current-voltage relationship and conductance to TREK-1. It was easily activated by low pH solution (pH 6.3), negative pressure, and riluzole. Immunofluorescence images showed the expression of TREK-1 was stronger compared to TREK-2 on IB4 positive neurons. These results suggest that modulation of the TREK-1 channel may have beneficial analgesic effects in neuropathic pain patients.

  15. Temporal MRI characterization, neurobiochemical and neurobehavioral changes in a mouse repetitive concussive head injury model

    PubMed Central

    Yang, Zhihui; Wang, Ping; Morgan, Drake; Lin, Dan; Pan, Jianchun; Lin, Fan; Strang, Kevin H.; Selig, Tyler M.; Perez, Pablo D.; Febo, Marcelo; Chang, Binggong; Rubenstein, Richard; Wang, Kevin K.W.

    2015-01-01

    Single and repeated sports-related mild traumatic brain injury (mTBI), also referred to as concussion, can result in chronic post-concussive syndrome (PCS), neuropsychological and cognitive deficits, or chronic traumatic encephalopathy (CTE). However PCS is often difficult to diagnose using routine clinical, neuroimaging or laboratory evaluations, while CTE currently only can be definitively diagnosed postmortem. We sought to develop an animal model to simulate human repetitive concussive head injury for systematic study. In this study, mice received single or multiple head impacts by a stereotaxic impact device with a custom-made rubber tip-fitted impactor. Dynamic changes in MRI, neurobiochemical markers (Tau hyperphosphorylation and glia activation in brain tissues) and neurobehavioral functions such as anxiety, depression, motor function and cognitive function at various acute/subacute (1-7 day post-injury) and chronic (14-60 days post-injury) time points were examined. To explore the potential biomarkers of rCHI, serum levels of total Tau (T-Tau) and phosphorylated Tau (P-Tau) were also monitored at various time points. Our results show temporal dynamics of MRI consistent with structural perturbation in the acute phase and neurobiochemical changes (P-Tau and GFAP induction) in the subacute and chronic phase as well as development of chronic neurobehavioral changes, which resemble those observed in mTBI patients. PMID:26058556

  16. Vismodegib suppresses TRAIL-mediated liver injury in a mouse model of nonalcoholic steatohepatitis.

    PubMed

    Hirsova, Petra; Ibrahim, Samar H; Bronk, Steven F; Yagita, Hideo; Gores, Gregory J

    2013-01-01

    Hedgehog signaling pathway activation has been implicated in the pathogenesis of NASH. Despite this concept, hedgehog pathway inhibitors have not been explored. Thus, we examined the effect of vismodegib, a hedgehog signaling pathway inhibitor, in a diet-induced model of NASH. C57BL/6 mice were placed on 3-month chow or FFC (high saturated fats, fructose, and cholesterol) diet. One week prior to sacrifice, mice were treated with vismodegib or vehicle. Mice fed the FFC diet developed significant steatosis, which was unchanged by vismodegib therapy. In contrast, vismodegib significantly attenuated FFC-induced liver injury as manifested by reduced serum ALT and hepatic TUNEL-positive cells. In line with the decreased apoptosis, vismodegib prevented FFC-induced strong upregulation of death receptor DR5 and its ligand TRAIL. In addition, FFC-fed mice, but not chow-fed animals, underwent significant liver injury and apoptosis following treatment with a DR5 agonist; however, this injury was prevented by pre-treatment with vismodegib. Consistent with a reduction in liver injury, vismodegib normalized FFC-induced markers of inflammation including mRNA for TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 and a variety of macrophage markers. Furthermore, vismodegib in FFC-fed mice abrogated indices of hepatic fibrogenesis. In conclusion, inhibition of hedgehog signaling with vismodegib appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition may be salutary in human NASH.

  17. Joint loads resulting in ACL rupture: Effects of age, sex, and body mass on injury load and mode of failure in a mouse model.

    PubMed

    Blaker, Carina L; Little, Christopher B; Clarke, Elizabeth C

    2016-09-07

    Anterior cruciate ligament (ACL) tears are a common knee injury with a known but poorly understood association with secondary joint injuries and post-traumatic osteoarthritis (OA). Female sex and age are known risk factors for ACL injury but these variables are rarely explored in mouse models of injury. This study aimed to further characterize a non-surgical ACL injury model to determine its clinical relevance across a wider range of mouse specifications. Cadaveric and anesthetized C57BL/6 mice (9-52 weeks of age) underwent joint loading to investigate the effects of age, sex, and body mass on ACL injury mechanisms. The ACL injury load (whole joint load required to rupture the ACL) was measured from force-displacement data, and mode of failure was assessed using micro-dissection and histology. ACL injury load was found to increase with body mass and age (p < 0.001) but age was not significant when controlling for mass. Sex had no effect. In contrast, the mode of ACL failure varied with both age and sex groups. Avulsion fractures (complete or mixed with mid-substance tears) were common in all age groups but the proportion of mixed and mid-substance failures increased with age. Females were more likely than males to have a major avulsion relative to a mid-substance tear (p < 0.01). This data compliments studies in human cadaveric knees, and provides a basis for determining the severity of joint injury relative to a major ACL tear in mice, and for selecting joint loading conditions in future experiments using this model. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

  18. The Immune Response to Skin Trauma Is Dependent on the Etiology of Injury in a Mouse Model of Burn and Excision.

    PubMed

    Valvis, Samantha M; Waithman, Jason; Wood, Fiona M; Fear, Mark W; Fear, Vanessa S

    2015-08-01

    Skin trauma has many different causes including incision, blunt force, and burn. All of these traumas trigger an immune response. However, it is currently unclear whether the immune response is specific to the etiology of the injury. This study was established to determine whether the immune response to excision and burn injury of equivalent extent was the same. Using a mouse model of a full-thickness 19 mm diameter excision or 19 mm diameter full-thickness burn injury, we examined the innate immune response at the level of serum cytokine induction, whole-blood lymphocyte populations, dendritic cell function/phenotype, and the ensuing adaptive immune responses of CD4 and CD8 T-cell populations. Strikingly, both the innate and adaptive immune system responses differed between the burn and excision injuries. Acute cytokine induction was faster and different in profile to that of excision injury, leading to changes in systemic monocyte and neutrophil levels. Differences in the immune profile between burn and excision were also noted up to day 84 post injury, suggesting that the etiology of injury leads to sustained changes in the response. This may in part underlie clinical observations of differences in patient morbidity and mortality in response to different skin injury types.

  19. Longitudinal evaluation of mouse hind limb bone loss after spinal cord injury using novel, in vivo, methodology.

    PubMed

    McManus, Madonna M; Grill, Raymond J

    2011-12-07

    Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse, aberrant neuronal signaling and hormonal changes. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies. Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis. An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (naïve) mice (13% decrease, p < 0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal

  20. Tauroursodeoxycholic Acid Attenuates Renal Tubular Injury in a Mouse Model of Type 2 Diabetes

    PubMed Central

    Zhang, Jing; Fan, Ying; Zeng, Chuchu; He, Li; Wang, Niansong

    2016-01-01

    Renal tubular injury is a critical factor in the pathogenesis of diabetic nephropathy (DN). Endoplasmic reticulum (ER) stress is involved in diabetic nephropathy. Tauroursodeoxycholic acid (TUDCA) is an effective inhibitor of ER stress. Here, we investigated the role of TUDCA in the progression of tubular injury in DN. For eight weeks, being treated with TUDCA at 250 mg/kg intraperitoneal injection (i.p.) twice a day, diabetic db/db mice had significantly reduced blood glucose, albuminuria and attenuated renal histopathology. These changes were associated with a significant decreased expression of ER stress markers. At the same time, diabetic db/db mice had more TUNEL-positive nuclei in the renal tubule, which were attenuated by TUDCA treatment, along with decreases in ER stress–associated apoptotic markers in the kidneys. In summary, the effect of TUDCA on tubular injury, in part, is associated with inhibition of ER stress in the kidneys of diabetic db/db mice. TUDCA shows potential as a therapeutic target for the prevention and treatment of DN. PMID:27669287

  1. Activation of NOD1 by DAP contributes to myocardial ischemia/reperfusion injury via multiple signaling pathways.

    PubMed

    Yang, Hui; Li, Nan; Song, Li-Na; Wang, Lei; Tian, Cui; Tang, Chao-Shu; Du, Jie; Li, Hui-Hua; Yu, Xiao-Hong; Wang, Hong-Xia

    2015-04-01

    NOD1 is a member of nucleotide-binding oligomerization domain-like receptors family that participates in many inflammatory processes. Previous studies demonstrated that NOD1 plays an important role in inflammatory cardiovascular diseases. However, its role in myocardial ischemia/reperfusion (I/R) injury remains unknown. The present study investigate whether NOD1 is involved in the pathogenesis of mouse myocardial I/R injury and the underlying mechanisms. Administration of NOD1 ligand (DAP) significantly enhanced myocardial I/R injury, as demonstrated by increased infarct size, the number of TUNEL-positive nuclei, caspase-3 activity, the infiltration of Mac-2- and IL-6-positive cells as compared with untreated heart or cardiomyocytes after I/R injury. In contrast, knockdown of NOD1 by siRNA markedly attenuated mimetic I/R induced cardiomyocyte apoptosis in vitro, indicating that NOD1 enhanced myocardial I/R injury partially through direct heart effects. These effects were partially associated with activation of JNK, p38 MAPK and NF-κB signaling pathways. Taken together, these results provide the first evidence that activation of intracellular sensor NOD1 enhances myocardial I/R injury and may provide novel therapeutic target for ameliorating the ischemic heart diseases.

  2. Enhanced currents through L-type calcium channels in cardiomyocytes disturb the electrophysiology of the dystrophic heart.

    PubMed

    Koenig, Xaver; Rubi, Lena; Obermair, Gerald J; Cervenka, Rene; Dang, Xuan B; Lukacs, Peter; Kummer, Stefan; Bittner, Reginald E; Kubista, Helmut; Todt, Hannes; Hilber, Karlheinz

    2014-02-15

    Duchenne muscular dystrophy (DMD), induced by mutations in the gene encoding for the cytoskeletal protein dystrophin, is an inherited disease characterized by progressive muscle weakness. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with cardiac complications. These include cardiomyopathy development and cardiac arrhythmias. The current understanding of the pathomechanisms in the heart is very limited, but recent research indicates that dysfunctional ion channels in dystrophic cardiomyocytes play a role. The aim of the present study was to characterize abnormalities in L-type calcium channel function in adult dystrophic ventricular cardiomyocytes. By using the whole cell patch-clamp technique, the properties of currents through calcium channels in ventricular cardiomyocytes isolated from the hearts of normal and dystrophic adult mice were compared. Besides the commonly used dystrophin-deficient mdx mouse model for human DMD, we also used mdx-utr mice, which are both dystrophin- and utrophin-deficient. We found that calcium channel currents were significantly increased, and channel inactivation was reduced in dystrophic cardiomyocytes. Both effects enhance the calcium influx during an action potential (AP). Whereas the AP in dystrophic mouse cardiomyocytes was nearly normal, implementation of the enhanced dystrophic calcium conductance in a computer model of a human ventricular cardiomyocyte considerably prolonged the AP. Finally, the described dystrophic calcium channel abnormalities entailed alterations in the electrocardiograms of dystrophic mice. We conclude that gain of function in cardiac L-type calcium channels may disturb the electrophysiology of the dystrophic heart and thereby cause arrhythmias.

  3. Cardiac-specific overexpression of E3 ligase Nrdp1 increases ischemia and reperfusion-induced cardiac injury.

    PubMed

    Zhang, Yuan; Zeng, Yong; Wang, Min; Tian, Cui; Ma, Xu; Chen, Houzao; Fang, Quan; Jia, Lixin; Du, Jie; Li, Huihua

    2011-05-01

    Cardiomyocyte death is a major event of myocardial infarction. Previously, we and others have shown that E3 ligase-mediated protein turnover plays a critical role in cardiac injury. In this study, we sought to determine the role of a newly identified E3 ligase, neuregulin receptor degradation protein-1 (Nrdp1), on cardiac ischemia/reperfusion (I/R) injury. I/R injury markedly upregulated Nrdp1 expression in heart tissue. To elucidate the role of Nrdp1 in I/R-induced cardiac injury, neonatal cardiomyocytes were infected with adenoviral constructs expressing wild-type, dominant-negative Nrdp1 genes. Increased Nrdp1 expression enhanced I/R-induced cardiomyocyte apoptosis and inflammation as compared with the green fluorescent protein (GFP) control; these effects were attenuated by overexpression of a dominant-negative Nrdp1 (C34S/H36Q). Furthermore, cardiac-specific Nrdp1 overexpression in vivo in mouse significantly increased infarct size, the number of TUNEL-positive nuclei and inflammatory cells, as well as mortality, as compared with wild-type mice after I/R injury. The mechanisms underlying these effects were associated with the downregulation of an Nrdp1 substrate, ErbB3, accompanied by suppression of its downstream targets AKT, ERK1/2, and activation of p38 and JNK1/2. Together, these results provide evidence for an important role for Nrdp1 in regulating I/R-induced cardiac injury. Nrdp1 may constitute a new therapeutic target for ameliorating the I/R-induced cardiac injury.

  4. Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes

    PubMed Central

    Tan, Xian Wen; Bhave, Mrinal; Fong, Alan Yean Yip; Matsuura, Eiji; Kobayashi, Kazuko; Shen, Lian Hua; Hwang, Siaw San

    2016-01-01

    This study was aimed at preliminarily assessing the cytoprotective and antioxidative effects of rice bran extracts (RBEs) from a Sarawak local rice variety (local name: “BJLN”) and a commercial rice variety, “MR219,” on oxidative stress in rat H9c2(2-1) cardiomyocytes. The cardiomyocytes were incubated with different concentrations of RBE and hydrogen peroxide (H2O2), respectively, to identify their respective IC50 values and safe dose ranges. Two nonlethal and close-to-IC50 doses of RBE were selected to evaluate their respective effects on H2O2 induced oxidative stress in cardiomyocytes. Both RBEs showed dose-dependent cytotoxicity effects on cardiomyocytes. H2O2 induction of cardiomyocytes pretreated with RBE further revealed the dose-dependent cytoprotective and antioxidative effects of RBE via an increase in IC50 values of H2O2. Preliminary analyses of induction effects of RBE and H2O2 on cellular antioxidant enzyme, catalase (CAT), also revealed their potential in regulating these activities and expression profile of related gene on oxidative stress in cardiomyocytes. Pretreated cardiomyocytes significantly upregulated the enzymatic activity and expression level of CAT under the exposure of H2O2 induced oxidative stress. This preliminary study has demonstrated the potential antioxidant effects of RBE in alleviating H2O2-mediated oxidative injuries via upregulation in enzymatic activities and expression levels of CAT. PMID:27239253

  5. Dataset of integrin-linked kinase protein: Protein interactions in cardiomyocytes identified by mass spectrometry.

    PubMed

    Traister, Alexandra; Lu, Mingliang; Coles, John G; Maynes, Jason T

    2016-06-01

    Using hearts from mice overexpressing integrin linked kinase (ILK) behind the cardiac specific promoter αMHC, we have performed immunoprecipitation and mass spectrometry to identify novel ILK protein:protein interactions that regulate cardiomyocyte activity and calcium flux. Integrin linked kinase complexes were captured from mouse heart lysates using a commercial antibody, with subsequent liquid chromatography tandem mass spectral analysis. Interacting partners were identified using the MASCOT server, and important interactions verified using reverse immunoprecipitation and mass spectrometry. All ILK interacting proteins were identified in a non-biased manner, and are stored in the ProteomeXchange Consortium via the PRIDE partner repository (reference ID PRIDE: PXD001053). The functional role of identified ILK interactions in cardiomyocyte function and arrhythmia were subsequently confirmed in human iPSC-cardiomyocytes.

  6. Neuroglobin Overexpression Improves Sensorimotor Outcomes in a Mouse Model of Traumatic Brain Injury

    PubMed Central

    Taylor, Jordan M.; Kelley, Brian; Gregory, Eugene J.; Berman, Nancy E.J.

    2014-01-01

    There is a significant need for novel treatments that will improve traumatic brain injury (TBI) outcomes. One potential neuroprotective mechanism is to increase oxygen binding proteins such as neuroglobin. Neuroglobin has a high affinity for oxygen, is an effective free radical scavenger, and is neuroprotective within the brain following hypoxia and ischemia. The purpose of this study was to determine whether neuroglobin overexpression improves sensorimotor outcomes following TBI in transgenic neuroglobin overexpressing (NGB) mice. Additional study aims were to determine if and when an endogenous neuroglobin response occurred following TBI in wild-type (WT) mice, and in what brain regions and cell types the response occurred. Controlled cortical impact (CCI) was performed in adult (5 month) C57/BL6 WT mice, and NGB mice constitutively overexpressing neuroglobin via the chicken beta actin promoter coupled with the cytomegalovirus distal enhancer. The gridwalk task was used for sensorimotor testing of both WT and NGB mice, prior to injury, and at 2, 3, and 7 days post-TBI. NGB mice displayed significant reductions in the average number of foot faults per minute walking at 2, 3, and 7 days post-TBI when compared to WT mice at each time point. Neuroglobin mRNA expression was assessed in the injured cortex of WT mice prior to injury, and at 1, 3, 7, and 14 days post-TBI using quantitative real time polymerase chain reaction (qRT-PCR). Neuroglobin mRNA was significantly increased at 7 days post-TBI. Immunostaining showed neuroglobin primarily localized to neurons and glial cells in the injured cortex and ipsilateral hippocampus of WT mice, while neuroglobin was present in all brain regions of NGB mice at 7 days post-TBI. These results showed that overexpression of neuroglobin reduced sensorimotor deficits following TBI, and that an endogenous increase in neuroglobin expression occurs during the subacute period. Increasing neuroglobin expression through novel therapeutic

  7. Hepatic NK cell-mediated hypersensitivity to ConA-induced liver injury in mouse liver expressing hepatitis C virus polyprotein.

    PubMed

    Fu, Qiuxia; Yan, Shaoduo; Wang, Licui; Duan, Xiangguo; Wang, Lei; Wang, Yue; Wu, Tao; Wang, Xiaohui; An, Jie; Zhang, Yulong; Zhou, Qianqian; Zhan, Linsheng

    2016-08-04

    The role of hepatic NK cells in the pathogenesis of HCV-associated hepatic failure is incompletely understood. In this study, we investigated the effect of HCV on ConA-induced immunological hepatic injury and the influence of HCV on hepatic NK cell activation in the liver after ConA administration. An immunocompetent HCV mouse model that encodes the entire viral polyprotein in a liver-specific manner based on hydrodynamic injection and φC31o integrase was used to study the role of hepatic NK cells. Interestingly, the frequency of hepatic NK cells was reduced in HCV mice, whereas the levels of other intrahepatic lymphocytes remained unaltered. Next, we investigated whether the reduction in NK cells within HCV mouse livers might elicit an effect on immune-mediated liver injury. HCV mice were subjected to acute liver injury models upon ConA administration. We observed that HCV mice developed more severe ConA-induced immune-mediated hepatitis, which was dependent on the accumulated intrahepatic NK cells. Our results indicated that after the administration of ConA, NK cells not only mediated liver injury through the production of immunoregulatory cytokines (IFN-γ, TNF-α and perforin) with direct antiviral activity, but they also killed target cells directly through the TRAIL/DR5 and NKG2D/NKG2D ligand signaling pathway in HCV mice. Our findings suggest a critical role for NK cells in oversensitive liver injury during chronic HCV infection.

  8. Proteolysis regulates cardiomyocyte maturation and tissue integration

    PubMed Central

    Fukuda, Ryuichi; Gunawan, Felix; Beisaw, Arica; Jimenez-Amilburu, Vanesa; Maischein, Hans-Martin; Kostin, Sawa; Kawakami, Koichi; Stainier, Didier Y. R.

    2017-01-01

    Tissue integrity is critical for organ formation and function. During heart development, cardiomyocytes differentiate and integrate to form a coherent tissue that contracts synchronously. However, the molecular mechanisms regulating cardiac tissue integrity are poorly understood. Here we show that proteolysis, via the E3 ubiquitin ligase ASB2, regulates cardiomyocyte maturation and tissue integrity. Cardiomyocytes in asb2b zebrafish mutants fail to terminally differentiate, resulting in reduced cardiac contractility and output. Mosaic analyses reveal a cell-autonomous requirement for Asb2b in cardiomyocytes for their integration as asb2b mutant cardiomyocytes are unable to meld into wild-type myocardial tissue. In vitro and in vivo data indicate that ASB2 negatively regulates TCF3, a bHLH transcription factor. TCF3 must be degraded for cardiomyocyte maturation, as TCF3 gain-of-function causes a number of phenotypes associated with cardiomyocyte dedifferentiation. Overall, our results show that proteolysis has an important role in cardiomyocyte maturation and the formation of a coherent myocardial tissue. PMID:28211472

  9. Transplantation of Mouse Embryonic Stem Cells into the Cochlea of an Auditory-Neuropathy Animal Model: Effects of Timing after Injury

    PubMed Central

    Schulte, Bradley A.; Goddard, John C.; Hedrick, Michelle; Schulte, Jason B.; Wei, Ling; Schmiedt, Richard A.

    2008-01-01

    Application of ouabain to the round window membrane of the gerbil selectively induces the death of most spiral ganglion neurons and thus provides an excellent model for investigating the survival and differentiation of embryonic stem cells (ESCs) introduced into the inner ear. In this study, mouse ESCs were pretreated with a neural-induction protocol and transplanted into Rosenthal’s canal (RC), perilymph, or endolymph of Mongolian gerbils either 1–3 days (early post-injury transplant group) or 7 days or longer (late post-injury transplant group) after ouabain injury. Overall, ESC survival in RC and perilymphatic spaces was significantly greater in the early post-injury microenvironment as compared to the later post-injury condition. Viable clusters of ESCs within RC and perilymphatic spaces appeared to be associated with neovascularization in the early post-injury group. A small number of ESCs transplanted within RC stained for mature neuronal or glial cell markers. ESCs introduced into perilymph survived in several locations, but most differentiated into glia-like cells. ESCs transplanted into endolymph survived poorly if at all. These experiments demonstrate that there is an optimal time window for engraftment and survival of ESCs that occurs in the early post-injury period. PMID:18449604

  10. Improvement of cold injury-induced mouse brain edema by endothelin ETB antagonists is accompanied by decreases in matrixmetalloproteinase 9 and vascular endothelial growth factor-A.

    PubMed

    Michinaga, Shotaro; Seno, Naoki; Fuka, Mayu; Yamamoto, Yui; Minami, Shizuho; Kimura, Akimasa; Hatanaka, Shunichi; Nagase, Marina; Matsuyama, Emi; Yamanaka, Daisuke; Koyama, Yutaka

    2015-09-01

    Brain edema is a potentially fatal pathological state that often occurs after brain injuries such as ischemia and trauma. However, therapeutic agents that fundamentally treat brain edema have not yet been established. We previously found that endothelin ETB receptor antagonists attenuate the formation and maintenance of vasogenic brain edema after cold injury in mice. In this study, the effects of ETB antagonists on matrixmetalloproteinase (MMP)9 and vascular endothelial growth factor (VEGF)-A expression were examined in the cold injury model. Cold injury was performed in the left brain of male ddY mice (5-6 weeks old) for the induction of vasogenic edema. Expression of MMP9 and VEGF-A mRNA in the mouse cerebrum was increased by cold injury. Immunohistochemical observations showed that the MMP9 and VEGF-A were mainly produced in reactive astrocytes in the damaged cerebrum. Intracerebroventricular administration of BQ788 (10 μg) or IRL-2500 (10 μg) (selective ETB antagonists) attenuated brain edema and disruption of the blood-brain barrier after cold injury. BQ788 and IRL-2500 reversed the cold injury-induced increases in MMP9 and VEGF-A expression. The induction of reactive astrocytes producing MMP9 and VEGF-A in the damaged cerebrum was attenuated by BQ788 and IRL-2500. These results suggest that attenuations of astrocytic MMP9 and VEGF-A expression by ETB antagonists may be involved in the amelioration of vasogenic brain edema.

  11. Some cell kinetic effects of combined injury with ionizing radiation and cyclophosphamide on mouse bladder urothelium.

    PubMed

    Reitan, J B

    1985-01-01

    Cyclophosphamide was given intraperitoneally to groups of eight female mice 48 h after local electron irradiation to the bladder with 0, 10 and 20 Gy respectively. The reactions in the urothelium were monitored by histology, incorporation of tritiated thymidine and flow cytometry. A wave of increased thymidine incorporation combined with an increase in the proportion of diploid S-phase cells was seen in the unirradiated bladders 24 h after the drug treatment, followed by normalization after 1 week. This response was significantly less pronounced in the irradiated animals. In the unirradiated animals a similar wave characterized by an increased proportion of octaploid cells was also seen, but this wave occurred later in the irradiated animals. Severe injury was observed in the rectum of the 20 Gy-irradiated animals. Irradiation prior to drug treatment led to only small effects, but a decreased ability for regenerative DNA synthesis after drug injury seems to persist. This affects both proliferation and the building up of polyploidy.

  12. Manganese Superoxide Dismutase Protects against 6-Hydroxydopamine Injury in Mouse Brains*

    PubMed Central

    Callio, Jason; Oury, Tim D.; Chu, Charleen T.

    2007-01-01

    Dopaminergic neurons of the substantia nigra are susceptible to toxin-based insults. Intrastriatal injection of 6-hydroxydopamine results in selective toxicity to these neurons. A mechanistic role for reactive oxygen species is supported by observations that antioxidants confer protection from 6-hydroxydopamine. Although cell culture studies have suggested extracellular or nonmitochondrial mechanisms in 6-hydroxydopamine toxicity, the compartmentalization of oxidative injury mechanisms is incompletely defined in vivo. Transgenic mice overexpressing mitochondrial manganese superoxide dismutase or extracellular superoxide dismutase received unilateral intrastriatal injections of 6-hydroxydopamine. Mice that overexpress manganese superoxide dismutase showed significantly smaller striatal lesions than littermate controls. There were no differences in nonspecific striatal injury associated with contralateral vehicle injection. Manganese superoxide dismutase overexpression also protected against loss of neuronal cell bodies in the substantia nigra. In contrast, mice overexpressing extracellular superoxide dismutase showed no protection from 6-hydroxydopamine toxicity in either brain region. Protection of the nigrostriatal system by overexpression of manganese super-oxide dismutase supports a role for mitochondrially derived superoxide in 6-hydroxydopamine toxicity. Mitochondrial oxidative stress appears to be a common mechanism among diverse models of Parkinson disease, whether involving toxins, mutated genes, or cybrid cells containing patient mitochondria. Antioxidant therapies that target this subcellular compartment may prove promising. PMID:15755737

  13. Stanniocalcin-1 Protects a Mouse Model from Renal Ischemia-Reperfusion Injury by Affecting ROS-Mediated Multiple Signaling Pathways

    PubMed Central

    Liu, Dajun; Shang, Huiping; Liu, Ying

    2016-01-01

    Stanniocalcin-1 (STC-1) protects against renal ischemia-reperfusion injury (RIRI). However, the molecular mechanisms remain widely unknown. STC-1 inhibits reactive oxygen species (ROS), whereas most ROS-mediated pathways are associated with ischemic injury. Therefore, to explore the mechanism, the effects of STC-1 on ROS-medicated pathways were studied. Non-traumatic vascular clamps were used to establish RIRI mouse models. The serum levels of STC-1, interleukin-6 (IL-6), interferon (IFN) γ, P53, and capase-3 were measured by ELISA kits. Superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by fluorescence spectrofluorometer. All these molecules changed significantly in a RIRI model mouse when compared with those in a sham control. Kidney cells were isolated from sham and model mice. STC-1 was overexpressed or knockout in these kidney cells. The molecules in ROS-medicated pathways were measured by real-time quantitative PCR and Western blot. The results showed that STC-1 is an effective ROS scavenger. The serum levels of STC-1, MDA and SOD activity were increased while the serum levels of IL-6, iIFN-γ, P53, and capase-3 were decreased in a model group when compared with a sham control (p < 0.05). Furthermore, the levels of STC-1,p53, phosphorylated mitogen-activated protein kinase kinase (p-MEKK-1), c-Jun N-terminal kinase (p-JNK), extracellular signal-regulated kinase (p-ERK), IkB kinase (p-IKK), nuclear factor (NF) κB, apoptosis signal-regulating kinase 1 (ASK-1) and caspase-3 changed significantly in kidney cells isolated from a RIRI model when compared to those isolated from a sham control (p < 0.05). Meanwhile, STC-1 overexpression or silence caused significant changes of the levels of these ROS-mediated molecules. Therefore, STC-1 maybe improve anti-inflammation, anti-oxidant and anti-apoptosis activities by affecting ROS-mediated pathways, especially the phospho-modifications of the respective proteins, resulting in the increase of SOD and

  14. Evidence for Cardiomyocyte Renewal in Humans

    SciTech Connect

    Bergmann, O; Bhardwaj, R D; Bernard, S; Zdunek, S; Barnabe-Heider, F; Walsh, S; Zupicich, J; Alkass, K; Buchholz, B A; Druid, H; Jovinge, S; Frisen, J

    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 in 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.

  15. Evidence for cardiomyocyte renewal in humans.

    PubMed

    Bergmann, Olaf; Bhardwaj, Ratan D; Bernard, Samuel; Zdunek, Sofia; Barnabé-Heider, Fanie; Walsh, Stuart; Zupicich, Joel; Alkass, Kanar; Buchholz, Bruce A; Druid, Henrik; Jovinge, Stefan; Frisén, Jonas

    2009-04-03

    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 carbon-14, 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 25 to 0.45% at the age of 75. Fewer than 50% of cardiomyocytes are exchanged during a normal life span. The capacity to generate cardiomyocytes in the adult human heart suggests that it may be rational to work toward the development of therapeutic strategies aimed at stimulating this process in cardiac pathologies.

  16. mTORC1 and mTORC2 play different roles in regulating cardiomyocyte differentiation from embryonic stem cells.

    PubMed

    Zheng, Bei; Wang, Jiadan; Tang, Leilei; Shi, Jiana; Zhu, Danyan

    2017-01-01

    Mammalian target of rapamycin (mTOR) is a serine/threonine kinase and functions through two distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2), with their key components Raptor and Rictor, to play crucial roles in cellular survival and growth. However, the roles of mTORC1 and mTORC2 in regulating cardiomyocyte differentiation from mouse embryonic stem (mES) cells are not clear. In this study, we performed Raptor or Rictor knockdown experiments to investigate the roles of mTORC1 and mTORC2 in cardiomyocyte differentiation. Ablation of Raptor markedly increased the number of cardiomyocytes derived from mES cells with well-organized myofilaments. Expression levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein), and α-Actinin (cardiomyocyte marker) were increased in Raptor knockdown cells. In contrast, loss of Rictor prevented cardiomyocyte differentiation. The dual ablation of Raptor and Rictor also decreased the number of cardiomyocytes. The two complexes exerted a regulatory mechanism in such a manner that knockdown of Raptor/mTORC1 resulted in a decreased phosphorylation of Rictor (Thr1135), which subsequently activated Rictor/mTORC2 in the differentiation of mES cells into cardiomyocytes. In conclusion, mTORC1 and mTORC2 played different roles in cardiomyocyte differentiation from mES cells in vitro. The activation of Rictor/mTORC2 was critical for facilitating cardiomyocyte differentiation from mES cells. Thus, this complex may be a promising target for regulating myocardial differentiation from embryonic stem cells or induced pluripotent stem cells.

  17. Palmitoylethanolamide Reduces Neuropsychiatric Behaviors by Restoring Cortical Electrophysiological Activity in a Mouse Model of Mild Traumatic Brain Injury

    PubMed Central

    Guida, Francesca; Boccella, Serena; Iannotta, Monica; De Gregorio, Danilo; Giordano, Catia; Belardo, Carmela; Romano, Rosaria; Palazzo, Enza; Scafuro, Maria A.; Serra, Nicola; de Novellis, Vito; Rossi, Francesco; Maione, Sabatino; Luongo, Livio

    2017-01-01

    Traumatic brain injury (TBI) represents a major public health problem, which is associated with neurological dysfunction. In severe or moderate cases of TBI, in addition to its high mortality rate, subjects may encounter diverse behavioral dysfunctions. Previous reports suggest that an association between TBI and chronic pain syndromes tends to be more common in patients with mild forms of brain injury. Despite causing minimal brain damage, mild TBI (mTBI) often leads to persistent psychologically debilitating symptoms, which can include anxiety, various forms of memory and learning deficits, and depression. At present, no effective treatment options are available for these symptoms, and little is known about the complex cellular activity affecting neuronal activity that occurs in response to TBI during its late phase. Here, we used a mouse model to investigate the effect of Palmitoylethanolamide (PEA) on both the sensorial and neuropsychiatric dysfunctions associated with mTBI through behavioral, electrophysiological, and biomolecular approaches. Fourteen-day mTBI mice developed anxious, aggressive, and reckless behavior, whilst depressive-like behavior and impaired social interactions were observed from the 60th day onward. Altered behavior was associated with changes in interleukin 1 beta (IL-1β) expression levels and neuronal firing activity in the medial prefrontal cortex. Compared with vehicle, PEA restored the behavioral phenotype and partially normalized the biochemical and functional changes occurring at the supraspinal level. In conclusion, our findings reveal some of the supraspinal modifications responsible for the behavioral alterations associated with mTBI and suggest PEA as a pharmacological tool to ameliorate neurological dysfunction induced by the trauma. PMID:28321191

  18. Palmitoylethanolamide Reduces Neuropsychiatric Behaviors by Restoring Cortical Electrophysiological Activity in a Mouse Model of Mild Traumatic Brain Injury.

    PubMed

    Guida, Francesca; Boccella, Serena; Iannotta, Monica; De Gregorio, Danilo; Giordano, Catia; Belardo, Carmela; Romano, Rosaria; Palazzo, Enza; Scafuro, Maria A; Serra, Nicola; de Novellis, Vito; Rossi, Francesco; Maione, Sabatino; Luongo, Livio

    2017-01-01

    Traumatic brain injury (TBI) represents a major public health problem, which is associated with neurological dysfunction. In severe or moderate cases of TBI, in addition to its high mortality rate, subjects may encounter diverse behavioral dysfunctions. Previous reports suggest that an association between TBI and chronic pain syndromes tends to be more common in patients with mild forms of brain injury. Despite causing minimal brain damage, mild TBI (mTBI) often leads to persistent psychologically debilitating symptoms, which can include anxiety, various forms of memory and learning deficits, and depression. At present, no effective treatment options are available for these symptoms, and little is known about the complex cellular activity affecting neuronal activity that occurs in response to TBI during its late phase. Here, we used a mouse model to investigate the effect of Palmitoylethanolamide (PEA) on both the sensorial and neuropsychiatric dysfunctions associated with mTBI through behavioral, electrophysiological, and biomolecular approaches. Fourteen-day mTBI mice developed anxious, aggressive, and reckless behavior, whilst depressive-like behavior and impaired social interactions were observed from the 60th day onward. Altered behavior was associated with changes in interleukin 1 beta (IL-1β) expression levels and neuronal firing activity in the medial prefrontal cortex. Compared with vehicle, PEA restored the behavioral phenotype and partially normalized the biochemical and functional changes occurring at the supraspinal level. In conclusion, our findings reveal some of the supraspinal modifications responsible for the behavioral alterations associated with mTBI and suggest PEA as a pharmacological tool to ameliorate neurological dysfunction induced by the trauma.

  19. Optical Detection of Early Damage in Retinal Ganglion Cells in a Mouse Model of Partial Optic Nerve Crush Injury

    PubMed Central

    Yi, Ji; Puyang, Zhen; Feng, Liang; Duan, Lian; Liang, Peiji; Backman, Vadim; Liu, Xiaorong; Zhang, Hao F.

    2016-01-01

    Purpose Elastic light backscattering spectroscopy (ELBS) has exquisite sensitivity to the ultrastructural properties of tissue and thus has been applied to detect various diseases associated with ultrastructural alterations in their early stages. This study aims to test whether ELBS can detect early damage in retinal ganglion cells (RGCs). Methods We used a mouse model of partial optic nerve crush (pONC) to induce rapid RGC death. We confirmed RGC loss by axon counting and characterized the changes in retinal morphology by optical coherence tomography (OCT) and in retinal function by full-field electroretinogram (ERG), respectively. To quantify the ultrastructural properties, elastic backscattering spectroscopic analysis was implemented in the wavelength-dependent images recorded by reflectance confocal microscopy. Results At 3 days post-pONC injury, no significant change was found in the thickness of the RGC layer or in the mean amplitude of the oscillatory potentials measured by OCT and ERG, respectively; however, we did observe a significantly decreased number of axons compared with the controls. At 3 days post-pONC, we used ELBS to calculate the ultrastructural marker (D), the shape factor quantifying the shape of the local mass density correlation functions. It was significantly reduced in the crushed eyes compared with the controls, indicating the ultrastructural fragmentation in the crushed eyes. Conclusions Elastic light backscattering spectroscopy detected ultrastructural neuronal damage in RGCs following the pONC injury when OCT and ERG tests appeared normal. Our study suggests a potential clinical method for detecting early neuronal damage prior to anatomical alterations in the nerve fiber and ganglion cell layers. PMID:27784071

  20. The Spectrum of Neurobehavioral Sequelae after Repetitive Mild Traumatic Brain Injury: A Novel Mouse Model of Chronic Traumatic Encephalopathy

    PubMed Central

    Plog, Benjamin A.; Dayawansa, Samantha; Chen, Michael; Dashnaw, Matthew L.; Czerniecka, Katarzyna; Walker, Corey T.; Viterise, Tyler; Hyrien, Ollivier; Iliff, Jeffrey J.; Deane, Rashid; Nedergaard, Maiken; Huang, Jason H.

    2014-01-01

    Abstract There has been an increased focus on the neurological sequelae of repetitive mild traumatic brain injury (TBI), particularly neurodegenerative syndromes, such as chronic traumatic encephalopathy (CTE); however, no animal model exists that captures the behavioral spectrum of this phenomenon. We sought to develop an animal model of CTE. Our novel model is a modification and fusion of two of the most popular models of TBI and allows for controlled closed-head impacts to unanesthetized mice. Two-hundred and eighty 12-week-old mice were divided into control, single mild TBI (mTBI), and repetitive mTBI groups. Repetitive mTBI mice received six concussive impacts daily for 7 days. Behavior was assessed at various time points. Neurological Severity Score (NSS) was computed and vestibulomotor function tested with the wire grip test (WGT). Cognitive function was assessed with the Morris water maze (MWM), anxiety/risk-taking behavior with the elevated plus maze, and depression-like behavior with the forced swim/tail suspension tests. Sleep electroencephalogram/electromyography studies were performed at 1 month. NSS was elevated, compared to controls, in both TBI groups and improved over time. Repetitive mTBI mice demonstrated transient vestibulomotor deficits on WGT. Repetitive mTBI mice also demonstrated deficits in MWM testing. Both mTBI groups demonstrated increased anxiety at 2 weeks, but repetitive mTBI mice developed increased risk-taking behaviors at 1 month that persist at 6 months. Repetitive mTBI mice exhibit depression-like behavior at 1 month. Both groups demonstrate sleep disturbances. We describe the neurological sequelae of repetitive mTBI in a novel mouse model, which resemble several of the neuropsychiatric behaviors observed clinically in patients sustaining repetitive mild head injury. PMID:24766454

  1. Sub-Chronic Neuropathological and Biochemical Changes in Mouse Visual System after Repetitive Mild Traumatic Brain Injury

    PubMed Central

    Tzekov, Radouil; Dawson, Clint; Orlando, Megan; Mouzon, Benoit; Reed, Jon; Evans, James; Crynen, Gogce; Mullan, Michael; Crawford, Fiona

    2016-01-01

    Repetitive mild traumatic brain injury (r-mTBI) results in neuropathological and biochemical consequences in the human visual system. Using a recently developed mouse model of r-mTBI, with control mice receiving repetitive anesthesia alone (r-sham) we assessed the effects on the retina and optic nerve using histology, immunohistochemistry, proteomic and lipidomic analyses at 3 weeks post injury. Retina tissue was used to determine retinal ganglion cell (RGC) number, while optic nerve tissue was examined for cellularity, myelin content, protein and lipid changes. Increased cellularity and areas of demyelination were clearly detectable in optic nerves in r-mTBI, but not in r-sham. These changes were accompanied by a ~25% decrease in the total number of Brn3a-positive RGCs. Proteomic analysis of the optic nerves demonstrated various changes consistent with a negative effect of r-mTBI on major cellular processes like depolymerization of microtubules, disassembly of filaments and loss of neurons, manifested by decrease of several proteins, including neurofilaments (NEFH, NEFM, NEFL), tubulin (TUBB2A, TUBA4A), microtubule-associated proteins (MAP1A, MAP1B), collagen (COL6A1, COL6A3) and increased expression of other proteins, including heat shock proteins (HSP90B1, HSPB1), APOE and cathepsin D. Lipidomic analysis showed quantitative changes in a number of phospholipid species, including a significant increase in the total amount of lysophosphatidylcholine (LPC), including the molecular species 16:0, a known demyelinating agent. The overall amount of some ether phospholipids, like ether LPC, ether phosphatidylcholine and ether lysophosphatidylethanolamine were also increased, while the majority of individual molecular species of ester phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were decreased. Results from the biochemical analysis correlate well with changes detected by histological and immunohistochemical methods and indicate the involvement of

  2. Sub-Chronic Neuropathological and Biochemical Changes in Mouse Visual System after Repetitive Mild Traumatic Brain Injury.

    PubMed

    Tzekov, Radouil; Dawson, Clint; Orlando, Megan; Mouzon, Benoit; Reed, Jon; Evans, James; Crynen, Gogce; Mullan, Michael; Crawford, Fiona

    2016-01-01

    Repetitive mild traumatic brain injury (r-mTBI) results in neuropathological and biochemical consequences in the human visual system. Using a recently developed mouse model of r-mTBI, with control mice receiving repetitive anesthesia alone (r-sham) we assessed the effects on the retina and optic nerve using histology, immunohistochemistry, proteomic and lipidomic analyses at 3 weeks post injury. Retina tissue was used to determine retinal ganglion cell (RGC) number, while optic nerve tissue was examined for cellularity, myelin content, protein and lipid changes. Increased cellularity and areas of demyelination were clearly detectable in optic nerves in r-mTBI, but not in r-sham. These changes were accompanied by a ~25% decrease in the total number of Brn3a-positive RGCs. Proteomic analysis of the optic nerves demonstrated various changes consistent with a negative effect of r-mTBI on major cellular processes like depolymerization of microtubules, disassembly of filaments and loss of neurons, manifested by decrease of several proteins, including neurofilaments (NEFH, NEFM, NEFL), tubulin (TUBB2A, TUBA4A), microtubule-associated proteins (MAP1A, MAP1B), collagen (COL6A1, COL6A3) and increased expression of other proteins, including heat shock proteins (HSP90B1, HSPB1), APOE and cathepsin D. Lipidomic analysis showed quantitative changes in a number of phospholipid species, including a significant increase in the total amount of lysophosphatidylcholine (LPC), including the molecular species 16:0, a known demyelinating agent. The overall amount of some ether phospholipids, like ether LPC, ether phosphatidylcholine and ether lysophosphatidylethanolamine were also increased, while the majority of individual molecular species of ester phospholipids, like phosphatidylcholine and phosphatidylethanolamine, were decreased. Results from the biochemical analysis correlate well with changes detected by histological and immunohistochemical methods and indicate the involvement of

  3. The spectrum of neurobehavioral sequelae after repetitive mild traumatic brain injury: a novel mouse model of chronic traumatic encephalopathy.

    PubMed

    Petraglia, Anthony L; Plog, Benjamin A; Dayawansa, Samantha; Chen, Michael; Dashnaw, Matthew L; Czerniecka, Katarzyna; Walker, Corey T; Viterise, Tyler; Hyrien, Ollivier; Iliff, Jeffrey J; Deane, Rashid; Nedergaard, Maiken; Huang, Jason H

    2014-07-01

    There has been an increased focus on the neurological sequelae of repetitive mild traumatic brain injury (TBI), particularly neurodegenerative syndromes, such as chronic traumatic encephalopathy (CTE); however, no animal model exists that captures the behavioral spectrum of this phenomenon. We sought to develop an animal model of CTE. Our novel model is a modification and fusion of two of the most popular models of TBI and allows for controlled closed-head impacts to unanesthetized mice. Two-hundred and eighty 12-week-old mice were divided into control, single mild TBI (mTBI), and repetitive mTBI groups. Repetitive mTBI mice received six concussive impacts daily for 7 days. Behavior was assessed at various time points. Neurological Severity Score (NSS) was computed and vestibulomotor function tested with the wire grip test (WGT). Cognitive function was assessed with the Morris water maze (MWM), anxiety/risk-taking behavior with the elevated plus maze, and depression-like behavior with the forced swim/tail suspension tests. Sleep electroencephalogram/electromyography studies were performed at 1 month. NSS was elevated, compared to controls, in both TBI groups and improved over time. Repetitive mTBI mice demonstrated transient vestibulomotor deficits on WGT. Repetitive mTBI mice also demonstrated deficits in MWM testing. Both mTBI groups demonstrated increased anxiety at 2 weeks, but repetitive mTBI mice developed increased risk-taking behaviors at 1 month that persist at 6 months. Repetitive mTBI mice exhibit depression-like behavior at 1 month. Both groups demonstrate sleep disturbances. We describe the neurological sequelae of repetitive mTBI in a novel mouse model, which resemble several of the neuropsychiatric behaviors observed clinically in patients sustaining repetitive mild head injury.

  4. Cardiac fibroblast–derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy

    PubMed Central

    Bang, Claudia; Batkai, Sandor; Dangwal, Seema; Gupta, Shashi Kumar; Foinquinos, Ariana; Holzmann, Angelika; Just, Annette; Remke, Janet; Zimmer, Karina; Zeug, Andre; Ponimaskin, Evgeni; Schmiedl, Andreas; Yin, Xiaoke; Mayr, Manuel; Halder, Rashi; Fischer, Andre; Engelhardt, Stefan; Wei, Yuanyuan; Schober, Andreas; Fiedler, Jan; Thum, Thomas

    2014-01-01

    In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast–derived exosomes revealed a relatively high abundance of many miRNA passenger strands (“star” miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal–derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II–induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA–enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target. PMID:24743145

  5. VEGF-C and aortic cardiomyocytes guide coronary artery stem development

    PubMed Central

    Chen, Heidi I.; Poduri, Aruna; Numi, Harri; Kivela, Riikka; Saharinen, Pipsa; McKay, Andrew S.; Raftrey, Brian; Churko, Jared; Tian, Xueying; Zhou, Bin; Wu, Joseph C.; Alitalo, Kari; Red-Horse, Kristy

    2014-01-01

    Coronary arteries (CAs) stem from the aorta at 2 highly stereotyped locations, deviations from which can cause myocardial ischemia and death. CA stems form during embryogenesis when peritruncal blood vessels encircle the cardiac outflow tract and invade the aorta, but the underlying patterning mechanisms are poorly understood. Here, using murine models, we demonstrated that VEGF-C–deficient hearts have severely hypoplastic peritruncal vessels, resulting in delayed and abnormally positioned CA stems. We observed that VEGF-C is widely expressed in the outflow tract, while cardiomyocytes develop specifically within the aorta at stem sites where they surround maturing CAs in both mouse and human hearts. Mice heterozygous for islet 1 (Isl1) exhibited decreased aortic cardiomyocytes and abnormally low CA stems. In hearts with outflow tract rotation defects, misplaced stems were associated with shifted aortic cardiomyocytes, and myocardium induced ectopic connections with the pulmonary artery in culture. These data support a model in which CA stem development first requires VEGF-C to stimulate vessel growth around the outflow tract. Then, aortic cardiomyocytes facilitate interactions between peritruncal vessels and the aorta. Derangement of either step can lead to mispatterned CA stems. Studying this niche for cardiomyocyte development, and its relationship with CAs, has the potential to identify methods for stimulating vascular regrowth as a treatment for cardiovascular disease. PMID:25271623

  6. Prostaglandin E₂ promotes post-infarction cardiomyocyte replenishment by endogenous stem cells.

    PubMed

    Hsueh, Ying-Chang; Wu, Jasmine M F; Yu, Chun-Keung; Wu, Kenneth K; Hsieh, Patrick C H

    2014-04-01

    Although self-renewal ability of adult mammalian heart has been reported, few pharmacological treatments are known to promote cardiomyocyte regeneration after injury. In this study, we demonstrate that the critical period of stem/progenitor cell-mediated cardiomyocyte replenishment is initiated within 7 days and saturates on day 10 post-infarction. Moreover, blocking the inflammatory reaction with COX-2 inhibitors may also reduce the capability of endogenous stem/progenitor cells to repopulate lost cells. Injection of the COX-2 product PGE2 enhances cardiomyocyte replenishment in young mice and recovers cell renewal through attenuating TGF-β1 signaling in aged mice. Further analyses suggest that cardiac stem cells are PGE2-responsive and that PGE2 may regulate stem cell activity directly through the EP2 receptor or indirectly by modulating its micro-environment in vivo. Our findings provide evidence that PGE2 holds great potential for cardiac regeneration.

  7. Trichloroethylene exposure reduces liver injury in a mouse model of primary biliary cholangitis.

    PubMed

    Ray, Jessica L; Kopec, Anna K; Joshi, Nikita; Cline-Fedewa, Holly; Lash, Lawrence H; Williams, Kurt J; Leung, Patrick S; Gershwin, M Eric; Luyendyk, James P

    2017-01-23

    Trichloroethylene (TCE) is a persistent environmental contaminant proposed to contribute to autoimmune disease. Experimental studies in lupus-prone MRL(+/+) mice have suggested that TCE exposure can trigger autoimmune hepatitis. The vast majority of studies examining the connection between TCE and autoimmunity utilize this model, and the impact of TCE exposure in other established models of autoimmune liver disease is not known. We tested the hypothesis that TCE exposure exacerbates experimental hepatic autoimmunity in dominant negative transforming growth factor beta receptor type II (dnTGFBRII) mice, which develop serological and histological features resembling human primary biliary cholangitis. Female 8-week-old wild-type and dnTGFBRII mice were exposed to TCE (0.5 mg/ml) or vehicle (1% ethoxylated castor oil) in the drinking water for 12 or 22 weeks. Liver histopathology in 20- and 30-week-old wild-type mice was unremarkable irrespective of treatment. Mild portal inflammation was observed in vehicle-exposed 20-week-old dnTGFBRII mice and was not exacerbated by TCE exposure. Vehicle-exposed 30-week-old dnTGFBRII mice developed anti-mitochondrial antibodies, marked hepatic inflammation with necrosis, and hepatic accumulation of both B and T lymphocytes. To our surprise, TCE exposure dramatically reduced hepatic parenchymal inflammation and injury in 30-week-old dnTGFBRII mice, reflected by changes in hepatic proinflammatory gene expression, serum chemistry, and histopathology. Interestingly, TCE did not affect hepatic B cell accumulation or induction of the anti-inflammatory cytokine IL10. These data indicate that TCE exposure reduces autoimmune liver injury in female dnTGFBRII mice and suggests that the precise effect of environmental chemicals in autoimmunity depends on the experimental model.

  8. Development of a novel mouse model of amodiaquine-induced liver injury with a delayed onset.

    PubMed

    Metushi, Imir G; Cai, Ping; Dervovic, Dzana; Liu, Feng; Lobach, Alexandra; Nakagawa, Tetsuya; Uetrecht, Jack

    2015-01-01

    Amodiaquine (AQ) treatment is associated with a high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. Evidence suggests that AQ-induced IDILI is immune mediated. A significant impediment to mechanistic studies of IDILI is the lack of valid animal models. This study reports the first animal model of IDILI with characteristics similar to mild IDILI in humans. Treatment of female C57BL/6 mice with AQ led to liver injury with delayed onset, which resolved despite continued treatment. Covalent binding of AQ was detected in the liver, which was greater in female than in male mice, and higher in the liver than in other organs. Covalent binding in the liver was maximal by Day 3, which did not explain the delayed onset of alanine aminotransferase (ALT) elevation. However, coincident with the elevated serum ALT, infiltration of liver and splenic mononuclear cells and activation of CD8 T-cells within the liver were identified. By Week 7, when ALT levels had returned close to normal, down-regulation of several inflammatory cytokines and up-regulation of PD-1 on T-cells suggested induction of immune tolerance. Treatment of Rag1(-/-) mice with AQ resulted in higher ALT activities than C57BL/6 mice, which suggested that the adaptive immune response was responsible for immune tolerance. In contrast, depletion of NK cells significantly attenuated the increase in ALT, which implied a role for NK cells in mild AQ-induced IDILI. This is the first example of a delayed-onset animal model of IDILI that appears to be immune-mediated.

  9. Tolerance to ischaemic injury in remodelled mouse hearts: less ischaemic glycogenolysis and preserved metabolic efficiency

    PubMed Central

    Masoud, Waleed G.T.; Abo Al-Rob, Osama; Yang, Yang; Lopaschuk, Gary D.; Clanachan, Alexander S.

    2015-01-01

    Aims Post-infarction remodelled failing hearts have reduced metabolic efficiency. Paradoxically, they have increased tolerance to further ischaemic injury. This study was designed to investigate the metabolic mechanisms that may contribute to this phenomenon and to examine the relationship between ischaemic tolerance and metabolic efficiency during post-ischaemic reperfusion. Methods and results Male C57BL/6 mice were subjected to coronary artery ligation (CAL) or SHAM surgery. After 4 weeks, in vivo mechanical function was assessed by echocardiography, and then isolated working hearts were perfused in this sequence: 45 min aerobic, 15 min global no-flow ischaemia, and 30 min aerobic reperfusion. Left ventricular (LV) function, metabolic rates, and metabolic efficiency were measured. Relative to SHAM, both in vivo and in vitro CAL hearts had depressed cardiac function under aerobic conditions (45 and 36%, respectively), but they had a greater recovery of LV function during post-ischaemic reperfusion (67 vs. 49%, P < 0.05). While metabolic efficiency (LV work per ATP produced) was 50% lower during reperfusion of SHAM hearts, metabolic efficiency in CAL hearts did not decrease. During ischaemia, glycogenolysis was 28% lower in CAL hearts, indicative of lower ischaemic proton production. There were no differences in mitochondrial abundance, calcium handling proteins, or key metabolic enzymes. Conclusion Compared with SHAM, remodelled CAL hearts are more tolerant to ischaemic injury and undergo no further deterioration of metabolic efficiency during reperfusion. Less glycogen utilization in CAL hearts during ischaemia may contribute to increased ischaemic tolerance by limiting ischaemic proton production that may improve ion homeostasis during early reperfusion. PMID:26150203

  10. Hepatogenic differentiation from human adipose-derived stem cells and application for mouse acute liver injury.

    PubMed

    Guo, De-Liang; Wang, Zhi-Gang; Xiong, Liang-Kun; Pan, Le-Yu; Zhu, Qian; Yuan, Yu-Feng; Liu, Zhi-Su

    2017-03-01

    Adipose-derived stem cells (ADSCs) derived from adipose tissue have the capacity to differentiate into endodermal, mesoderm, and ectodermal cell lineages in vitro, which are an ideal engraft in tissue-engineered repair. In this study, human ADSCs were isolated from subcutaneous fat. The markers of ADSCs, CD13, CD71, CD73, CD90, CD105, CD166, CYP3A4, and ALB were detected by immunofluorescence assays. Human ADSCs were cultured in a specific hepatogenesis differentiation medium containing HGF, bFGF, nicotinamide, ITS, and oncostatin M for hepatogenic differentiation. The hepatocyte markers were analyzed using immunofluorescence and real-time PCR after dramatic changes in morphology. Hepatocytes derived from ADSCs or ADSCs were transplanted into the mice of liver injury for observation cells colonization and therapy in liver tissue. The result demonstrated that human ADSCs were positive for the CD13, CD71, CD73, CD90, CD105, and CD166 but negative for hepatocyte markers, ALB and CYP3A4. After hepatogenic differentiation, the hepatocytes were positive for liver special markers, gene expression level showed a time-lapse increase with induction time. Human ADSCs or ADSCs-derived hepatocyte injected into the vein could improve liver function repair and functionally rescue the CCl4-treated mice with liver injury, but the ADSCs transplantation was better than ADSCs-derived hepatocyte transplantation. In conclusion, our research shows that a population of hepatocyte can be specifically generated from human ADSCs and that cells may allow for participation in tissue-repair.

  11. PTEN-β-Catenin Signaling Modulates Regulatory T Cells and Inflammatory Responses in Mouse Liver Ischemia and Reperfusion Injury.

    PubMed

    Zhu, Qiang; Li, Changyong; Wang, Kunpeng; Yue, Shi; Jiang, Longfeng; Ke, Michael; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Zhang, Feng; Lu, Ling; Ke, Bibo

    2017-02-02

    The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays an important role in regulating T cell activation during inflammatory response. Activation of β-catenin is crucial for maintaining immune homeostasis. This study investigates the functional roles and molecular mechanisms by which PTEN-β-catenin signaling promotes regulatory T cell (Treg) induction in a mouse model of liver ischemia and reperfusion injury (IRI). We found that mice with myeloid specific PTEN knockout (PTEN(M-KO) ) exhibited reduced liver damage as evidenced by decreased levels of serum ALT, intrahepatic macrophage trafficking, and pro-inflammatory mediators compared to the PTEN-proficient (PTEN(FL/FL) ) controls. Disruption of myeloid PTEN activated β-catenin, which in turn promoted PPARγ-mediated Jagged-1/Notch signaling and induced Foxp3(+) Tregs while inhibiting Th17 cells. However, blocking of Notch signaling by inhibiting γ-secretase reversed myeloid PTEN deficiency-mediated protection in IR-triggered liver inflammation with reduced Foxp3(+) and increased RORγt-mediated IL-17A expression in ischemic livers. Moreover, knockdown of β-catenin or PPARγ in PTEN-deficient macrophages inhibited Jagged-1/Notch activation and reduced Foxp3(+) Treg induction, leading to increased proinflammatory mediators in macrophage/T cell co-cultures. In conclusion, our findings demonstrate that PTEN-β-catenin signaling is a novel regulator involved in modulating Treg development and provides a potential therapeutic target in liver IRI. This article is protected by copyright. All rights reserved.

  12. Single-cell transcriptome and epigenomic reprogramming of cardiomyocyte-derived cardiac progenitor cells

    PubMed Central

    Chen, Xin; Chakravarty, Tushar; Zhang, Yiqiang; Li, Xiaojin; Zhong, Jiang F.; Wang, Charles

    2016-01-01

    The molecular basis underlying the dedifferentiation of mammalian adult cardiomyocytes (ACMs) into myocyte-derived cardiac progenitor cells (mCPCs) during cardiac tissue regeneration is poorly understood. We present data integrating single-cell transcriptome and whole-genome DNA methylome analyses of mouse mCPCs to understand the epigenomic reprogramming governing their intrinsic cellular plasticity. Compared to parental cardiomyocytes, mCPCs display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlating well with the methylome, our single-cell transcriptomic data show 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, implanting mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. This dataset suggests that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration. Understanding cardiomyocyte epigenomic reprogramming may enable the design of future clinical therapies that induce cardiac regeneration, and prevent heart failure. PMID:27622691

  13. Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes.

    PubMed

    Raulf, Alexandra; Voeltz, Nadine; Korzus, Daniel; Fleischmann, Bernd K; Hesse, Michael

    2017-02-24

    Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

  14. Gene-Expression Changes in Cerium Chloride-Induced Injury of Mouse Hippocampus

    PubMed Central

    Sheng, Lei; Zhu, Liyuan; Guan, Ning; Gui, Suxin; Sang, Xuezi; Zhao, Xiaoyang; Sun, Qingqing; Wang, Ling; Cheng, Jie; Hu, Renping; Hong, Fashui

    2013-01-01

    Cerium is widely used in many aspects of modern society, including agriculture, industry and medicine. It has been demonstrated to enter the ecological environment, is then transferred to humans through food chains, and causes toxic actions in several organs including the brain of animals. However, the neurotoxic molecular mechanisms are not clearly understood. In this study, mice were exposed to 0.5, 1, and 2 mg/kg BW cerium chloride (CeCl3) for 90 consecutive days, and their learning and memory ability as well as hippocampal gene expression profile were investigated. Our findings suggested that exposure to CeCl3 led to hippocampal lesions, apoptosis, oxidative stress and impairment of spatial recognition memory. Furthermore, microarray data showed marked alterations in the expression of 154 genes involved in learning and memory, immunity and inflammation, signal transduction, apoptosis and response to stress in the 2 mg/kg CeCl3 exposed hippocampi. Specifically, the significant up-regulation of Axud1, Cdc37, and Ube2v1 caused severe apoptosis, and great suppression of Adcy8, Fos, and Slc5a7 expression led to impairment of mouse cognitive ability. Therefore, Axud1, Cdc37, Ube2v1, Adcy8, Fos, and Slc5a7 may be potential biomarkers of hippocampal toxicity caused by CeCl3 exposure. PMID:23573234

  15. Thalidomide Ameliorates Inflammation and Vascular Injury but Aggravates Tubular Damage in the Irradiated Mouse Kidney

    SciTech Connect

    Scharpfenecker, Marion; Floot, Ben; Russell, Nicola S.; Coppes, Rob P.; Stewart, Fiona A.

    2014-07-01

    Purpose: The late side effects of kidney irradiation include vascular damage and fibrosis, which are promoted by an irradiation-induced inflammatory response. We therefore treated kidney-irradiated mice with the anti-inflammatory and angiogenesis-modulating drug thalidomide in an attempt to prevent the development of late normal tissue damage and radiation nephropathy in the mouse kidney. Methods and Materials: Kidneys of C57Bl/6 mice were irradiated with a single dose of 14 Gy. Starting from week 16 after irradiation, the mice were fed with thalidomide-containing chow (100 mg/kg body weight/day). Gene expression and kidney histology were analyzed at 40 weeks and blood samples at 10, 20, 30, and 40 weeks after irradiation. Results: Thalidomide improved the vascular structure and vessel perfusion after irradiation, associated with a normalization of pericyte coverage. The drug also reduced infiltration of inflammatory cells but could not suppress the development of fibrosis. Irradiation-induced changes in hematocrit and blood urea nitrogen levels were not rescued by thalidomide. Moreover, thalidomide worsened tubular damage after irradiation and also negatively affected basal tubular function. Conclusions: Thalidomide improved the inflammatory and vascular side effects of kidney irradiation but could not reverse tubular toxicity, which probably prevented preservation of kidney function.

  16. Dexmedetomidine attenuates acute lung injury induced by lipopolysaccharide in mouse through inhibition of MAPK pathway.

    PubMed

    Xu, Yingzhen; Zhang, Ruyi; Li, Chunli; Yin, Xue; Lv, Changjun; Wang, Yaoqi; Zhao, Wenxiang; Zhang, Xiuli

    2015-10-01

    Dexmedetomidine (Dex) is widely used for sedation in intensive care units and can be used as an adjunct to anesthetics. Previous studies have demonstrated that Dex has anti-inflammatory property. In this study, we aim to explore the potential therapeutic effects and mechanisms of Dex on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. To induce ALI, mice were intraperitoneally injected with LPS, while Dex was treated 1 h before LPS injection. The inflammation of lung tissues was evaluated by HE stain, and bronchoalveolar lavage fluid (BALF) was obtained after 6 h to measure protein concentrations. We also used an enzyme-linked immunosorbent assay to detect the secretion levels of proinflammatory cytokines in the serum. Western blotting method was adopted to observe changes in mitogen-activated protein kinases and downstream nuclear transcription factors. The results showed that pretreatment with Dex considerably reduced neutrophil infiltration and pulmonary edema, and significantly reduced protein concentrations in the BALF, as well as suppressed LPS-induced elevation of proinflammatory cytokines (TNF-α and IL-1β) in the serum. In addition, we observed that the molecular mechanism of Dex-mediated anti-inflammation is associated with decreasing phosphorylation of MKK4, MMK3/6, ERK1/2, p38MAPK, and JNK, and diminishing activation of Elk-1, c-Jun, and ATF-2. Dex could attenuate ALI induced by LPS in mice, and this effect may be mediated through the inhibition of MAPK pathway.

  17. Injected matrix stimulates myogenesis and regeneration of mouse skeletal muscle after ischaemic injury.

    PubMed

    Kuraitis, D; Ebadi, D; Zhang, P; Rizzuto, E; Vulesevic, B; Padavan, D T; Al Madhoun, A; McEwan, K A; Sofrenovic, T; Nicholson, K; Whitman, S C; Mesana, T G; Skerjanc, I S; Musarò, A; Ruel, M; Suuronen, E J

    2012-09-12

    Biomaterial-guided regeneration represents a novel approach for the treatment of myopathies. Revascularisation and the intramuscular extracellular matrix are important factors in stimulating myogenesis and regenerating muscle damaged by ischaemia. In this study, we used an injectable collagen matrix, enhanced with sialyl LewisX (sLeX), to guide skeletal muscle differentiation and regeneration. The elastic properties of collagen and sLeX-collagen matrices were similar to those of skeletal muscle, and culture of pluripotent mESCs on the matrices promoted their differentiation into myocyte-like cells expressing Pax3, MHC3, myogenin and Myf5. The regenerative properties of matrices were evaluated in ischaemic mouse hind-limbs. Treatment with the sLeX-matrix augmented the production of myogenic-mediated factors insulin-like growth factor (IGF)-1, and IGF binding protein-2 and -5 after 3 days. This was followed by muscle regeneration, including a greater number of regenerating myofibres and increased transcription of Six1, M-cadherin, myogenin and Myf5 after 10 days. Simultaneously, the sLeX-matrix promoted increased mobilisation and engraftment of bone marrow-derived progenitor cells, the development of larger arterioles and the restoration of tissue perfusion. Both matrix treatments tended to reduce maximal forces of ischaemic solei muscles, but sLeX-matrix lessened this loss of force and also prevented muscle fatigue. Only sLeX-matrix treatment improved mobility of mice on a treadmill. Together, these results suggest a novel approach for regenerative myogenesis, whereby treatment only with a matrix, which possesses an inherent ability to guide myogenic differentiation of pluripotent stem cells, can enhance the endogenous vascular and myogenic regeneration of skeletal muscle, thus holding promise for future clinical use.

  18. A Major Effect QTL on Chromosome 18 for Noise Injury to the Mouse Cochlear Lateral Wall

    PubMed Central

    Ohlemiller, Kevin K.; Rosen, Allyson D.; Gagnon, Patricia M.

    2009-01-01

    We recently demonstrated a striking difference among inbred mouse strains in the effects of a single noise exposure, whereby CBA/J and CBA/CaJ (CBA) mice show moderate reversible reduction in the endocochlear potential (EP) while C57BL/6J (B6) mice do not (Ohlemiller, K.K., Gagnon, P.M. 2007. Genetic dependence of cochlear cells and structures injured by noise. Hearing Res. 224, 34-50). Acute EP reduction in CBA was reliably associated with characteristic pathology of the spiral ligament and stria vascularis, both immediately after noise and 8 weeks later. Analysis of B6×CBA F1 hybrid mice indicated that EP reduction and its anatomic correlates are co-inherited in an autosomal dominant manner. Further analysis of N2 mice resulting from the backcross of F1 hybrids to B6 mice led us to suggest that the EP reduction phenotype principally reflects the influence of a small number of quantitative trait loci (QTLs). Here we report the results of QTL mapping of the EP reduction phenotype in CBA/J using 106 N2 mice from a (CBA×B6) × B6 backcross. Correlation of acute post-noise EP with 135 markers distributed throughout the genome revealed a single major effect QTL on chromosome 18 (12.5 cM, LOD 3.57) (Nirep, for Noise-induced reduction in EP QTL), and two marginally significant QTLs on chromosomes 5 and 16 (LOD 1.43 and 1.73, respectively). Our results underscore that fact that different cochlear structures may possess different susceptibilities to noise through the influence of non-overlapping genes. While Nirep and similar-acting QTLs do not appear to influence the extent of permanent hearing loss from a single noise exposure, they could reduce the homeostatic ‘reserve’ of the lateral wall in protracted or continual exposures, and thereby influence long term threshold stability. PMID:19913606

  19. The yellow fluorescent protein (YFP-H) mouse reveals neuroprotection as a novel mechanism underlying Chondroitinase ABC-mediated repair following spinal cord injury

    PubMed Central

    Carter, Lucy M.; Starkey, Michelle L.; Akrimi, Sonia F.; Davies, Meirion; McMahon, Stephen B.; Bradbury, Elizabeth J.

    2012-01-01

    Chondroitinase ABC (ChABC) represents a promising therapeutic strategy for the treatment of spinal cord injury due to its potent effects on restoring function to spinal injured adult mammals. However, there is limited mechanistic insight as to the underlying effects of ChABC treatment, where the effects are mediated, and which signalling pathways are involved in ChABC-mediated repair. Here we utilise a transgenic (YFP-H) mouse to demonstrate that cortical layer V projection neurons undergo severe atrophy four weeks following thoracic dorsal column injury and that ChABC is neuroprotective for these neurons following ICV infusion. ChABC also prevented cell atrophy following localised delivery to the spinal cord, suggesting a possible retrograde neuroprotective effect mediated at the injury site. Furthermore, neuroprotection of corticospinal cell somata coincided with increased axonal sprouting in the spinal cord. In addition, Western blot analysis of a number of kinases important in survival and growth signalling revealed a significant increase in phosphorylated ERK1 at the spinal injury site following in vivo ChABC treatment, indicating that activated ERK may play a role in downstream repair processes following ChABC treatment. Total forms of PKC and AKT were also elevated, indicating that modification of the glial scar by ChABC promotes long-lasting signalling changes at the lesion site. Thus, using the YFP-H mouse as a novel tool to study degenerative changes and repair following spinal cord injury we demonstrate, for the first time, that ChABC treatment regulates multiple signalling cascades at the injury site and exerts protective effects on axotomised corticospinal projection neurons. PMID:19109493

  20. Regulation of cardiomyocyte autophagy by calcium.

    PubMed

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

    2016-04-15

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

  1. Characterization of a nose-only inhaled phosgene acute lung injury mouse model

    PubMed Central

    Plahovinsak, Jennifer L.; Perry, Mark R.; Knostman, Katherine A.; Segal, Robert; Babin, Michael C.

    2016-01-01

    Context Phosgene’s primary mode of action is as a pulmonary irritant characterized by its early latent phase where life-threatening, non-cardiogenic pulmonary edema is typically observed 6–24 h post-exposure. Objective To develop an inhaled phosgene acute lung injury (ALI) model in C57BL/6 mice that can be used to screen potential medical countermeasures. Methods A Cannon style nose-only inhalation exposure tower was used to expose mice to phosgene (8 ppm) or air (sham). An inhalation lethality study was conducted to determine the 8 ppm median lethal exposure (LCt50) at 24 and 48 h post-exposure. The model was then developed at 1.2 times the 24 h LCt50. At predetermined serial sacrifice time points, survivors were euthanized, body and lung weights collected, and lung tissues processed for histopathology. Additionally, post-exposure clinical observations were used to assess quality of life. Results and discussion The 24-hour LCt50 was 226ppm*min (8 ppm for 28.2 min) and the 48-hour LCt50 was 215ppm*min (8 ppm for 26.9 min). The phosgene exposed animals had a distinct progression of clinical signs, histopathological changes and increased lung/body weight ratios. Early indicators of a 1.2 times the 24-hour LCt50 phosgene exposure were significant changes in the lung-to-body weight ratios by 4 h post-exposure. The progression of clinical signs and histopathological changes were important endpoints for characterizing phosgene-induced ALI for future countermeasure studies. Conclusion An 8 ppm phosgene exposure for 34 min (1.2 × LCt50) is the minimum challenge recommended for evaluating therapeutic interventions. The predicted higher mortality in the phosgene-only controls will help demonstrate efficacy of candidate treatments and increase the probability that a change in survival rate is statistically significant PMID:26671199

  2. Involvement of Mouse Constitutive Androstane Receptor in Acifluorfen-Induced Liver Injury and Subsequent Tumor Development.

    PubMed

    Kuwata, Kazunori; Inoue, Kaoru; Ichimura, Ryohei; Takahashi, Miwa; Kodama, Yukio; Shibutani, Makoto; Yoshida, Midori

    2016-06-01

    Acifluorfen (ACI), a protoporphyrinogen oxidase (PROTOX) inhibitor herbicide, promotes the accumulation of protoporphyrin IX (PPIX), and induces tumors in the rodent liver. Porphyria is a risk factor for liver tumors in humans; however, the specific mechanisms through which ACI induces hepatocarcinogenesis in rodents are unclear. Here, we investigated the mode of action of ACI-induced hepatocarcinogenesis, focusing on constitutive androstane receptor (CAR, NR1I3), which is essential for the development of rodent liver tumors in response to certain cytochrome P450 (CYP) 2B inducers. Dietary treatment with 2500 ppm ACI for up to 13 weeks increased Cyp2b10 expression in the livers of wild-type (WT) mice, but not in CAR-knockout (CARKO) mice. Microscopically, ACI treatment-induced cytotoxic changes, including hepatocellular necrosis and inflammation, and caused regenerative changes accompanied by prolonged increases in the numbers of proliferating cell nuclear antigen-positive hepatocytes in WT mice. In contrast, these cytotoxic and regenerative changes in hepatocytes were significantly attenuated, but still observed, in CARKO mice. ACI treatment also increased liver PPIX levels similarly in both genotypes; however, no morphological evidence of porphyrin deposition was found in hepatocytes from either genotype. Treatment with 2500 ppm ACI for 26 weeks after initiation with diethylnitrosamine increased the incidence and multiplicities of altered foci and adenomas in hepatocytes from WT mice; these effects were significantly reduced in CARKO mice. These results indicated that prolonged cytotoxicity in the liver was a key factor for ACI-induced hepatocarcinogenesis, and that CAR played an important role in ACI-induced liver injury and tumor development in mice.

  3. Lentiviral vector PLV-PI3KCG gene transfer inhibits hypoxic cardiomyocytes apoptosis

    PubMed Central

    Li, Yan-Yan; Zhang, Hui; Lu, Xin-Zheng

    2015-01-01

    The PI3K/Akt signal pathway was suggested to be associated with apoptosis. However, it was still unclear whether activated PI3K/Akt signaling pathway could inhibit hypoxic cardiomyocytes apoptosis. In this research, the recombinant PI3KCG lentiviral vector plasmid (PLV-PI3KCG) was constructed and transfected into neonatal rat hypoxia/reoxygenation (H/R) injury cardiomyocytes models which were randomly divided into five groups as the normal control group, H/R group, HR empty plasmid group (HRE group), HR PLV-PI3KCG transfection preconditioning group (HRP group), and HR PLV-PI3KCG transfection + LY294002 group (HRPL group). Compared with the H/R, HRE and HRPL groups, the cardiomyocytes beat frequency and survival rate in the HRP group were significantly increased (P<0.05) and the released LDH were significantly decreased (P<0.05). The Bcl-2/Bax ratio was significantly lower in H/R, HRE and HRPL groups than that in HRP group (P<0.05). Activated PI3K/Akt signaling pathway could play a protection role in the cardiomyocytes H/R injury process which could be inhibited by LY294002. PMID:26884933

  4. Nuclear Magnetic Resonance Metabolomic Profiling of Mouse Kidney, Urine and Serum Following Renal Ischemia/Reperfusion Injury

    PubMed Central

    Leenders, Justine; Poma, Laurence; Defraigne, Jean-Olivier; Krzesinski, Jean-Marie; de Tullio, Pascal

    2016-01-01

    Background Ischemia/reperfusion (I/R) is the most common cause of acute kidney injury (AKI). Its pathophysiology remains unclear. Metabolomics is dedicated to identify metabolites involved in (patho)physiological changes of integrated living systems. Here, we performed 1H-Nuclear Magnetic Resonance metabolomics using urine, serum and kidney samples from a mouse model of renal I/R. Methods Renal 30-min ischemia was induced in 12-week-old C57BL/6J male mice by bilaterally clamping vascular pedicles, and was followed by 6, 24 or 48-hour reperfusion (n = 12/group). Sham-operated mice were used as controls. Statistical discriminant analyses, i.e. principal component analysis and orthogonal projections to latent structures (OPLS-DA), were performed on urine, serum and kidney lysates at each time-point. Multivariate receiver operating characteristic (ROC) curves were drawn, and sensitivity and specificity were calculated from ROC confusion matrix (with averaged class probabilities across 100 cross-validations). Results Urine OPLS-DA analysis showed a net separation between I/R and sham groups, with significant variations in levels of taurine, di- and tri-methylamine, creatine and lactate. Such changes were observed as early as 6 hours post reperfusion. Major metabolome modifications occurred at 24h post reperfusion. At this time-point, correlation coefficients between urine spectra and conventional AKI biomarkers, i.e. serum creatinine and urea levels, reached 0.94 and 0.95, respectively. The area under ROC curve at 6h, 24h and 48h post surgery were 0.73, 0.98 and 0.97, respectively. Similar discriminations were found in kidney samples, with changes in levels of lactate, fatty acids, choline and taurine. By contrast, serum OPLS-DA analysis could not discriminate sham-operated from I/R-exposed animals. Conclusions Our study demonstrates that renal I/R in mouse causes early and sustained metabolomic changes in urine and kidney composition. The most implicated pathways at 6h

  5. Prediction of Liver Injury Induced by Chemicals in Human With a Multiparametric Assay on Isolated Mouse Liver Mitochondria

    PubMed Central

    Porceddu, Mathieu; Buron, Nelly; Borgne-Sanchez, Annie

    2012-01-01

    Drug-induced liver injury (DILI) in humans is difficult to predict using classical in vitro cytotoxicity screening and regulatory animal studies. This explains why numerous compounds are stopped during clinical trials or withdrawn from the market due to hepatotoxicity. Thus, it is important to improve early prediction of DILI in human. In this study, we hypothesized that this goal could be achieved by investigating drug-induced mitochondrial dysfunction as this toxic effect is a major mechanism of DILI. To this end, we developed a high-throughput screening platform using isolated mouse liver mitochondria. Our broad spectrum multiparametric assay was designed to detect the global mitochondrial membrane permeabilization (swelling), inner membrane permeabilization (transmembrane potential), outer membrane permeabilization (cytochrome c release), and alteration of mitochondrial respiration driven by succinate or malate/glutamate. A pool of 124 chemicals (mainly drugs) was selected, including 87 with documented DILI and 37 without reported clinical hepatotoxicity. Our screening assay revealed an excellent sensitivity for clinical outcome of DILI (94 or 92% depending on cutoff) and a high positive predictive value (89 or 82%). A highly significant relationship between drug-induced mitochondrial toxicity and DILI occurrence in patients was calculated (p < 0.001). Moreover, this multiparametric assay allowed identifying several compounds for which mitochondrial toxicity had never been described before and even helped to clarify mechanisms with some drugs already known to be mitochondriotoxic. Investigation of drug-induced loss of mitochondrial integrity and function with this multiparametric assay should be considered for integration into basic screening processes at early stage to select drug candidates with lower risk of DILI in human. This assay is also a valuable tool for assessing the mitochondrial toxicity profile and investigating the mechanism of action of new

  6. Defects in cytokine-mediated neuroprotective glial responses to excitotoxic hippocampal injury in senescence-accelerated mouse.

    PubMed

    Hasegawa-Ishii, Sanae; Takei, Shiro; Inaba, Muneo; Umegaki, Hiroyuki; Chiba, Yoichi; Furukawa, Ayako; Kawamura, Noriko; Hosokawa, Masanori; Shimada, Atsuyoshi

    2011-01-01

    Aging is a result of damage accumulation, and understanding of the mechanisms of aging requires exploration of the cellular and molecular systems functioning to control damage. Senescence-accelerated mouse prone 10 (SAMP10) has been established as an inbred strain exhibiting accelerated aging with an earlier onset of cognitive impairment due to neurodegeneration than the senescence-resistant control (SAMR1) strain. We hypothesized that tissue-protective responses of glial cells are impaired in SAMP10 mice. We injected kainic acid (KA) to induce hippocampal injury and studied how cytokines were upregulated on Day 3 using 3-month-old SAMP10 and SAMR1 mice. Following microarray-based screening for upregulated genes, we performed real-time RT-PCR and immunohistochemistry. Results indicated well-orchestrated cytokine-mediated glial interactions in the injured hippocampus of SAMR1 mice, in which microglia-derived interferon (IFN)-γ stimulated astrocytes via IFN-γ receptor and thereby induced expression of CXCL10 and macrophage inflammatory protein (MIP)-1α, and activated microglia produced granulocyte-macrophage colony-stimulating factor (GM-CSF) and osteopontin (OPN). OPN was the most strongly upregulated cytokine. CD44, an OPN receptor, was also strongly upregulated in the neuropil, especially on neurons and astrocytes. KA-induced hippocampal upregulation of these cytokines was strikingly reduced in SAMP10 mice compared to SAMR1 mice. On Day 30 after KA injection, SAMP10 but not SAMR1 mice exhibited hippocampal layer atrophy. Since the OPN-CD44 system is essential for neuroprotection and remodeling, these findings highlight the defects of SAMP10 mice in cytokine-mediated neuroprotective glia-neuron interactions, which may be associated with the mechanism underlying the vulnerability of SAMP10 mice to age-related neurodegeneration.

  7. Redox mechanisms of cardiomyocyte mitochondrial protection

    PubMed Central

    Bartz, Raquel R.; Suliman, Hagir B.; Piantadosi, Claude A.

    2015-01-01

    Oxidative and nitrosative stress are primary contributors to the loss of myocardial tissue in insults ranging from ischemia/reperfusion injury from coronary artery disease and heart transplantation to sepsis-induced myocardial dysfunction and drug-induced myocardial damage. This cell damage caused by oxidative and nitrosative stress leads to mitochondrial protein, DNA, and lipid modifications, which inhibits energy production and contractile function, potentially leading to cell necrosis and/or apoptosis. However, cardiomyocytes have evolved an elegant set of redox-sensitive mechanisms that respond to and contain oxidative and nitrosative damage. These responses include the rapid induction of antioxidant enzymes, mitochondrial DNA repair mechanisms, selective mitochondrial autophagy (mitophagy), and mitochondrial biogenesis. Coordinated cytoplasmic to nuclear cell-signaling and mitochondrial transcriptional responses to the presence of elevated cytoplasmic oxidant production, e.g., H2O2, allows nuclear translocation of the Nfe2l2 transcription factor and up-regulation of downstream cytoprotective genes such as heme oxygenase-1 which generates physiologic signals, such as CO that up-regulates Nfe212 gene transcription. Simultaneously, a number of other DNA binding transcription factors are expressed and/or activated under redox control, such as Nuclear Respiratory Factor-1 (NRF-1), and lead to the induction of genes involved in both intracellular and mitochondria-specific repair mechanisms. The same insults, particularly those related to vascular stress and inflammation also produce elevated levels of nitric oxide, which also has mitochondrial protein thiol-protective functions and induces mitochondrial biogenesis through cyclic GMP-dependent and perhaps other pathways. This brief review provides an overview of these pathways and interconnected cardiac repair mechanisms. PMID:26578967

  8. Nrf2 is critical in defense against high glucose-induced oxidative damage in cardiomyocytes.

    PubMed

    He, Xiaoqing; Kan, Hong; Cai, Lu; Ma, Qiang

    2009-01-01

    Exposure to high levels of glucose induces the production of reactive oxygen species (ROS) in cardiomyocytes that may contribute to the development of cardiomyopathy in diabetes. Nuclear factor erythroid 2-related factor 2 (Nrf2) controls the antioxidant response element (ARE)-dependent gene regulation in response to oxidative stress. The role of Nrf2 in defense against high glucose-induced oxidative damage in cardiomyocytes was investigated. Glucose at high concentrations induced ROS production in both primary neonatal and adult cardiomyocytes from the Nrf2 wild type (WT) mouse heart, whereas, in Nrf2 knockout (KO) cells, ROS was significantly higher under basal conditions and high glucose markedly further increased ROS production in concentration and time-dependent manners. Concomitantly, high glucose induced significantly higher levels of apoptosis at lower concentrations and in shorter time in Nrf2 KO cells than in WT cells. Primary adult cardiomyocytes from control and diabetic mice also showed dependence on Nrf2 function for isoproterenol-stimulated contraction. Additionally, cardiomyocytes from Nrf2 KO mice exhibited increased sensitivity to 3-nitropropionic acid, an inhibitor of mitochondrial respiratory complex II, for both ROS production and apoptosis compared with Nrf2 WT cells, further emphasizing the role of Nrf2 in ROS defense in the cells. Mechanistically, Nrf2 was shown to mediate the basal expression and induction of ARE-controlled cytoprotective genes, Nqo1 and Ho1, at both mRNA and protein levels in cardiomyocytes, as both the basal and inducible expressions of the genes were lost in Nrf2 KO cells or largely reduced by Nrf2 SiRNA. The findings, for the first time, established Nrf2 as a critical regulator of defense against ROS in normal and diabetic hearts.

  9. The effect of focal brain injury on beta-amyloid plaque deposition, inflammation and synapses in the APP/PS1 mouse model of Alzheimer's disease.

    PubMed

    Collins, Jessica M; King, Anna E; Woodhouse, Adele; Kirkcaldie, Matthew T K; Vickers, James C

    2015-05-01

    Traumatic brain injury is a risk factor for Alzheimer's disease (AD), however the effect of such neural damage on the onset and progression of beta-amyloid (Aβ) plaque pathology is not well understood. This study utilized an in vivo model of focal brain injury to examine how localized damage may acutely affect the onset and progression of Aβ plaque deposition as well as inflammatory and synaptic changes, in the APP/PS1 (APPSWE, PSEN1dE9) transgenic model of AD relative to wild-type (Wt) mice. Acute focal brain injury in 3- and 9-month-old APP/PS1 and Wt mice was induced by insertion of a needle into the somatosensory neocortex, as compared to sham surgery, and examined at 24h and 7d post-injury (PI). Focal brain injury did not induce thioflavine-S stained or (pan-Aβ antibody) MOAB-2-labeled plaques at either 24h or 7d PI in 3-month-old APP/PS1 mice or Wt mice. Nine-month-old APP/PS1 mice demonstrate cortical Aβ plaques but focal injury had no statistically significant (p>0.05) effect on thioflavine-S or MOAB-2 plaque load surrounding the injury site at 24h PI or 7d PI. There was a significant (p<0.001) increase in cross-sectional cortical area occupied by Iba-1 positive microglia in injured mice compared to sham animals, however this response did not differ between APP/PS1 and Wt mice (p>0.05). For both Wt and APP/PS1 mice alike, synaptophysin puncta near the injury site were significantly reduced 24h PI (compared to sites distant to the injury and the corresponding area in sham mice; p<0.01), but not after 7d PI (p>0.05). There was no significant effect of genotype on this response (p>0.05). These results indicate that focal brain injury and the associated microglial response do not acutely alter Aβ plaque deposition in the APP/PS1 mouse model. Furthermore the current study demonstrated that the brains of both Wt and APP/PS1 mice are capable of recovering lost synaptophysin immunoreactivity post-injury, the latter in the presence of Aβ plaque pathology that

  10. Mitochondrial delivery of Coenzyme Q10 via systemic administration using a MITO-Porter prevents ischemia/reperfusion injury in the mouse liver.

    PubMed

    Yamada, Yuma; Nakamura, Kohei; Abe, Jiro; Hyodo, Mamoru; Haga, Sanae; Ozaki, Michitaka; Harashima, Hideyoshi

    2015-09-10

    We herein report on a mitochondrial therapeutic effect based on the delivery of coenzyme Q10 (CoQ10), an anti-oxidant, to in vivo mitochondria using a MITO-Porter, a liposome-based mitochondrial delivery system that functions via membrane fusion. To evaluate the effects, we used a mouse liver ischemia/reperfusion injury (I/R injury) model, in which mitochondrial reactive oxygen species are overexpressed. We packaged CoQ10 in the lipid phase of a MITO-Porter and optimized the mitochondrial fusogenic activities to produce the CoQ10-MITO-Porter. A histological observation of the carriers in the liver by confocal laser scanning microscopy was done and the accumulation of the carrier labeled with a radio isotope in the liver confirmed that the CoQ10-MITO-Porter was delivered to liver mitochondria via systemic injection. These analytical results permitted us to optimize the compositions of the CoQ10-MITO-Porter so as to permit it to efficiently accumulate in mouse liver mitochondria. Finally, we applied the optimized CoQ10-MITO-Porter to mice via tail vein injection, and hepatic I/R injury was then induced, followed by measuring serum alanine aminotransferase (ALT) levels, a marker of liver injury. We confirmed that the use of the CoQ10-MITO-Porter resulted in a significant decrease in serum ALT levels, indicating that in vivo mitochondrial delivery of the CoQ10 via MITO-Porter prevents I/R injury in mice livers. This provides a demonstration of the potential use of such a delivery system in mitochondrial therapies.

  11. Recovery of Neurological Function Despite Immediate Sleep Disruption Following Diffuse Brain Injury in the Mouse: Clinical Relevance to Medically Untreated Concussion

    PubMed Central

    Rowe, Rachel K.; Harrison, Jordan L.; O'Hara, Bruce F.; Lifshitz, Jonathan

    2014-01-01

    Study Objective: We investigated the relationship between immediate disruption of posttraumatic sleep and functional outcome in the diffuse brain-injured mouse. Design: Adult male C57BL/6 mice were subjected to moderate midline fluid percussion injury (n = 65; 1.4 atm; 6-10 min righting reflex time) or sham injury (n = 44). Cohorts received either intentional sleep disruption (minimally stressful gentle handling) or no sleep disruption for 6 h following injury. Following disruption, serum corticosterone levels (enzyme-linked immunosorbent assay) and posttraumatic sleep (noninvasive piezoelectric sleep cages) were measured. For 1-7 days postinjury, sensorimotor outcome was assessed by Rotarod and a modified Neurological Severity Score (NSS). Cognitive function was measured using Novel Object Recognition (NOR) and Morris water maze (MWM) in the first week postinjury. Setting: Neurotrauma research laboratory. Measurements and Results: Disrupting posttraumatic sleep for 6 h did not affect serum corticosterone levels or functional outcome. In the hour following the first dark onset, sleep-disrupted mice exhibited a significant increase in sleep; however, this increase was not sustained and there was no rebound of lost sleep. Regardless of sleep disruption, mice showed a time-dependent improvement in Rotarod performance, with brain-injured mice having significantly shorter latencies on day 7 compared to sham. Further, brain-injured mice, regardless of sleep disruption, had significantly higher NSS scores postinjury compared with sham. Cognitive behavioral testing showed no group differences among any treatment group measured by MWM and NOR. Conclusion: Short-duration disruption of posttraumatic sleep did not affect functional outcome, measured by motor and cognitive performance. These data raise uncertainty about posttraumatic sleep as a mechanism of recovery from diffuse brain injury. Citation: Rowe RK; Harrison JL; O'Hara BF; Lifshitz J. Recovery of neurological

  12. Hepatoprotective Effects of Antrodia cinnamomea: The Modulation of Oxidative Stress Signaling in a Mouse Model of Alcohol-Induced Acute Liver Injury

    PubMed Central

    Liu, Yange; Wang, Juan; Li, Lanzhou; Hu, Wenji; Qu, Yidi; Ding, Yipei; Meng, Lina

    2017-01-01

    In the present study, the components of A. cinnamomea (AC) mycelia were systematically analyzed. Subsequently, its hepatoprotective effects and the underlying mechanisms were explored using a mouse model of acute alcohol-induced liver injury. AC contained 25 types of fatty acid, 16 types of amino acid, 3 types of nucleotide, and 8 types of mineral. The hepatoprotective effects were observed after 2 weeks of AC treatment at doses of 75 mg/kg, 225 mg/kg, and 675 mg/kg in the mouse model. These effects were indicated by the changes in the levels of aspartate aminotransferase, alanine aminotransferase, several oxidation-related factors, and inflammatory cytokines in serum and/or liver samples. AC reduced the incidence rate of necrosis, inflammatory infiltration, fatty droplets formation, and cell apoptosis in liver detecting via histological and TUNEL assay. In addition, AC reduced the expression of cleaved caspase-3, -8, and -9 and the levels of phosphor-protein kinase B (Akt) and phosphor-nuclear factor-κB (NF-κB) in the liver samples. Collectively, AC-mediated hepatoprotective effects in a mouse model of acute alcohol-induced liver injury are the result of reduction in oxidative stress. This may be associated with Akt/NF-κB signaling. These results provide valuable evidence to support the use of A. cinnamomea as a functional food and/or medicine. PMID:28337253

  13. Cardiomyocyte ultrastructural damage in β-thalassaemic mice

    PubMed Central

    Sanyear, Chanita; Butthep, Punnee; Nithipongvanich, Ramaneeya; Sirankapracha, Pornpan; Winichagoon, Pranee; Fucharoen, Suthat; Svasti, Saovaros

    2013-01-01

    β-thalassaemia is a hereditary anaemia resulting from the absence or reduction in β-globin chain production. Heart complications related to iron overload are the most serious cause of death in these patients. In this report cardiac pathology of β-thalassaemic mice was evaluated by light and electron microscopy. The study was carried out in thalassaemic mice carrying human β-thalassaemia mutation, IVSII-654 (654), transgenic mice carrying human βE-globin transgene insertion (E4), thalassaemic mice with human βE-globin transgene insertion (654/E4) and homozygous thalassaemic mice rescued by the human βE-globin transgene (R), which is generated by cross-breeding between the 654 and E4 mice. Histology showed iron deposition in cardiac myocytes of 654 and R mice, but the ultrastructural damage was observed only in the R mice when compared with the wild type, 654, E4 and 654/E4 mice. Histopathological changes in the cardiomyocytes of the R mice included mitochondrial swelling, loss of myofilaments and the presence of lipofuscin, related to the increased level of tissue iron content. The progressive ultrastructural pathology in R mice cardiomyocytes is consistent with the ultrastructural pathology previously studied in patients with thalassaemia. Thus, this R thalassaemic mouse model is suitable for in vivo pathophysiological study of thalassaemic heart. PMID:24020406

  14. Noninvasive in vivo magnetic resonance imaging of injury-induced neointima formation in the carotid artery of the apolipoprotein-E null mouse.

    PubMed

    Manka, D R; Gilson, W; Sarembock, I; Ley, K; Berr, S S

    2000-11-01

    Mice deficient in apolipoprotein-E (apoE) experience severe hypercholesterolemia, are prone to atherosclerosis, and recently have emerged as a powerful tool in the study of plaque formation. In this study, we developed magnetic resonance (MR) imaging methods to detect the progression of atherosclerosis noninvasively in a mouse model of arterial injury. Four 14-week-old apoE-deficient mice were imaged 5 weeks after beginning an atherogenic Western diet and 4 weeks after wire denudation injury of the left common carotid artery (LCCA). Information from several images was combined into high-information content images using methods previously developed. The image resolution was 47 x 47 x 750 microm(3). We acquired T1-, T2-, and proton density (PD)-weighted images (TR/TE 650/14, 2000/60, and 2000/14 msec, respectively). Each 8-bit image was placed in a separate color channel to produce a 24-bit color image (red = T1, green = PD, and blue = T2). The composite image created contrast between different tissue types that was superior to that of any single image and revealed significant luminal narrowing of the LCCA, but not the uninjured RCCA. MR images were compared with corresponding histopathology cross sections and luminal area measurements from each method correlated(r2= 0.61). Atherosclerotic luminal narrowing was successfully detected through MR imaging in a mouse model of arterial injury that is small, reproduces quickly, and lends itself to genetic analysis and manipulation.

  15. 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.

  16. PEDF improves cardiac function in rats with acute myocardial infarction via inhibiting vascular permeability and cardiomyocyte apoptosis.

    PubMed

    Zhang, Hao; Wang, Zheng; Feng, Shou-Jie; Xu, Lei; Shi, He-Xian; Chen, Li-Li; Yuan, Guang-Da; Yan, Wei; Zhuang, Wei; Zhang, Yi-Qian; Zhang, Zhong-Ming; Dong, Hong-Yan

    2015-03-11

    Pigment epithelium-derived factor (PEDF) is a pleiotropic gene with anti-inflammatory, antioxidant and anti-angiogenic properties. However, recent reports about the effects of PEDF on cardiomyocytes are controversial, and it is not known whether and how PEDF acts to inhibit hypoxic or ischemic endothelial injury in the heart. In the present study, adult Sprague-Dawley rat models of acute myocardial infarction (AMI) were surgically established. PEDF-small interfering RNA (siRNA)-lentivirus (PEDF-RNAi-LV) or PEDF-LV was delivered into the myocardium along the infarct border to knockdown or overexpress PEDF, respectively. Vascular permeability, cardiomyocyte apoptosis, myocardial infarct size and animal cardiac function were analyzed. We also evaluated PEDF's effect on the suppression of the endothelial permeability and cardiomyocyte apoptosis under hypoxia in vitro. The results indicated that PEDF significantly suppressed the vascular permeability and inhibited hypoxia-induced endothelial permeability through PPARγ-dependent tight junction (TJ) production. PEDF protected cardiomyocytes against ischemia or hypoxia-induced cell apoptosis both in vivo and in vitro via preventing the activation of caspase-3. We also found that PEDF significantly reduced myocardial infarct size and enhanced cardiac function in rats with AMI. These data suggest that PEDF could protect cardiac function from ischemic injury, at least by means of reducing vascular permeability, cardiomyocyte apoptosis and myocardial infarct size.

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

    PubMed Central

    Liu, Ju; Zhang, Hui

    2016-01-01

    Ischaemia/reperfusion (I/R) injury will cause additional death of cardiomyocytes in ischaemic heart disease. Recent studies revealed that renalase was involved in the I/R injury. So, the myocardial tissue-specific knockdown mouse models were needed for the investigations of renalase. To establish the mouse models, intramyocardial injection of siRNAs targeting renalase was performed in mice. The wild distribution and high transfection efficiency of the siRNAs were approved. And the renalase expression was efficiently suppressed in myocardial tissue. Compared with the high cost, time consumption, and genetic compensation risk of the Cre/loxP technology, RNA interference (RNAi) technology is much cheaper and less time-consuming. Among the RNAi technologies, injection of siRNAs is safer than virus. And considering the properties of the I/R injury mouse models, the efficiency and durability of injection with siRNAs are acceptable for the studies. Altogether, intramyocardial injection of siRNAs targeting renalase is an economical, safe, and efficient method to establish myocardial tissue-specific renalase knockdown mouse models. PMID:27868059

  18. Impact of mitochondria on nitrite metabolism in HL-1 cardiomyocytes

    PubMed Central

    Dungel, Peter; Teuschl, Andreas H.; Banerjee, Asmita; Paier-Pourani, Jamile; Redl, Heinz; Kozlov, Andrey V.

    2013-01-01

    Apart from ATP synthesis mitochondria have many other functions, one being nitrite reductase activity. Nitric oxide (NO) released from nitrite has been shown to protect the heart from ischemia/reperfusion (I/R) injury in a cGMP-dependent manner. However, the exact impact of mitochondria on the release of NO from nitrite in cardiomyocytes is not completely understood. Besides mitochondria, a number of non-mitochondrial metalloproteins have been suggested to facilitate this process. The aim of this study was to investigate the impact of mitochondria on the bioactivation of nitrite in HL-1 cardiomyocytes. The levels of nitrosyl complexes of hemoglobin (NO-Hb) and cGMP levels were measured by electron spin resonance spectroscopy and enzyme immunoassay. In addition the formation of free NO was determined by confocal microscopy as well as intracellular nitrite and S-nitrosothiols by chemoluminescence analysis. NO was released from nitrite in cell culture in an oxygen-dependent manner. Application of specific inhibitors of the respiratory chain, p450, NO synthases (NOS) and xanthine oxidoreductase (XOR) showed that all four enzymatic systems are involved in the release of NO, but more than 50% of NO is released via the mitochondrial pathway. Only NO released by mitochondria activated cGMP synthesis. Cardiomyocytes co-cultured with red blood cells (RBC) competed with RBC for nitrite, but free NO was detected only in HL-1 cells suggesting that RBC are not a source of NO in this model. Apart from activation of cGMP synthesis, NO formed in HL-1 cells diffused out of the cells and formed NO-Hb complexes. In addition nitrite was converted by HL-1 cells to S-nitrosyl complexes. In HL-1 cardiomyocytes, several enzymatic systems are involved in nitrite reduction to NO but only the mitochondrial pathway of NO release activates cGMP synthesis. Our data suggest that this pathway may be a key regulator of myocardial contractility especially under hypoxic conditions. PMID:23730288

  19. LOX-1 dependent overexpression of immunoglobulins in cardiomyocytes in response to angiotensin II.

    PubMed

    Kang, Bum-Yong; Hu, Changping; Prayaga, Sastry; Khaidakov, Magomed; Sawamura, Tatsuya; Seung, Ki-Bae; Mehta, Jawahar L

    2009-02-06

    LOX-1, a cell surface lectin-like receptor, is upregulated by oxidized low-density lipoprotein (ox-LDL) and angiotensin II (Ang II), and plays an important role in host defense. The specific C-type lectin domain on LOX-1 is essential for ox-LDL binding and internalization, generation of oxidant species and eliciting immune response. Here, we show that LOX-1 deletion alters genes that relate to immune response. Microarray (and qPCR) analysis of cardiac tissues showed downregulated expression of several immunoglobulins (Igk-V8, Igk-C, Igh-6, Igj, Ighg, Igh, and Igl-V1) in the LOX-1 knockout (KO) mice [p<0.05 vs. the wild-type (WT) mice]. The expression of these immunoglobulins was upregulated several-fold in the LOX-1 KO mice hearts when these mice were infused with Ang II (p<0.05, vs. WT mice). Importantly, cultured mouse HL-1 cardiomyocytes expressed these immunoglobulins, and pretreatment of cardiomyocytes with a specific anti-LOX-1 antibody enhanced the generation of immunoglobulins upon subsequent exposure to Ang II. These observations mirrored the data obtained from WT and LOX-1 KO mice hearts in the resting state and following Ang II infusion. This study provides first set of data on immunoglobulin expression in cardiac tissues of WT and LOX-1 KO mice and in cultured HL-1 cardiomyocytes, and demonstrates that LOX-1 inactivation leads to upregulation of immunoglobulins in cardiomyocytes upon challenge with Ang II.

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

    PubMed

    Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G; Bosnjak, Zeljko J

    2014-10-31

    Myocardial ischemia-reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of mitochondrial fission; and (2) the increased mitochondrial fission is resulted from both increased activation and decreased inactivation of Drp1 through Cdk1, PKCδ, and calcineurin-mediated pathways, respectively.

  1. Neural stem/progenitor cells differentiate into oligodendrocytes, reduce inflammation, and ameliorate learning deficits after transplantation in a mouse model of traumatic brain injury.

    PubMed

    Koutsoudaki, Paraskevi N; Papastefanaki, Florentia; Stamatakis, Antonios; Kouroupi, Georgia; Xingi, Evangelia; Stylianopoulou, Fotini; Matsas, Rebecca

    2016-05-01

    The central nervous system has limited capacity for regeneration after traumatic injury. Transplantation of neural stem/progenitor cells (NPCs) has been proposed as a potential therapeutic approach while insulin-like growth factor I (IGF-I) has neuroprotective properties following various experimental insults to the nervous system. We have previously shown that NPCs transduced with a lentiviral vector for IGF-I overexpression have an enhanced ability to give rise to neurons in vitro but also in vivo, upon transplantation in a mouse model of temporal lobe epilepsy. Here we studied the regenerative potential of NPCs, IGF-I-transduced or not, in a mouse model of hippocampal mechanical injury. NPC transplantation, with or without IGF-I transduction, rescued the injury-induced spatial learning deficits as revealed in the Morris Water Maze. Moreover, it had beneficial effects on the host tissue by reducing astroglial activation and microglial/macrophage accumulation while enhancing generation of endogenous oligodendrocyte precursor cells. One or two months after transplantation the grafted NPCs had migrated towards the lesion site and in the neighboring myelin-rich regions. Transplanted cells differentiated toward the oligodendroglial, but not the neuronal or astrocytic lineages, expressing the early and late oligodendrocyte markers NG2, Olig2, and CNPase. The newly generated oligodendrocytes reached maturity and formed myelin internodes. Our current and previous observations illustrate the high plasticity of transplanted NPCs which can acquire injury-dependent phenotypes within the host CNS, supporting the fact that reciprocal interactions between transplanted cells and the host tissue are an important factor to be considered when designing prospective cell-based therapies for CNS degenerative conditions.

  2. Role of cardiomyocyte circadian clock in myocardial metabolic adaptation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  3. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

    PubMed

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

    2016-02-01

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

  4. Platelet-derived growth factor (PDGF) signaling directs cardiomyocyte movement toward the midline during heart tube assembly.

    PubMed

    Bloomekatz, Joshua; Singh, Reena; Prall, Owen Wj; Dunn, Ariel C; Vaughan, Megan; Loo, Chin-San; Harvey, Richard P; Yelon, Deborah

    2017-01-18

    Communication between neighboring tissues plays a central role in guiding organ morphogenesis. During heart tube assembly, interactions with the adjacent endoderm control the medial movement of cardiomyocytes, a process referred to as cardiac fusion. However, the molecular underpinnings of this endodermal-myocardial relationship remain unclear. Here, we show an essential role for platelet-derived growth factor receptor alpha (Pdgfra) in directing cardiac fusion. Mutation of pdgfra disrupts heart tube assembly in both zebrafish and mouse. Timelapse analysis of individual cardiomyocyte trajectories reveals misdirected cells in zebrafish pdgfra mutants, suggesting that PDGF signaling steers cardiomyocytes toward the midline during cardiac fusion. Intriguingly, the ligand pdgfaa is expressed in the endoderm medial to the pdgfra-expressing myocardial precursors. Ectopic expression of pdgfaa interferes with cardiac fusion, consistent with an instructive role for PDGF signaling. Together, these data uncover a novel mechanism through which endodermal-myocardial communication can guide the cell movements that initiate cardiac morphogenesis.

  5. In situ mechanical analysis of cardiomyocytes at nano scales

    NASA Astrophysics Data System (ADS)

    Liu, Yuansheng; Feng, Jiantao; Shi, Liang; Niu, Ruibin; Sun, Quanmei; Liu, Hao; Li, Jing; Guo, Jihong; Zhu, Jihong; Han, Dong

    2011-12-01

    Nanomechanical behaviors of single living cardiomyocytes are quantitatively observed using calculated torsions and deflections of an AFM cantilever. The lateral contractions are related to the calcium intensity within rather than the vertical beating power of the cardiomyocytes. Drug-induced nanomechanical changes of cardiomyocytes were further investigated by measuring lateral contractions in real time.

  6. Resolvins AT-D1 and E1 differentially impact functional outcome, post-traumatic sleep, and microglial activation following diffuse brain injury in the mouse.

    PubMed

    Harrison, Jordan L; Rowe, Rachel K; Ellis, Timothy W; Yee, Nicole S; O'Hara, Bruce F; Adelson, P David; Lifshitz, Jonathan

    2015-07-01

    Traumatic brain injury (TBI) is induced by mechanical forces which initiate a cascade of secondary injury processes, including inflammation. Therapies which resolve the inflammatory response may promote neural repair without exacerbating the primary injury. Specific derivatives of omega-3 fatty acids loosely grouped as specialized pro-resolving lipid mediators (SPMs) and termed resolvins promote the active resolution of inflammation. In the current study, we investigate the effect of two resolvin molecules, RvE1 and AT-RvD1, on post-traumatic sleep and functional outcome following diffuse TBI through modulation of the inflammatory response. Adult, male C57BL/6 mice were injured using a midline fluid percussion injury (mFPI) model (6-10min righting reflex time for brain-injured mice). Experimental groups included mFPI administered RvE1 (100ng daily), AT-RvD1 (100ng daily), or vehicle (sterile saline) and counterbalanced with uninjured sham mice. Resolvins or saline were administered daily for seven consecutive days beginning 3days prior to TBI to evaluate proof-of-principle to improve outcome. Immediately following diffuse TBI, post-traumatic sleep was recorded for 24h post-injury. For days 1-7 post-injury, motor outcome was assessed by rotarod. Cognitive function was measured at 6days post-injury using novel object recognition (NOR). At 7days post-injury, microglial activation was quantified using immunohistochemistry for Iba-1. In the diffuse brain-injured mouse, AT-RvD1 treatment, but not RvE1, mitigated motor and cognitive deficits. RvE1 treatment significantly increased post-traumatic sleep in brain-injured mice compared to all other groups. RvE1 treated mice displayed a higher proportion of ramified microglia and lower proportion of activated rod microglia in the cortex compared to saline or AT-RvD1 treated brain-injured mice. Thus, RvE1 treatment modulated post-traumatic sleep and the inflammatory response to TBI, albeit independently of improvement in motor

  7. Synergistic Interaction of Light Alcohol Administration in the Presence of Mild Iron Overload in a Mouse Model of Liver Injury: Involvement of Triosephosphate Isomerase Nitration and Inactivation

    PubMed Central

    Gao, Wanxia; Zhao, Jie; Gao, Zhonghong

    2017-01-01

    It is well known that iron overload promotes alcoholic liver injury, but the doses of iron or alcohol used in studies are usually able to induce liver injury independently. Little attention has been paid to the coexistence of low alcohol consumption and mild iron overload when either of them is insufficient to cause obvious liver damage, although this situation is very common among some people. We studied the interactive effects and the underlining mechanism of mild doses of iron and alcohol on liver injury in a mouse model. Forty eight male Kunming mice were randomly divided into four groups: control, iron (300 mg/kg iron dextran, i.p.), alcohol (2 g/kg/day ethanol for four weeks i.g.), and iron plus alcohol group. After 4 weeks of treatment, mice were sacrificed and blood and livers were collected for biochemical analysis. Protein nitration level in liver tissue was determined by immunoprecipitation and Western blot analysis. Although neither iron overload nor alcohol consumption at our tested doses can cause severe liver injury, it was found that co-administration of the same doses of alcohol and iron resulted in liver injury and hepatic dysfunction, accompanied with elevated ratio of NADH/NAD+, reduced antioxidant ability, increased oxidative stress, and subsequent elevated protein nitration level. Further study revealed that triosephosphate isomerase, an important glycolytic enzyme, was one of the targets to be oxidized and nitrated, which was responsible for its inactivation. These data indicate that even under low alcohol intake, a certain amount of iron overload can cause significant liver oxidative damage, and the modification of triosephosphate isomerasemight be the important underlining mechanism of hepatic dysfunction. PMID:28103293

  8. Correlation of in vivo and ex vivo 1H-MRI with histology in two severities of mouse spinal cord injury

    PubMed Central

    Noristani, Harun N.; Lonjon, Nicolas; Cardoso, Maïda; Le Corre, Marine; Chan-Seng, Emilie; Captier, Guillaume; Privat, Alain; Coillot, Christophe; Goze-Bac, Christophe; Perrin, Florence E.

    2015-01-01

    Spinal cord injury (SCI) is a debilitating neuropathology with no effective treatment. Magnetic resonance imaging (MRI) technology is the only method used to assess the impact of an injury on the structure and function of the human spinal cord. Moreover, in pre-clinical SCI research, MRI is a non-invasive method with great translational potential since it provides relevant longitudinal assessment of anatomical and structural alterations induced by an injury. It is only recently that MRI techniques have been effectively used for the follow-up of SCI in rodents. However, the vast majority of these studies have been carried out on rats and when conducted in mice, the contusion injury model was predominantly chosen. Due to the remarkable potential of transgenic mice for studying the pathophysiology of SCI, we examined the use of both in and ex vivo 1H-MRI (9.4 T) in two severities of the mouse SCI (hemisection and over-hemisection) and documented their correlation with histological assessments. We demonstrated that a clear distinction between the two injury severities is possible using in and ex vivo 1H-MRI and that ex vivo MR images closely correlate with histology. Moreover, tissue modifications at a remote location from the lesion epicenter were identified by conventional ex vivo MRI analysis. Therefore, in vivo MRI has the potential to accurately identify in mice the progression of tissue alterations induced by SCI and is successfully implemented by ex vivo MRI examination. This combination of in and ex vivo MRI follow-up associated with histopathological assessment provides a valuable approach for further studies intended to evaluate therapeutic strategies on SCI. PMID:25798092

  9. RhoA signaling in cardiomyocytes protects against stress-induced heart failure but facilitates cardiac fibrosis.

    PubMed

    Lauriol, Jessica; Keith, Kimberly; Jaffré, Fabrice; Couvillon, Anthony; Saci, Abdel; Goonasekera, Sanjeewa A; McCarthy, Jason R; Kessinger, Chase W; Wang, Jianxun; Ke, Qingen; Kang, Peter M; Molkentin, Jeffery D; Carpenter, Christopher; Kontaridis, Maria I

    2014-10-21

    The Ras-related guanosine triphosphatase RhoA mediates pathological cardiac hypertrophy, but also promotes cell survival and is cardioprotective after ischemia/reperfusion injury. To understand how RhoA mediates these opposing roles in the myocardium, we generated mice with a cardiomyocyte-specific deletion of RhoA. Under normal conditions, the hearts from these mice showed functional, structural, and growth parameters similar to control mice. Additionally, the hearts of the cardiomyocyte-specific, RhoA-deficient mice subjected to transverse aortic constriction (TAC)-a procedure that induces pressure overload and, if prolonged, heart failure-exhibited a similar amount of hypertrophy as those of the wild-type mice subjected to TAC. Thus, neither normal cardiac homeostasis nor the initiation of compensatory hypertrophy required RhoA in cardiomyocytes. However, in response to chronic TAC, hearts from mice with cardiomyocyte-specific deletion of RhoA showed greater dilation, with thinner ventricular walls and larger chamber dimensions, and more impaired contractile function than those from control mice subjected to chronic TAC. These effects were associated with aberrant calcium signaling, as well as decreased activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and AKT. In addition, hearts from mice with cardiomyocyte-specific RhoA deficiency also showed less fibrosis in response to chronic TAC, with decreased transcriptional activation of genes involved in fibrosis, including myocardin response transcription factor (MRTF) and serum response factor (SRF), suggesting that the fibrotic response to stress in the heart depends on cardiomyocyte-specific RhoA signaling. Our data indicated that RhoA regulates multiple pathways in cardiomyocytes, mediating both cardioprotective (hypertrophy without dilation) and cardio-deleterious effects (fibrosis).

  10. The infarcted cardiac microenvironment cannot selectively promote embryonic stem cell differentiation into cardiomyocytes.

    PubMed

    Chen, You-Ren; Li, Yang; Chen, Li; Yang, Xin-Chun; Su, Pi-Xiong; Cai, Jun

    2011-01-01

    Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in industrialized countries. It is controversial whether embryonic stem cells are feasible sources for in situ cardiac regeneration in infarcted hearts. In order to investigate whether the infarcted cardiac microenvironment could selectively promote embryonic stem cell differentiation into cardiomyocytes, we assessed the cardiac differentiation potential of mouse embryonic stem cells (mESCs) injected into normal (n=16) or acutely infarcted rat hearts (n=18). We found that the transplanted 4',6-diamidino-2-phenylindole (DAPI)-labeled mESCs were able to survive and form stable intracardiac grafts both in normal and infarcted hearts, along with macrophages found specifically in the engraftment area. Two to four weeks after mESC transplantation, we found that more DAPI-positive mESCs differentiated into cardiomyocytes, marked by cardiac troponin T (cTnT), in normal than those in infarcted hearts (2.67±0.79% vs. 1.06±0.52%, P<.01). However, the discrepancy between the percentage of DAPI-positive cells that express cTnT in normal and that in infarcted hearts was diminished after 4 weeks (1.17±0.98% vs. 1.07±1.02%, P>.05), when the transverse striation began to present in the mESCs-derived cardiomyocytes. In addition, mESCs differentiated into vimentin-positive cardiac fibroblasts in normal and infracted hearts. Our results indicated that transplanted mESCs cannot only survive but differentiate into cardiomyocytes in infarcted rat hearts. However, the infarcted cardiac microenvironment cannot selectively promote mESCs differentiation into cardiomyocytes.

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

    PubMed

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

    2004-01-01

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

  12. Downregulation of miRNA-134 protects neural cells against ischemic injury in N2A cells and mouse brain with ischemic stroke by targeting HSPA12B.

    PubMed

    Chi, W; Meng, F; Li, Y; Wang, Q; Wang, G; Han, S; Wang, P; Li, J

    2014-09-26

    MicroRNAs (miRNAs) have emerged as a major regulator in neurological diseases, and understanding their molecular mechanism in modulating cerebral ischemic injury may provide potential therapeutic targets for ischemic stroke. However, as one of 19 differentially expressed miRNAs in mouse brain with middle cerebral artery occlusion (MCAO), the role of miR-134 in ischemic injury is not well understood. In this study, the miR-134 expression level was manipulated both in oxygen-glucose deprivation (OGD)-treated N2A neuroblastoma cells in vitro and mouse brain with MCAO-induced ischemic stroke in vivo, and its possible targets of heat shock protein A5 (HSPA5) and HSPA12B were determined by bioinformatics analysis and dual luciferase assay. The results showed that overexpression of miR-134 exacerbated cell death and apoptosis both in vitro and in vivo. Conversely, downregulating miR-134 levels reduced cell death and apoptosis. Furthermore, non-expression of miR-134 enhanced HSPA12B protein levels in OGD-treated N2A cells as well as in the ischemic region. It could attenuate brain infarction size and neural cell damage, and improve neurological outcomes in mice with ischemic stroke, whereas upregulation of miR-134 had the opposite effect. In addition, HSPA12B was validated to be a target of miR-134 and its short interfering RNAs (siRNAs) could block miR-134 inhibitor-induced neuroprotection in OGD-treated N2A cells. In conclusion, downregulation of miR-134 could induce neuroprotection against ischemic injury in vitro and in vivo by negatively upregulating HSPA12B protein expression.

  13. Up-Regulation of IL-1β, IL-6, and CCL-2 by a Novel Mouse Model of Pancreatic Ischemia-Reperfusion Injury1, 2, 3

    PubMed Central

    Lunsford, Keri E.; Baird, Brandon Jackson; Sempowski, Gregory D.; Cardona, Diana M.; Li, Zhiguo; Weinhold, Kent J.; Sudan, Debra L.; Brennan, Todd V.

    2013-01-01

    Little is known about the immunologic events surrounding pancreatic ischemia-reperfusion injury (IRI) due to a lack of established experimental models. The purpose of the current study was to develop a mouse model for pancreatic IRI to serve as a basis for immunologic characterization of pancreatic organ damage at transplantation. Methods Reversible ischemia was surgically induced by vascular isolation of the distal pancreas for 0, 10, 20, or 30 minutes. Mice receiving laparotomy without clamping served as sham-operated controls. Following reperfusion, mice were serially assayed for biochemical and histologic evidence of inflammation, proinflammatory cytokine and chemokine production, and inflammatory gene up-regulation. Results Following induction of pancreatic IRI, serum amylase and LDH peaked at 6 hours and returned to baseline by 120 hours following injury in all groups. Mice undergoing 30 minutes of IRI demonstrated greatest biochemical evidence of inflammation. Histologic scoring similarly demonstrated marked inflammation in mice subjected to 30 minutes IRI compared to controls. Serum cytokine/chemokine analysis demonstrated significant up-regulation of G-CSF, IFN-γ, TNF-α, IL-2, IL-1β, IL-6, CCL-2, CCL-5, CXCL-1, and MIP-2. Similar up-regulation of ccl2, il1b, il6, fos, hspa1a, hspd1, and cd14 gene expression was detected by real-time PCR analysis of pancreatic tissue. Conclusions This novel model of distal pancreatic IRI in the mouse demonstrates time-limited pancreatic inflammation and injury by histologic and biochemical indices. Inflammation may be, in part, a result of the immunologic effects of IL-1β, IL-6, and CCL-2. This model provides a method by which immunologic mechanisms of pancreatic IRI can be elucidated. PMID:23416685

  14. Interleukin-6 and interleukin-6 receptor expression, localization, and involvement in pain-sensing neuron activation in a mouse intervertebral disc injury model.

    PubMed

    Sainoh, Takeshi; Orita, Sumihisa; Miyagi, Masayuki; Sakuma, Yoshihiro; Yamauchi, Kazuyo; Suzuki, Miyako; Kubota, Go; Oikawa, Yasuhiro; Inage, Kazuhide; Sato, Jun; Fujimoto, Kazuki; Shiga, Yasuhiro; Inoue, Gen; Aoki, Yasuchika; Takahashi, Kazuhisa; Ohtori, Seiji

    2015-10-01

    The pathological mechanism of intractable low back pain is unclear. However, intervertebral disc (IVD) degeneration is a primary cause of low back pain, and pain-related mediators, such as interleukin-6 (IL-6), have been correlated with discogenic pain. The objective of this study is to elucidate the mechanism of local IL-6 and IL-6 receptor (IL-6R) expression after IVD injury as well as determine the involvement of IL-6/IL-6 signaling in discogenic pain. To do this, quantitative and immunohistological analyses in a mouse model of IVD injury were performed. Firstly, we measured the local expression levels of IL-6 and IL-6R in IVDs by enzyme-linked immunosorbent assay (ELISA). Secondly, we immunohistochemically confirmed their localization in injured IVDs. Lastly, we evaluated the effects of intradiscal injection of an IL-6 inhibitor by evaluating pain-related protein, calcitonin gene-related peptide (CGRP), expression in dorsal root ganglia (DRG) neurons that innervate IVDs. Injured IVDs showed increased production of IL-6 and IL-6R. IL-6 and IL-6R expression in the injured IVD were predominantly localized in the annulus fibrosus and endplate, and intradiscal injection of the IL-6 inhibitor suppressed CGRP expression in the DRG neurons. These results show that IL-6 and IL-6R expression levels are responsive to IVD injury and that inhibition of IL-6/IL-6R signaling may be a promising analgesic treatment for degenerative disc diseases.

  15. The comparison of mouse full metallothionein-1 versus alpha and beta domains and metallothionein-1-to-3 mutation following traumatic brain injury reveals different biological motifs.

    PubMed

    Manso, Yasmina; Serra, Montserrat; Comes, Gemma; Giralt, Mercedes; Carrasco, Javier; Cols, Neus; Vasák, Milan; González-Duarte, Pilar; Hidalgo, Juan

    2010-06-01

    Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

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

    PubMed

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

    2016-11-01

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

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

    PubMed Central

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

    2011-01-01

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

  18. Mechanochemotransduction During Cardiomyocyte Contraction Is Mediated by Localized Nitric Oxide Signaling

    PubMed Central

    Jian, Zhong; Han, Huilan; Zhang, Tieqiao; Puglisi, Jose; Izu, Leighton T.; Shaw, John A.; Onofiok, Ekama; Erickson, Jeffery R.; Chen, Yi-Je; Horvath, Balazs; Shimkunas, Rafael; Xiao, Wenwu; Li, Yuanpei; Pan, Tingrui; Chan, James; Banyasz, Tamas; Tardiff, Jil C.; Chiamvimonvat, Nipavan; Bers, Donald M.; Lam, Kit S.; Chen-Izu, Ye

    2014-01-01

    Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca2+ dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca2+ transient, which enhanced contractility to counter mechanical load, but also caused spontaneous Ca2+ sparks during diastole that could be arrhythmogenic. The increases in the Ca2+ transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2+ in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2+ sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca2+-calmodulin–dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca2+ sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca2+ sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2+ sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress–induced Ca2+ dysregulation, arrhythmias, and cardiomyopathy. PMID:24643800

  19. Mechanochemotransduction during cardiomyocyte contraction is mediated by localized nitric oxide signaling.

    PubMed

    Jian, Zhong; Han, Huilan; Zhang, Tieqiao; Puglisi, Jose; Izu, Leighton T; Shaw, John A; Onofiok, Ekama; Erickson, Jeffery R; Chen, Yi-Je; Horvath, Balazs; Shimkunas, Rafael; Xiao, Wenwu; Li, Yuanpei; Pan, Tingrui; Chan, James; Banyasz, Tamas; Tardiff, Jil C; Chiamvimonvat, Nipavan; Bers, Donald M; Lam, Kit S; Chen-Izu, Ye

    2014-03-18

    Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca(2+) dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca(2+) transient, which enhanced contractility to counter mechanical load but also caused spontaneous Ca(2+) sparks during diastole that could be arrhythmogenic. The increases in the Ca(2+) transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2(+) in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2(+) sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca(2+) sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca(2+) sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2(+) sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress-induced Ca(2+) dysregulation, arrhythmias, and cardiomyopathy.

  20. Neuroprotective effect of gadolinium: a stretch-activated calcium channel blocker in mouse model of ischemia-reperfusion injury.

    PubMed

    Gulati, Puja; Muthuraman, Arunachalam; Jaggi, Amteshwar S; Singh, Nirmal

    2013-03-01

    The present study was designed to investigate the potential of gadolinium, a stretch-activated calcium channel blocker in ischemic reperfusion (I/R)-induced brain injury in mice. Bilateral carotid artery occlusion of 12 min followed by reperfusion for 24 h was given to induce cerebral injury in male Swiss mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was assessed using Morris water maze test and motor incoordination was evaluated using rota-rod, lateral push, and inclined beam walking tests. In addition, total calcium, thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH), and acetylcholinesterase (AChE) activity were also estimated in brain tissue. I/R injury produced a significant increase in cerebral infarct size. A significant loss of memory along with impairment of motor performance was also noted. Furthermore, I/R injury also produced a significant increase in levels of TBARS, total calcium, AChE activity, and a decrease in GSH levels. Pretreatment of gadolinium significantly attenuated I/R-induced infarct size, behavioral and biochemical changes. On the basis of the present findings, we can suggest that opening of stretch-activated calcium channel may play a critical role in ischemic reperfusion-induced brain injury and that gadolinium has neuroprotective potential in I/R-induced injury.

  1. Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

    PubMed

    Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

    2016-08-05

    Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune

  2. Nuclear receptor CAR (NR1I3) is essential for DDC-induced liver injury and oval cell proliferation in mouse liver.

    PubMed

    Yamazaki, Yuichi; Moore, Rick; Negishi, Masahiko

    2011-11-01

    The liver is endowed with the ability to regenerate hepatocytes in response to injury. When this regeneration ability is impaired during liver injury, oval cells, which are considered to be postnatal hepatic progenitors, proliferate and differentiate into hepatocytes. Here we have demonstrated that 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) activates the nuclear receptor constitutive active/androstane receptor (CAR), resulting in proliferation of oval cells in mouse liver. Activation of CAR by DDC was shown by hepatic nuclear CAR accumulation and cytochrome P450 (CYP)2B10 mRNA induction after feeding a 0.1% DDC-containing diet to Car(+/+) mice. After being fed the DDC diet, Car(+/+), but not Car(-/-) mice, developed severe liver injury and an A6 antibody-stained ductular reaction in an area around the portal tract. Oval cell proliferation was confirmed by laser capture microdissection and real-time PCR; mRNAs for the two oval cell markers epithelial cell adhesion molecule and TROP2 were specifically induced in the periportal region of DDC diet-fed Car(+/+), but not Car(-/-) mice. Although rates of both hepatocyte growth and death were initially enhanced only in DDC diet-fed Car(+/+) mice, growth was attenuated when oval cells proliferated, whereas death continued unabated. DDC-induced liver injury, which differs from other CAR activators such as phenobarbital, occurred in the periportal region where cells developed hypertrophy, accumulated porphyrin crystals and inflammation developed, all in association with the proliferation of oval cells. Thus, CAR provides an excellent experimental model for further investigations into its roles in liver regeneration, as well as the development of diseases such as hepatocellular carcinoma.

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

    SciTech Connect

    Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G.; Bosnjak, Zeljko J.

    2014-10-31

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

  4. Peripheral nerve injury is associated with chronic, reversible changes in global DNA methylation in the mouse prefrontal cortex.

    PubMed

    Tajerian, Maral; Alvarado, Sebastian; Millecamps, Magali; Vachon, Pascal; Crosby, Cecilia; Bushnell, M Catherine; Szyf, Moshe; Stone, Laura S

    2013-01-01

    Changes in brain structure and cortical function are associated with many chronic pain conditions including low back pain and fibromyalgia. The magnitude of these changes correlates with the duration and/or the intensity of chronic pain. Most studies report changes in common areas involved in pain modulation, including the prefrontal cortex (PFC), and pain-related pathological changes in the PFC can be reversed with effective treatment. While the mechanisms underlying these changes are unknown, they must be dynamically regulated. Epigenetic modulation of gene expression in response to experience and environment is reversible and dynamic. Epigenetic modulation by DNA methylation is associated with abnormal behavior and pathological gene expression in the central nervous system. DNA methylation might also be involved in mediating the pathologies associated with chronic pain in the brain. We therefore tested a) whether alterations in DNA methylation are found in the brain long after chronic neuropathic pain is induced in the periphery using the spared nerve injury modal and b) whether these injury-associated changes are reversible by interventions that reverse the pathologies associated with chronic pain. Six months following peripheral nerve injury, abnormal sensory thresholds and increased anxiety were accompanied by decreased global methylation in the PFC and the amygdala but not in the visual cortex or the thalamus. Environmental enrichment attenuated nerve injury-induced hypersensitivity and reversed the changes in global PFC methylation. Furthermore, global PFC methylation correlated with mechanical and thermal sensitivity in neuropathic mice. In summary, induction of chronic pain by peripheral nerve injury is associated with epigenetic changes in the brain. These changes are detected long after the original injury, at a long distance from the site of injury and are reversible with environmental manipulation. Changes in brain structure and cortical function that

  5. A dominantly negative mutation in cardiac troponin I at the interface with troponin T causes early remodeling in ventricular cardiomyocytes.

    PubMed

    Wei, Hongguang; Jin, J-P

    2014-08-15

    We previously reported a point mutation substituting Cys for Arg(111) in the highly conserved troponin T (TnT)-contacting helix of cardiac troponin I (cTnI) in wild turkey hearts (Biesiadecki et al. J Biol Chem 279: 13825-13832, 2004). This dominantly negative TnI-TnT interface mutation decreases the binding affinity of cTnI for TnT, impairs diastolic function, and blunts the β-adrenergic response of cardiac muscle (Wei et al. J Biol Chem 285: 27806-27816, 2010). Here we further investigate cellular phenotypes of transgenic mouse cardiomyocytes expressing the equivalent mutation cTnI-K118C. Functional studies were performed on single adult cardiomyocytes after recovery in short-term culture from isolation stress. The amplitude of contraction and the velocities of shortening and relengthening were lower in cTnI-K118C cardiomyocytes than wild-type controls. The intracellular Ca(2+) transient was slower in cTnI-K118C cardiomyocytes than wild-type cells. cTnI-K118C cardiomyocytes also showed a weaker β-adrenergic response. The resting length of cTnI-K118C cardiomyocytes was significantly greater than that of age-matched wild-type cells, with no difference in cell width. The resting sarcomere was not longer, but slightly shorter, in cTnI-K118C cardiomyocytes than wild-type cells, indicating longitudinal addition of sarcomeres. More tri- and quadrinuclei cardiomyocytes were found in TnI-K118C than wild-type hearts, suggesting increased nuclear divisions. Whole-genome mRNA array and Western blots detected an increased expression of leukemia inhibitory factor receptor-β in the hearts of 2-mo-old cTnI-K118C mice, suggesting a signaling pathway responsible for the potent effect of cTnI-K118C mutation on early remodeling in cardiomyocytes.

  6. A dominantly negative mutation in cardiac troponin I at the interface with troponin T causes early remodeling in ventricular cardiomyocytes

    PubMed Central

    Wei, Hongguang

    2014-01-01

    We previously reported a point mutation substituting Cys for Arg111 in the highly conserved troponin T (TnT)-contacting helix of cardiac troponin I (cTnI) in wild turkey hearts (Biesiadecki et al. J Biol Chem 279: 13825–13832, 2004). This dominantly negative TnI-TnT interface mutation decreases the binding affinity of cTnI for TnT, impairs diastolic function, and blunts the β-adrenergic response of cardiac muscle (Wei et al. J Biol Chem 285: 27806–27816, 2010). Here we further investigate cellular phenotypes of transgenic mouse cardiomyocytes expressing the equivalent mutation cTnI-K118C. Functional studies were performed on single adult cardiomyocytes after recovery in short-term culture from isolation stress. The amplitude of contraction and the velocities of shortening and relengthening were lower in cTnI-K118C cardiomyocytes than wild-type controls. The intracellular Ca2+ transient was slower in cTnI-K118C cardiomyocytes than wild-type cells. cTnI-K118C cardiomyocytes also showed a weaker β-adrenergic response. The resting length of cTnI-K118C cardiomyocytes was significantly greater than that of age-matched wild-type cells, with no difference in cell width. The resting sarcomere was not longer, but slightly shorter, in cTnI-K118C cardiomyocytes than wild-type cells, indicating longitudinal addition of sarcomeres. More tri- and quadrinuclei cardiomyocytes were found in TnI-K118C than wild-type hearts, suggesting increased nuclear divisions. Whole-genome mRNA array and Western blots detected an increased expression of leukemia inhibitory factor receptor-β in the hearts of 2-mo-old cTnI-K118C mice, suggesting a signaling pathway responsible for the potent effect of cTnI-K118C mutation on early remodeling in cardiomyocytes. PMID:24898585

  7. Clematichinenoside (AR) Attenuates Hypoxia/Reoxygenation-Induced H9c2 Cardiomyocyte Apoptosis via a Mitochondria-Mediated Signaling Pathway.

    PubMed

    Ding, Haiyan; Han, Rong; Chen, Xueshan; Fang, Weirong; Liu, Meng; Wang, Xuemei; Wei, Qin; Kodithuwakku, Nandani Darshika; Li, Yunman

    2016-05-30

    Mitochondria-mediated cardiomyocyte apoptosis is involved in myocardial ischemia/reperfusion (MI/R) injury. Clematichinenoside (AR) is a triterpenoid saponin isolated from the roots of Clematis chinensis with antioxidant and anti-inflammatory cardioprotection effects against MI/R injury, yet the anti-apoptotic effect and underlying mechanisms of AR in MI/R injury remain unclear. We hypothesize that AR may improve mitochondrial function to inhibit MI/R-induced cardiomyocyte apoptosis. In this study, we replicated an in vitro H9c2 cardiomyocyte MI/R model by hypoxia/reoxygenation (H/R) treatment. The viability of H9c2 cardiomyocytes was determined by MTT assay; apoptosis was evaluated by flow cytometry and TUNEL experiments; mitochondrial permeability transition pore (mPTP) opening was analyzed by a calcein-cobalt quenching method; and mitochondrial membrane potential (ΔΨm) was detected by JC-1. Moreover, we used western blots to determine the mitochondrial cytochrome c translocation to cytosolic and the expression of caspase-3, Bcl-2, and Bax proteins. These results showed that the application of AR decreased the ratio of apoptosis and the extent of mPTP opening, but increased ΔΨm. AR also inhibited H/R-induced release of mitochondrial cytochrome c and decreased the expression of the caspase-3, Bax proteins. Conversely, it remarkably increased the expression of Bcl-2 protein. Taken together, these results revealed that AR protects H9c2 cardiomyocytes against H/R-induced apoptosis through mitochondrial-mediated apoptotic signaling pathway.

  8. Lipopolysaccharide-induced endotoxemia in corn oil-preloaded mice causes an extended course of lung injury and repair and pulmonary fibrosis: A translational mouse model of acute respiratory distress syndrome

    PubMed Central

    Wu, Chaomin; Evans, Colin E.; Dai, Zhiyu; Huang, Xiaojia; Zhang, Xianming; Jin, Hua; Hu, Guochang; Song, Yuanlin; Zhao, You-Yang

    2017-01-01

    Acute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia respiratory failure, bilateral pulmonary infiltrates, and pulmonary edema of non-cardiac origin. Effective treatments for ARDS patients may arise from experimental studies with translational mouse models of this disease that aim to delineate the mechanisms underlying the disease pathogenesis. Mouse models of ARDS, however, can be limited by their rapid progression from injured to recovery state, which is in contrast to the course of ARDS in humans. Furthermore, current mouse models of ARDS do not recapitulate certain prominent aspects of the pathogenesis of ARDS in humans. In this study, we developed an improved endotoxemic mouse model of ARDS resembling many features of clinical ARDS including extended courses of injury and recovery as well as development of fibrosis following i.p. injection of lipopolysaccharide (LPS) to corn oil-preloaded mice. Compared with mice receiving LPS alone, those receiving corn oil and LPS exhibited extended course of lung injury and repair that occurred over a period of >2 weeks instead of 3–5days. Importantly, LPS challenge of corn oil-preloaded mice resulted in pulmonary fibrosis during the repair phase as often seen in ARDS patients. In summary, this simple novel mouse model of ARDS could represent a valuable experimental tool to elucidate mechanisms that regulate lung injury and repair in ARDS patients. PMID:28333981

  9. Visualizing the neutrophil response to sterile tissue injury in mouse dermis reveals a three-phase cascade of events.

    PubMed

    Ng, Lai Guan; Qin, Jim S; Roediger, Ben; Wang, Yilin; Jain, Rohit; Cavanagh, Lois L; Smith, Adrian L; Jones, Cheryl A; de Veer, Michael; Grimbaldeston, Michele A; Meeusen, Els N; Weninger, Wolfgang

    2011-10-01

    Neutrophil granulocytes traffic into sites of organ injury in which they may not only participate in tissue repair and pathogen clearance but may also contribute to collateral cell damage through the release of noxious mediators. The dynamics and mechanisms of neutrophil migration in the extravascular space toward loci of tissue damage are not well understood. Here, we have used intravital multi-photon microscopy to dissect the behavior of neutrophils in response to tissue injury in the dermis of mice. We found that, following confined physical injury, initially rare scouting neutrophils migrated in a directional manner toward the damage focus. This was followed by the attraction of waves of additional neutrophils, and finally stabilization of the neutrophil cluster around the injury. Although neutrophil migration in the steady state and during the scouting phase depended on pertussis toxin-sensitive signals, the amplification phase was sensitive to interference with the cyclic adenosine diphosphate ribose pathway. We finally demonstrated that neutrophil scouts also transit through the non-inflamed dermis, suggesting immunosurveillance function by these cells. Together, our data unravel a three-step cascade of events that mediates the specific accumulation of neutrophils at sites of sterile tissue injury in the interstitial space.

  10. Octreotide Protects the Mouse Retina against Ischemic Reperfusion Injury through Regulation of Antioxidation and Activation of NF-κB

    PubMed Central

    Wang, Jun; Sun, Ziqiang; Shen, Junsheng; Wu, Dongdong; Liu, Fang; Yang, Ruisheng; Ji, Shaoping; Ji, Ailing; Li, Yanzhang

    2015-01-01

    Somatostatin (SST), an endogenous peptide, may exert anti-inflammatory and neuroprotective effects on retinal injury induced by ischemia. Retinal ischemic reperfusion (I/R) injury always produces many reactive oxygen species (ROS), which can aggravate the tissue damage. The effects of octreotide (OCT), a SST analogue, on retinal I/R injury and ROS formation, are not very clear. In this study, we observed the effects of OCT on morphological changes, oxidative stress, and cell death, induced by retinal I/R injury. The activation of nuclear factor κB (NF-κB) and intercellular adhesion molecule-1 (ICAM-1) were further evaluated in I/R retina treated with or without OCT. The retinal layer thickness was increased at 1 day after I/R and decreased at 7 days after I/R (P < 0.05). This effect was associated with increase in MDA and ROS levels (P < 0.05). The Tunel-positive cells increased and the number of ganglion cell layer (GCL) neurons decreased significantly after I/R injury. The expression of p-p65 and ICAM-1 increased significantly in I/R retinas (P < 0.05). Each effect was markedly attenuated by application of OCT. These data indicate that OCT protects the retina against retinal I/R damage, which could be through inhibition of oxidative stress and downregulation of NF-κB and ICAM-1 expression. PMID:26175842

  11. Analysis of cardiomyocyte movement in the developing murine heart

    SciTech Connect

    Hashimoto, Hisayuki; Yuasa, Shinsuke; Tabata, Hidenori; Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto; Nakajima, Kazunori; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Fukuda, Keiichi

    2015-09-04

    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 cell 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.

  12. Enhanced neurofibrillary tangle formation, cerebral atrophy, and cognitive deficits induced by repetitive mild brain injury in a transgenic tauopathy mouse model.

    PubMed

    Yoshiyama, Yasumasa; Uryu, Kunihiro; Higuchi, Makoto; Longhi, Luca; Hoover, Rachel; Fujimoto, Scott; McIntosh, Tracy; Lee, Virginia M-Y; Trojanowski, John Q

    2005-10-01

    Traumatic brain injury (TBI) is a risk factors for Alzheimer's disease (AD), and repetitive TBI (rTBI) may culminate in dementia pugilistica (DP), a syndrome characterized by progressive dementia, parkinsonism, and the hallmark brain lesions of AD, including neurofibrillary tangles (NFTs), formed by abnormal tau filaments and senile plaques (SPs) composed of Abeta fibrils. Previous study showed that mild rTBI (mrTBI) accelerated the deposition of Abeta in the brains of transgenic (Tg) mice (Tg2576) that over-express human Abeta precursor proteins with the familial AD Swedish mutations (APP695swe) and model of AD-like amyloidosis. Here, we report studies of the effects of mrTBI on AD-like tau pathologies in Tg mice expressing the shortest human tau isoform (T44) subjected to mrTBI, causing brain concussion without structural brain damage to simulate injuries linked to DP. Twelve-month-old Tg T44 (n = 18) and wild-type (WT; n = 24) mice were subjected to mrTBI (four times a day, 1 day per week, for 4 weeks; n = 24) or sham treatment (n = 18). Histopathological analysis of mice at 9 months after mrTBI revealed that one of the Tg T44 mice showed extensive telencephalic NFT and cerebral atrophy. Although statistical analysis of neurobehavioral tests at 6 months after mrTBI did not show any significant difference in any of groups of mice, the Tg T44 mouse with extensive NFT had an exceptionally low neurobehavioral score. The reasons for the augmentation of tau pathologies in only one T44 tau Tg mouse subjected to mrTBI remain to be elucidated.

  13. Resistance to cardiomyocyte hypertrophy in ae3 −/− mice, deficient in the AE3 Cl−/HCO3− exchanger

    PubMed Central

    2014-01-01

    Background Cardiac hypertrophy is central to the etiology of heart failure. Understanding the molecular pathways promoting cardiac hypertrophy may identify new targets for therapeutic intervention. Sodium-proton exchanger (NHE1) activity and expression levels in the heart are elevated in many models of hypertrophy through protein kinase C (PKC)/MAPK/ERK/p90RSK pathway stimulation. Sustained NHE1 activity, however, requires an acid-loading pathway. Evidence suggests that the Cl−/HCO3− exchanger, AE3, provides this acid load. Here we explored the role of AE3 in the hypertrophic growth cascade of cardiomyocytes. Methods AE3-deficient (ae3 −/− ) mice were compared to wildtype (WT) littermates to examine the role of AE3 protein in the development of cardiomyocyte hypertrophy. Mouse hearts were assessed by echocardiography. As well, responses of cultured cardiomyocytes to hypertrophic stimuli were measured. pH regulation capacity of ae3 −/− and WT cardiomyocytes was assessed in cultured cells loaded with the pH-sensitive dye, BCECF-AM. Results ae3 −/− mice were indistinguishable from wild type (WT) mice in terms of cardiovascular performance. Stimulation of ae3 −/− cardiomyocytes with hypertrophic agonists did not increase cardiac growth or reactivate the fetal gene program. ae3 −/− mice are thus protected from pro-hypertrophic stimulation. Steady state intracellular pH (pHi) in ae3 −/− cardiomyocytes was not significantly different from WT, but the rate of recovery of pHi from imposed alkalosis was significantly slower in ae3 −/− cardiomyocytes. Conclusions These data reveal the importance of AE3-mediated Cl−/HCO3− exchange in cardiovascular pH regulation and the development of cardiomyocyte hypertrophy. Pharmacological antagonism of AE3 is an attractive approach in the treatment of cardiac hypertrophy. PMID:25047106

  14. The protective effect of dopamine against OGD/R injury-induced cell death in HT22 mouse hippocampal cells.

    PubMed

    Wang, Wenzhu; Zhao, Lixi; Bai, Fan; Zhang, Tong; Dong, Hao; Liu, Lixu

    2016-03-01

    Previous studies have shown that levo-dopamine (L-dopa) can improve the consciousness of certain patients with prolonged coma after cerebral ischemia-reperfusion injury, and promote cell growth in vivo. This study aimed to investigate whether L-dopa, which is used clinically to treat Parkinson's disease, might also ameliorate ischemia-reperfusion injury-induced cell death. The oxygen-glucose deprivation and re-oxygenation (OGD/R) model was used to mimic the ischemia-reperfusion pathological process in vitro. HT22 cells were treated with dopamine hydrochloride at different times (i.e., 2 h prior to OGD, during the period of OGD, during the period of R, and throughout the period of OGD/R) and at different concentrations (i.e., 25 μM, 50 μM, 100 μM). Lactate dehydrogenase (LDH) release, flow cytometry-annexin V, and propidium iodide staining with light microscopy showed that dopamine hydrochloride (added during re-oxygenation) promoted cell proliferation and facilitated maintenance of normal cell morphology. However, when present during oxygen-glucose deprivation for 18 h and present throughout OGD/R, dopamine hydrochloride increased cell damage as manifested by shrinkage, rounding up, and reduced viability. In conclusion, dopamine protected HT22 cells from OGD/R injury-induced cell death only at a particular point in time, suggesting that it may be useful for treating severe ischemia-reperfusion brain injury.

  15. Atorvastatin protects cardiomyocytes from oxidative stress by inhibiting LOX-1 expression and cardiomyocyte apoptosis.

    PubMed

    Zhang, Lei; Cheng, Linfang; Wang, Qiqi; Zhou, Dongchen; Wu, Zhigang; Shen, Ling; Zhang, Li; Zhu, Jianhua

    2015-03-01

    Coronary artery disease (CAD) is a major health problem worldwide. The most severe form of CAD is acute coronary syndrome (ACS). Recent studies have demonstrated the beneficial role of atorvastatin in ACS; however, the mechanisms underlying this effect have not been fully clarified. Growing evidence indicates that activation of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) plays an important role in oxidative stress-induced cardiomyocyte apoptosis during ACS. In this study, we examined whether atorvastatin inhibits H2O2-induced LOX-1 expression and H9c2 cardiomyocyte apoptosis, and investigated the underlying signaling pathway. Treatment of H9c2 cardiomyocytes with H2O2 resulted in elevated expression of LOX-1 mRNA and protein, as well as increased caspase-3 and -9 protein expression and cell apoptosis. H2O2-induced LOX-1 expression, caspase protein expression, and cardiomyocyte apoptosis were attenuated by pretreatment with atorvastatin. Atorvastatin activated H2O2-inhibited phosphorylation of Akt in a concentration-dependent manner. The Akt inhibitor, LY294002, inhibited the effect of atorvastatin on inducing Akt phosphorylation and on suppressing H2O2-mediated caspase up-regulation and cell apoptosis. These findings indicate that atorvastatin protects cardiomyocyte from oxidative stress via inhibition of LOX-1 expression and apoptosis, and that activation of H2O2-inhibited phosphorylation of Akt may play an important role in the protective function of atorvastatin.

  16. Identifying the Long-Term Role of Inducible Nitric Oxide Synthase after Contusive Spinal Cord Injury Using a Transgenic Mouse Model

    PubMed Central

    Maggio, Dominic M.; Singh, Amanpreet; Iorgulescu, J. Bryan; Bleicher, Drew H.; Ghosh, Mousumi; Lopez, Michael M.; Tuesta, Luis M.; Flora, Govinder; Dietrich, W. Dalton; Pearse, Damien D.

    2017-01-01

    Inducible nitric oxide synthase (iNOS) is a potent mediator of oxidative stress during neuroinflammation triggered by neurotrauma or neurodegeneration. We previously demonstrated that acute iNOS inhibition attenuated iNOS levels and promoted neuroprotection and functional recovery after spinal cord injury (SCI). The present study investigated the effects of chronic iNOS ablation after SCI using inos-null mice. iNOS−/− knockout and wild-type (WT) control mice underwent a moderate thoracic (T8) contusive SCI. Locomotor function was assessed weekly, using the Basso Mouse Scale (BMS), and at the endpoint (six weeks), by footprint analysis. At the endpoint, the volume of preserved white and gray matter, as well as the number of dorsal column axons and perilesional blood vessels rostral to the injury, were quantified. At weeks two and three after SCI, iNOS−/− mice exhibited a significant locomotor improvement compared to WT controls, although a sustained improvement was not observed during later weeks. At the endpoint, iNOS−/− mice showed significantly less preserved white and gray matter, as well as fewer dorsal column axons and perilesional blood vessels, compared to WT controls. While short-term antagonism of iNOS provides histological and functional benefits, its long-term ablation after SCI may be deleterious, blocking protective or reparative processes important for angiogenesis and tissue preservation. PMID:28125047

  17. Sustainable antimicrobial effect of silver sulfadiazine-loaded nanosheets on infection in a mouse model of partial-thickness burn injury.

    PubMed

    Ito, Keisuke; Saito, Akihiro; Fujie, Toshinori; Nishiwaki, Keisuke; Miyazaki, Hiromi; Kinoshita, Manabu; Saitoh, Daizoh; Ohtsubo, Shinya; Takeoka, Shinji

    2015-09-01

    Partial-thickness burn injury has the potential for reepithelialization and heals within 3weeks. If the wound is infected by bacteria before reepithelization, however, the depth of disruption increases and the lesion easily progresses to the full-thickness dermal layers. In the treatment of partial-thickness burn injury, it is important to prevent the wound area from bacterial infection with an antimicrobial dressing. Here, we have tested the antimicrobial properties of polymeric ultra-thin films composed of poly(lactic acid) (termed "PLA nanosheets"), which have high flexibility, adhesive strength and transparency, and silver sulfadiazine (AgSD), which exhibits antimicrobial efficacy. The AgSD-loaded nanosheet released Ag(+) for more than 3days, and exerted antimicrobial efficacy against methicillin-resistant Staphylococcus aureus (MRSA) in an in vitro Kirby-Bauer test. By contrast, a cell viability assay indicated that the dose of AgSD used in the PLA nanosheets did not show significant cytotoxicity toward fibroblasts. In vivo evaluation using a mouse model of infection in a partial-thickness burn wound demonstrated that the nanosheet significantly reduced the number of MRSA bacteria on the lesion (more than 10(5)-fold) and suppressed the inflammatory reaction, thereby preventing a protracted wound healing process.

  18. Sesame oil improves functional recovery by attenuating nerve oxidative stress in a mouse model of acute peripheral nerve injury: role of Nrf-2.

    PubMed

    Hsu, Che-Chia; Huang, Hui-Cheng; Wu, Po-Ting; Tai, Ta-Wei; Jou, I-Ming

    2016-12-01

    Peripheral nervous injury (PNI) is a common form of trauma in modern society, especially in sport players. Despite the advance of therapy for PNI, the recovery of function can never reach the preinjury level after treatments. Recently, inhibiting neural oxidative stress shows a beneficial effect in improving functional recovery after PNI. In addition, sesame oil has been reported to possess the excellent antioxidative properties. However, whether sesame oil can improve the functional recovery after PNI by its antioxidative effect has never been investigated. Thirty mice were randomly divided into five groups of six: group I mice received sham operation; group II mice received sciatic nerve crush; and groups III-V mice daily ingested 0.5, 1 and 2 ml/kg of sesame oil for 6 days, respectively, after sciatic nerve crush. Oxidative stress, GAP43 and nuclear Nrf2 levels as well as spinal somatosensory evoked potentials were assessed on day 6, while paw withdrawal latency and sciatic function index were assessed on days 0, 3, and 6. Sesame oil significantly decreased lipid peroxidation and increased nuclear factor erythroid 2-related factor 2 and GAP43 expression in sciatic nerve. Furthermore, sesame oil improved electrophysiological and functional assessments in mice with sciatic nerve crush. In conclusion, sesame oil may improve nerve functional recovery by attenuating nerve oxidative stress in mouse acute peripheral nerve injury. Further, application of natural product sesame oil may be an alternative approach for improving nerve functional recovery in the clinical setting.

  19. Data on amyloid precursor protein accumulation, spontaneous physical activity, and motor learning after traumatic brain injury in the triple-transgenic mouse model of Alzheimer׳s disease.

    PubMed

    Kishimoto, Yasushi; Shishido, Hajime; Sawanishi, Mayumi; Toyota, Yasunori; Ueno, Masaki; Kubota, Takashi; Kirino, Yutaka; Tamiya, Takashi; Kawai, Nobuyuki

    2016-12-01

    This data article contains supporting information regarding the research article entitled "Traumatic brain injury accelerates amyloid-β deposition and impairs spatial learning in the triple-transgenic mouse model of Alzheimer׳s disease" (H. Shishido, Y. Kishimoto, N. Kawai, Y. Toyota, M. Ueno, T. Kubota, Y. Kirino, T. Tamiya, 2016) [1]. Triple-transgenic (3×Tg)-Alzheimer׳s disease (AD) model mice exhibited significantly poorer spatial learning than sham-treated 3×Tg-AD mice 28 days after traumatic brain injury (TBI). Correspondingly, amyloid-β (Aβ) deposition within the hippocampus was significantly greater in 3×Tg-AD mice 28 days after TBI. However, data regarding the short-term and long-term influences of TBI on amyloid precursor protein (APP) accumulation in AD model mice remain limited. Furthermore, there is little data showing whether physical activity and motor learning are affected by TBI in AD model mice. Here, we provide immunocytochemistry data confirming that TBI induces significant increases in APP accumulation in 3×Tg-AD mice at both 7 days and 28 days after TBI. Furthermore, 3×Tg-AD model mice exhibit a reduced ability to acquire conditioned responses (CRs) during delay eyeblink conditioning compared to sham-treated 3×Tg-AD model mice 28 days after TBI. However, physical activity and motor performance are not significantly changed in TBI-treated 3×Tg-AD model mice.

  20. Elastic interactions synchronize beating in cardiomyocytes.

    PubMed

    Cohen, Ohad; Safran, Samuel A

    2016-07-13

    Motivated by recent experimental results, we study theoretically the synchronization of the beating phase and frequency of two nearby cardiomyocyte cells. Each cell is represented as an oscillating force dipole in an infinite, viscoelastic medium and the propagation of the elastic signal within the medium is predicted. We examine the steady-state beating of two nearby cells, and show that elastic interactions result in forces that synchronize the phase and frequency of beating in a manner that depends on their mutual orientation. The theory predicts both in-phase and anti-phase steady-state beating depending on the relative cell orientations, as well as how synchronized beating varies with substrate elasticity and the inter-cell distance. These results suggest how mechanics plays a role in cardiac efficiency, and may be relevant for the design of cardiomyocyte based micro devices and other biomedical applications.

  1. RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury

    PubMed Central

    Newton, K; Dugger, D L; Maltzman, A; Greve, J M; Hedehus, M; Martin-McNulty, B; Carano, R A D; Cao, T C; van Bruggen, N; Bernstein, L; Lee, W P; Wu, X; DeVoss, J; Zhang, J; Jeet, S; Peng, I; McKenzie, B S; Roose-Girma, M; Caplazi, P; Diehl, L; Webster, J D; Vucic, D

    2016-01-01

    Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20−/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20−/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease. PMID:27177019

  2. Comparison of ribavirin and oseltamivir in reducing mortality and lung injury in mice infected with mouse adapted A/California/04/2009 (H1N1)

    PubMed Central

    Zarogiannis, Sotirios G.; Noah, James W.; Jurkuvenaite, Asta; Steele, Chad; Matalon, Sadis; Noah, Diana L.

    2016-01-01

    Aim To compare the efficacy of ribavirin and oseltamivir in reducing mortality, lung injury and cytokine response profile in pandemic influenza H1N1 (2009) infection. Main Methods We assessed survival, weight loss, lung viral load (by RT-PCR), lung injury (by protein content in bronchoalveolar lavage), and inflammation (cell counts, differentials and cytokines in the bronchoalveolar lavage) in BALB/c mice after infection with mouse-adapted pandemic influenza strain A/California/04/2009. Key Findings Our results indicate that ribavirin (80 mg kg−1) and oseltamivir (50 mg kg−1) are equally effective in improving survival (100% vs. 0% in water treated controls), while ribavirin proved to be more effective in significantly preventing weight loss. Both drugs diminished the injury of the alveolar-capillary barrier by decreasing the protein detected in the BAL to baseline levels, and they were also equally effective in reduction lung viral loads by 100-fold. Administration of either drug did not decrease the amount of inflammatory infiltrate in the lung, but ribavirin significantly reduced the percentage comprised of lymphocytes. This study shows that these antivirals differentially regulate inflammatory cytokines and chemokines with ribavirin significantly reducing most of the cytokines/chemokines measured. Ribavirin treatment leads to a Th1 cytokine response while oseltamivir leads to a Th2 cytokine response with significant increase in the levels of the anti-inflammatory cytokine IL-10. Significance This study reveals new mechanistic insights in the way that ribavirin and oseltamivir exert their antiviral activity and supports the theory that ribavirin could potentially serve as an efficacious therapeutic alternative for oseltamivir resistant pandemic H1N1 strains. PMID:22269828

  3. Hierarchically clustering to 1,033 genes differentially expressed in mouse superior colliculus in the courses of optic nerve development and injury.

    PubMed

    Mei, Qiang; Zhang, Yan-qi; Liu, Jian-jun; Li, Cheng-ren; Chen, Xing-shu; Li, Hong-li; Qin, Mao-lin; wu, Ya-zhou; Liu, Yun-lai; Cai, Wen-qin

    2013-11-01

    Tempo spatially specific expression of many development-related genes is the molecular basis for the formation of the central nervous system (CNS), especially those genes regulating the proliferation, differentiation, migration, axon growth, and orientation of nerve cells. The development-related genes are usually prominent during the embryonic and newborn stages, but rarely express during the adulthood. These genes are believed to be suitable target genes for promoting CNS regeneration, despite majority of which remains unknown. Hence, the aim of this study was to screen development-related genes which might contribute to CNS regeneration. In this study, 1,033 differentially-expressed genes of superior colliculus in the courses of mouse optic nerve development and injury, as previously identified by cDNA microarrays, were hierarchically clustered to display expression pattern of each gene and reveal the relationships among these genes, and infer the functions of some unknown genes based on function-identified genes with the similar expression patterns. Consequently, the expression patterns of 1,033 candidate genes were revealed at eight time points during optic nerve development or injury. According to the similarity among gene expression patterns, 1,033 genes were divided into seven groups. The potential function of genes in each group was inferred on the basis of the dynamic trend for mean gene expression values. Moreover, the expression patterns of six function-unidentified genes were extremely similar to that of the ptn gene which could promote and guide axonal extension. Therefore, these six genes are temporally regarded as candidate genes related to axon growth and guidance. The results may help to better understand the roles of function-identified genes in the stages of CNS development and injury, and offer useful clues to evaluate the functions of hundreds of unidentified genes.

  4. 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.

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

    PubMed Central

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

    2016-01-01

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

  6. A new monoclonal antibody to study mouse macrophage antigen during BHT-induced lung injury and repair.

    PubMed

    Kennel, S J; Lankford, T; Galloway, P; Witschi, H P

    1989-04-01

    A rat monoclonal antibody 133-13A to a mouse lung carcinoma cell line was found to react with macrophages in mouse lung [1]. This monoclonal antibody is different from previously described antibodies to macrophages. Immunogold electron-microscopy and immunoperoxidase light microscopy have been used to show that MoAb 133-13A binds specifically to macrophages in normal and in BHT treated mouse lungs. This MoAb recognizes a protein of approximately 100 kDa (P100) on cultured lung carcinoma cells and a 87 kDa protein on macrophages from lung or the peritoneal cavity which is different from other macrophage antigens. The surface glycoprotein has been purified from cultured cells using immunoaffinity chromatography. The purified protein was radioiodinated and MoAb 133-13A was used to develop a competition radioimmunoassay to quantitate P100. Spleen, intestines, lung, skin and uterus all have high levels of P100. P100 on peritoneal macrophages has been determined to be about 94,000 molecules/cell. Analyses of lung lavage and whole lung homogenates from mice treated with BHT, BHT plus 70% O2, and 70% O2 alone show that treated animals have elevated P100 content compared to corn oil treated mice.

  7. Cardio-protective effects of combined l-arginine and insulin: Mechanism and therapeutic actions in myocardial ischemia-reperfusion injury.

    PubMed

    Venardos, Kylie M; Rajapakse, Niwanthi W; Williams, David; Hoe, Louise S; Peart, Jason N; Kaye, David M

    2015-12-15

    Reduced nitric oxide (NO) bioavailability plays a central role in the pathogenesis of myocardial ischemia-reperfusion injury (I-R), and reduced l-arginine transport via cationic amino acid transporter-1 is a key contributor to the reduced NO levels. Insulin can increase NO levels by increasing the transport of its substrate l-arginine but insulin alone exerts minimal cardiac protection in I-R. We hypothesized that combined insulin and l-arginine may provide cardioprotective effects in the setting of myocardial I-R. The effect of supplemental insulin, l-arginine and the combination was examined in cardiomyocytes exposed to hypoxia/reoxygenation and in isolated perfused mouse hearts undergoing ischemia/reperfusion. When compared to controls, cardiomyocytes treated upon reoxygenation with 1nM insulin+1mM l-arginine exhibited significant (all P<0.05) improvements in NO generation and mitochondrial membrane potential, with a concomitant fall in reactive oxygen species production and LDH release. Insulin also increased l-arginine uptake following hypoxia-reoxygenation (P<0.05; n=4-6). In langendorff perfused isolated mouse hearts, combined l-arginine-insulin treatment upon reperfusion significantly (all P<0.05; n=9-11) improved recovery of left ventricular developed pressure, rate pressure product and end diastolic pressure following ischemia, independent of any changes in post-ischemic coronary flow, together with significantly lower LDH release. The observed improvements were greater than l-arginine or insulin treatment alone. In isolated cardiomyocytes (n=3-5), 1nM insulin caused cationic amino acid transporter-1 to redistribute to the cellular membrane from the cytosol and the effects of insulin on l-arginine uptake were partially dependent on the PI3K/Akt pathway. l-arginine-insulin treatment may be a novel strategy to ameliorate I-R injury.

  8. Cardiomyocyte death in doxorubicin-induced cardiotoxicity

    PubMed Central

    Zhang, Yi-Wei; Shi, Jianjian; Li, Yuan-Jian; Wei, Lei

    2009-01-01

    SUMMARY Doxorubicin (DOX) is one of the most widely used and successful antitumor drugs, but its cumulative and dose-dependent cardiac toxicity has been the major concern of oncologists in cancer therapeutic practice for decades. With the increasing population of cancer survivals, there is a growing need to develop preventive strategies and effective therapies against DOX-induced cardiotoxicity, in particular, the late onset cardiomyopathy. Although intensive investigations on the DOX-induced cardiotoxicity have been continued for decades, the underlying mechanisms responsible for DOX-induced cardiotoxicity have not been completely elucidated. A rapidly expanding body of evidence supports that cardiomyocyte death by apoptosis and necrosis is a primary mechanism of DOX-induced cardiomyopathy and other types of cell death, such as autophagy and senescence/aging, may participate in this process. In this review, we will focus on the current understanding of molecular mechanisms underlying DOX-induced cardiomyocyte death, including the major primary mechanism of excess production of reactive oxygen species (ROS) and other recently discovered ROS-independent mechanisms. Different sensitivity to DOX-induced cell death signals between adult and young cardiomyocytes will also be discussed. PMID:19866340

  9. Genetic deletion of neuronal pentraxin 1 expression prevents brain injury in a neonatal mouse model of cerebral hypoxia-ischemia.

    PubMed

    Thatipamula, Shabarish; Al Rahim, Md; Zhang, Jiangyang; Hossain, Mir Ahamed

    2015-03-01

    Neonatal hypoxic-ischemic (HI) brain injury is a leading cause of mortality and morbidity in infants and children for which there is no promising therapy at present. Previously, we reported induction of neuronal pentraxin 1 (NP1), a novel neuronal protein of the long-pentraxin family, following HI injury in neonatal brain. Here, we report that genetic deletion of NP1 expression prevents HI injury in neonatal brain. Elevated expression of NP1 was observed in neurons, not in astrocytes, of the ipsilateral cortical layers (I-IV) and in the hippocampal CA1 and CA3 areas of WT brains following hypoxia-ischemia; brain areas that developed infarcts (at 24-48 h), showed significantly increased numbers of TUNEL-(+) cells and tissue loss (at 7 days). In contrast, NP1-KO mice showed no evidence of brain infarction and tissue loss after HI. The immunofluorescence staining of brain sections with mitochondrial protein COX IV and subcellular fractionation analysis showed increased accumulation of NP1 in mitochondria, pro-death protein Bax activation and NP1 co-localization with activated caspase-3 in WT, but not in the NP1-KO brains; corroborating NP1 interactions with the mitochondria-derived pro-death pathways. Disruption of NP1 translocation to mitochondria by NP1-siRNA in primary cortical cultures significantly reduced ischemic neuronal death. NP1 was immunoprecipitated with activated Bax [6A7] proteins; HI caused increased interactions of NP1 with Bax, thereby, facilitating Bax translocation to mitochondrial and neuronal death. To further delineate the specificity of NPs, we found that NP1 but not the NP2 induction is specifically involved in brain injury mechanisms and that knockdown of NP1 only results in neuroprotection. Furthermore, live in vivo T2-weighted magnetic resonance imaging (MRI) including fractional anisotropy (FA) mapping showed no sign of delayed brain injury or tissue loss in the NP1-KO mice as compared to the WT at different post-HI periods (4-24 weeks

  10. Genetic deletion of neuronal pentraxin 1 expression prevents brain injury in a neonatal mouse model of cerebral hypoxia-ischemia

    PubMed Central

    Thatipamula, Shabarish; Rahim, Md Al; Zhang, Jiangyang; Hossain, Mir Ahamed

    2015-01-01

    Neonatal hypoxic-ischemic (HI) brain injury is a leading cause of mortality and morbidity in infants and children for which there is no promising therapy at present. Previously, we reported induction of neuronal pentraxin 1 (NP1), a novel neuronal protein of the long-pentraxin family, following HI injury in neonatal brain. Here, we report that genetic deletion of NP1 expression prevents HI injury in neonatal brain. Elevated expression of NP1 was observed in neurons, not in astrocytes, of the ipsilateral cortical layers (I–IV) and in the hippocampal CA1 and CA3 areas of WT brains following hypoxia-ischemia; brain areas that developed infarcts (at 24–48 h), showed significantly increased numbers of TUNEL-(+) cells and tissue loss (at 7 d). In contrast, NP1-KO mice showed no evidence of brain infarction and tissue loss after HI. The immunofluorescence staining of brain sections with mitochondrial protein COX IV and subcellular fractionation analysis showed increased accumulation of NP1 in mitochondria, pro-death protein Bax activation and NP1 co-localization with activated caspase-3 in WT, but not in the NP1-KO brains; corroborating NP1 interactions with the mitochondria-derived pro-death pathways. Disruption of NP1 translocation to mitochondria by NP1-siRNA in primary cortical cultures significantly reduced ischemic neuronal death. NP1 was immunoprecipitated with activated Bax[6A7] proteins; HI caused increased interactions of NP1 with Bax, thereby, facilitating Bax translocation to mitochondrial and neuronal death. To further delineate the specificity of NPs, we found that NP1 but not the NP2 induction is specifically involved in brain injury mechanisms and that knockdown of NP1 only results in neuroprotection. Furthermore, live in vivo T2-weighted magnetic resonance imaging (MRI) including fractional anisotropy (FA) mapping showed no sign of delayed brain injury or tissue loss in the NP1-KO mice as compared to the WT at different post-HI periods (4–24 weeks

  11. 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

  12. 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

  13. Methods for Assessing the Electromechanical Integration of Human Pluripotent Stem Cell-Derived Cardiomyocyte Grafts

    PubMed Central

    Zhu, Wei-Zhong; Filice, Dominic; Palpant, Nathan J.; Laflamme, Michael A.

    2014-01-01

    Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host–graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models. PMID:25070341

  14. Methods for assessing the electromechanical integration of human pluripotent stem cell-derived cardiomyocyte grafts.

    PubMed

    Zhu, Wei-Zhong; Filice, Dominic; Palpant, Nathan J; Laflamme, Michael A

    2014-01-01

    Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host-graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models.

  15. Inactivation of Sirt1 in mouse livers protects against endotoxemic liver injury by acetylating and activating NF-κB

    PubMed Central

    Cui, Xiaolan; Chen, Qian; Dong, Zhen; Xu, Longmei; Lu, Tianfei; Li, Dawei; Zhang, Jiangjun; Zhang, Ming; Xia, Qiang

    2016-01-01

    Sirtuin 1 (Sirt1) is a deacetylase that regulates many cellular processes in the liver, and so far its role in endotoxemic liver injury is elusive. So we conditionally inactivate Sirt1 in murine hepatocytes to determine its role in d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver damage, which is a well-established experimental model mimicking septic liver injury and fulminant hepatitis. Ablation of Sirt1 shows remarkable protection against GalN/LPS-induced liver injury, which is a result of enhanced NF-κB response because knockdown of RelA/p65 negates the protective effect of Sirt1 knockout. Mechanistically, NF-κB p65 is maintained in a hyperacetylated, DNA-binding competent state in tumor necrosis factor-α (TNF-α)-challenged albumin-Cre+ (AlbCre+) hepatocytes. Transfection of hepatocytes with a recombinant acetylated p65 expression construct replicates the protection afforded by Sirt1 knockout. Transfection of AlbCre+ hepatocytes with a recombinant wild-type Sirt1 construct, rather than a deacetylase-defective one, compromises NF-κB activation and resensitizes hepatocytes to TNF-α-induced apoptosis. Taken together, our results demonstrate that Sirt1 deacetylates p65 and compromises NF-κB activity in hepatocytes when confronted with LPS/TNF-α stimulation, leading to increased susceptibility to endotoxemic injury. These findings identify a possible protein effector to maneuver the hepatic NF-κB signaling pathway under inflammatory circumstances and a feasible way to increase hepatocellular resistance to endotoxin/TNF-α toxicity. PMID:27711079

  16. Age-related changes in lamin A/C expression in cardiomyocytes.

    PubMed

    Afilalo, Jonathan; Sebag, Igal A; Chalifour, Lorraine E; Rivas, Daniel; Akter, Rahima; Sharma, Kamal; Duque, Gustavo

    2007-09-01

    Lamin A and C (A/C) are type V intermediate filaments that form the nuclear lamina. Lamin A/C mutations lead to reduced expression of lamin A/C and diverse phenotypes such as familial cardiomyopathies and accelerated aging syndromes. Normal aging is associated with reduced expression of lamin A/C in osteoblasts and dermal fibroblasts but has never been assessed in cardiomyocytes. Our objective was to compare the expression of lamin A/C in cardiomyocytes of old (24 mo) versus young (4 mo) C57Bl/6J mice using a well-validated mouse model of aging. Lamin B1 was used as a control. Immunohistochemical and immunofluorescence analyses showed reduced expression of lamin A/C in cardiomyocyte nuclei of old mice (proportion of nuclei expressing lamin A/C, 9% vs. 62%, P < 0.001). Lamin A/C distribution was scattered peripherally and perinuclear in old mice, whereas it was homogeneous throughout the nuclei in young mice. Western blot analyses confirmed reduced expression of lamin A/C in nuclear extracts of old mice (ratio of lamin A/C to B1, 0.6 vs. 1.2, P < 0.01). Echocardiographic studies showed increased left ventricular wall thickness with preserved cavity size (concentric remodeling), increased left ventricular mass, and a slight reduction in fractional shortening in old mice. This is the first study to show that normal aging is associated with reduced expression and altered distribution of lamin A/C in nuclei of cardiomyocytes.

  17. Cardiomyocyte glucagon receptor signaling modulates outcomes in mice with experimental myocardial infarction

    PubMed Central

    Ali, Safina; Ussher, John R.; Baggio, Laurie L.; Kabir, M. Golam; Charron, Maureen J.; Ilkayeva, Olga; Newgard, Christopher B.; Drucker, Daniel J.

    2014-01-01

    Objective Glucagon is a hormone with metabolic actions that maintains normoglycemia during the fasting state. Strategies enabling either inhibition or activation of glucagon receptor (Gcgr) signaling are being explored for the treatment of diabetes or obesity. However, the cardiovascular consequences of manipulating glucagon action are poorly understood. Methods We assessed infarct size and the following outcomes following left anterior descending (LAD) coronary artery ligation; cardiac gene and protein expression, acylcarnitine profiles, and cardiomyocyte survival in normoglycemic non-obese wildtype mice, and in newly generated mice with selective inactivation of the cardiomyocyte Gcgr. Complementary experiments analyzed Gcgr signaling and cell survival in cardiomyocyte cultures and cell lines, in the presence or absence of exogenous glucagon. Results Exogenous glucagon administration directly impaired recovery of ventricular pressure in ischemic mouse hearts ex vivo, and increased mortality from myocardial infarction after LAD coronary artery ligation in mice in a p38 MAPK-dependent manner. In contrast, cardiomyocyte-specific reduction of glucagon action in adult GcgrCM−/− mice significantly improved survival, and reduced hypertrophy and infarct size following myocardial infarction. Metabolic profiling of hearts from GcgrCM−/− mice revealed a marked reduction in long chain acylcarnitines in both aerobic and ischemic hearts, and following high fat feeding, consistent with an essential role for Gcgr signaling in the control of cardiac fatty acid utilization. Conclusions Activation or reduction of cardiac Gcgr signaling in the ischemic heart produces substantial cardiac phenotypes, findings with implications for therapeutic strategies designed to augment or inhibit Gcgr signaling for the treatment of metabolic disorders. PMID:25685700

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

    SciTech Connect

    Xu, Aibin; Liu, Jingyi; Liu, Peilin; Jia, Min; Wang, Han; Tao, Ling

    2014-04-18

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

  19. Heme-induced contractile dysfunction in human cardiomyocytes caused by oxidant damage to thick filament proteins.

    PubMed

    Alvarado, Gerardo; Jeney, Viktória; Tóth, Attila; Csősz, Éva; Kalló, Gergő; Huynh, An T; Hajnal, Csaba; Kalász, Judit; Pásztor, Enikő T; Édes, István; Gram, Magnus; Akerström, Bo; Smith, Ann; Eaton, John W; Balla, György; Papp, Zoltán; Balla, József

    2015-12-01

    Intracellular free heme predisposes to oxidant-mediated tissue damage. We hypothesized that free heme causes alterations in myocardial contractility via disturbed structure and/or regulation of the contractile proteins. Isometric force production and its Ca(2+)-sensitivity (pCa50) were monitored in permeabilized human ventricular cardiomyocytes. Heme exposure altered cardiomyocyte morphology and evoked robust decreases in Ca(2+)-activated maximal active force (Fo) while increasing Ca(2+)-independent passive force (F passive). Heme treatments, either alone or in combination with H2O2, did not affect pCa50. The increase in F passive started at 3 µM heme exposure and could be partially reversed by the antioxidant dithiothreitol. Protein sulfhydryl (SH) groups of thick myofilament content decreased and sulfenic acid formation increased after treatment with heme. Partial restoration in the SH group content was observed in a protein running at 140 kDa after treatment with dithiothreitol, but not in other proteins, such as filamin C, myosin heavy chain, cardiac myosin binding protein C, and α-actinin. Importantly, binding of heme to hemopexin or alpha-1-microglobulin prevented its effects on cardiomyocyte contractility, suggesting an allosteric effect. In line with this, free heme directly bound to myosin light chain 1 in human cardiomyocytes. Our observations suggest that free heme modifies cardiac contractile proteins via posttranslational protein modifications and via binding to myosin light chain 1, leading to severe contractile dysfunction. This may contribute to systolic and diastolic cardiac dysfunctions in hemolytic diseases, heart failure, and myocardial ischemia-reperfusion injury.

  20. Spontaneously beating cardiomyocytes derived from white mature adipocytes

    PubMed Central

    Jumabay, Medet; Zhang, Rui; Yao, Yucheng; Goldhaber, Joshua I.; Boström, Kristina I.

    2010-01-01

    Aims Adipose stromal cells and dissociated brown adipose tissue have been shown to generate cardiomyocyte-like cells. However, it is not clear whether white mature adipocytes have the same potential, even though a close relationship has been found between adipocytes and vascular endothelial cells, another cardiovascular cell type. The objective of this study was to examine if white adipocytes would be able to supply cardiomyocytes. Methods and results We prepared a highly purified population of lipid-filled adipocytes from mice, 6–7 weeks of age. When allowed to lose lipids, the adipocytes assumed a fibroblast-like morphology, so-called dedifferentiated fat (DFAT) cells. Subsequently, 10–15% of the DFAT cells spontaneously differentiated into cardiomyocyte-like cells, in which the cardiomyocyte phenotype was identified by morphological observations, expression of cardiomyocyte-specific markers, and immunocytochemical staining. In addition, electrophysiological studies revealed pacemaker activity in these cells, and functional studies showed that a β-adrenergic agonist stimulated the beating rate, whereas a β-antagonist reduced it. In vitro treatment of newly isolated adipocytes or DFAT cells with inhibitors of bone morphogenetic proteins (BMP) and Wnt signalling promoted the development of the cardiomyocyte phenotype as determined by the number or beating colonies of cardiomyocyte-like cells and expression of troponin I, a cardiomyocyte-specific marker. Inhibition of BMP was most effective in promoting the cardiomyocyte phenotype in adipocytes, whereas Wnt-inhibition was most effective in DFAT cells. Conclusion White mature adipocytes can differentiate into cardiomyocyte-like cells, suggesting a link between adipocyte and cardiomyocyte differentiation. PMID:19643806

  1. Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity

    SciTech Connect

    Kashimshetty, Rohini; Desai, Varsha G.; Kale, Vijay M.; Lee, Taewon; Moland, Carrie L.; Branham, William S.; New, Lee S.; Chan, Eric C.Y.; Younis, Husam; Boelsterli, Urs A.

    2009-07-15

    Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities. Male wild-type or heterozygous Sod2{sup +/-} mice were injected intraperitoneously with flutamide (0, 30 or 100 mg/kg/day) for 28 days. A kinetic pilot study revealed that flutamide (100 mg/kg/day) caused approximately 10-fold greater exposure than the reported therapeutic mean plasma levels. Mutant (5/10), but not wild-type, mice in the high-dose group exhibited small foci of hepatocellular necrosis and an increased number of apoptotic hepatocytes. Hepatic GSSG/GSH, protein carbonyl levels, and serum lactate levels were significantly increased, suggesting oxidant stress and mitochondrial dysfunction. Measurement of mitochondrial superoxide in cultured hepatocytes demonstrated that mitochondria were a significant source of flutamide-enhanced oxidant stress. Indeed, mitochondria isolated from flutamide-treated Sod2{sup +/-} mice exhibited decreased aconitase activity as compared to vehicle controls. A transcriptomics analysis using MitoChips revealed that flutamide-treated Sod2{sup +/-} mice exhibited a selective decrease in the expression of all complexes I and III subunits encoded by mitochondrial DNA. In contrast, Sod2{sup +/-} mice receiving bicalutamide (50 mg/kg/day) did not reveal any hepatic changes. These results are compatible with our concept that flutamide targets hepatic mitochondria and exerts oxidant stress that can lead to overt hepatic injury in the presence of an underlying mitochondrial abnormality.

  2. Optimizing Mouse Surgery with Online Rectal Temperature Monitoring and Preoperative Heat Supply. Effects on Post-Ischemic Acute Kidney Injury.

    PubMed

    Marschner, Julian A; Schäfer, Hannah; Holderied, Alexander; Anders, Hans-Joachim

    2016-01-01

    Body temperature affects outcomes of tissue injury. We hypothesized that online body core temperature recording and selective interventions help to standardize peri-interventional temperature control and the reliability of outcomes in experimental renal ischemia reperfusion injury (IRI). We recorded core temperature in up to seven mice in parallel using a Thermes USB recorder and ret-3-iso rectal probes with three different protocols. Setup A: Heating pad during ischemia time; Setup B: Heating pad from incision to wound closure; Setup C: A ventilated heating chamber before surgery and during ischemia time with surgeries performed on a heating pad. Temperature profile recording displayed significant declines upon installing anesthesia. The profile of the baseline experimental setup A revealed that <1% of the temperature readings were within the target range of 36.5 to 38.5°C. Setup B and C increased the target range readings to 34.6 ± 28.0% and 99.3 ± 1.5%, respectively. Setup C significantly increased S3 tubular necrosis, neutrophil influx, and mRNA expression of kidney injury markers. In addition, using setup C different ischemia times generated a linear correlation with acute tubular necrosis parameters at a low variability, which further correlated with the degree of kidney atrophy 5 weeks after surgery. Changing temperature control setup A to C was equivalent to 10 minutes more ischemia time. We conclude that body temperature drops quickly in mice upon initiating anesthesia. Immediate heat supply, e.g. in a ventilated heating chamber, and online core temperature monitoring can help to standardize and optimize experimental outcomes.

  3. Chronic Exposure to Androgenic-Anabolic Steroids Exacerbates Axonal Injury and Microgliosis in the CHIMERA Mouse Model of Repetitive Concussion

    PubMed Central

    Namjoshi, Dhananjay R.; Cheng, Wai Hang; Carr, Michael; Martens, Kris M.; Zareyan, Shahab; Wilkinson, Anna; McInnes, Kurt A.; Cripton, Peter A.; Wellington, Cheryl L.

    2016-01-01

    Concussion is a serious health concern. Concussion in athletes is of particular interest with respect to the relationship of concussion exposure to risk of chronic traumatic encephalopathy (CTE), a neurodegenerative condition associated with altered cognitive and psychiatric functions and profound tauopathy. However, much remains to be learned about factors other than cumulative exposure that could influence concussion pathogenesis. Approximately 20% of CTE cases report a history of substance use including androgenic-anabolic steroids (AAS). How acute, chronic, or historical AAS use may affect the vulnerability of the brain to concussion is unknown. We therefore tested whether antecedent AAS exposure in young, male C57Bl/6 mice affects acute behavioral and neuropathological responses to mild traumatic brain injury (TBI) induced with the CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) platform. Male C57Bl/6 mice received either vehicle or a cocktail of three AAS (testosterone, nandrolone and 17α-methyltestosterone) from 8–16 weeks of age. At the end of the 7th week of treatment, mice underwent two closed-head TBI or sham procedures spaced 24 h apart using CHIMERA. Post-repetitive TBI (rTBI) behavior was assessed for 7 d followed by tissue collection. AAS treatment induced the expected physiological changes including increased body weight, testicular atrophy, aggression and downregulation of brain 5-HT1B receptor expression. rTBI induced behavioral deficits, widespread axonal injury and white matter microgliosis. While AAS treatment did not worsen post-rTBI behavioral changes, AAS-treated mice exhibited significantly exacerbated axonal injury and microgliosis, indicating that AAS exposure can alter neuronal and innate immune responses to concussive TBI. PMID:26784694

  4. Chronic Exposure to Androgenic-Anabolic Steroids Exacerbates Axonal Injury and Microgliosis in the CHIMERA Mouse Model of Repetitive Concussion.

    PubMed

    Namjoshi, Dhananjay R; Cheng, Wai Hang; Carr, Michael; Martens, Kris M; Zareyan, Shahab; Wilkinson, Anna; McInnes, Kurt A; Cripton, Peter A; Wellington, Cheryl L

    2016-01-01

    Concussion is a serious health concern. Concussion in athletes is of particular interest with respect to the relationship of concussion exposure to risk of chronic traumatic encephalopathy (CTE), a neurodegenerative condition associated with altered cognitive and psychiatric functions and profound tauopathy. However, much remains to be learned about factors other than cumulative exposure that could influence concussion pathogenesis. Approximately 20% of CTE cases report a history of substance use including androgenic-anabolic steroids (AAS). How acute, chronic, or historical AAS use may affect the vulnerability of the brain to concussion is unknown. We therefore tested whether antecedent AAS exposure in young, male C57Bl/6 mice affects acute behavioral and neuropathological responses to mild traumatic brain injury (TBI) induced with the CHIMERA (Closed Head Impact Model of Engineered Rotational Acceleration) platform. Male C57Bl/6 mice received either vehicle or a cocktail of three AAS (testosterone, nandrolone and 17α-methyltestosterone) from 8-16 weeks of age. At the end of the 7th week of treatment, mice underwent two closed-head TBI or sham procedures spaced 24 h apart using CHIMERA. Post-repetitive TBI (rTBI) behavior was assessed for 7 d followed by tissue collection. AAS treatment induced the expected physiological changes including increased body weight, testicular atrophy, aggression and downregulation of brain 5-HT1B receptor expression. rTBI induced behavioral deficits, widespread axonal injury and white matter microgliosis. While AAS treatment did not worsen post-rTBI behavioral changes, AAS-treated mice exhibited significantly exacerbated axonal injury and microgliosis, indicating that AAS exposure can alter neuronal and innate immune responses to concussive TBI.

  5. The effects of cilostazol on tissue oxygenation upon an ischemic-reperfusion injury in the mouse cerebrum.

    PubMed

    Morikawa, Takayuki; Hattori, Katsuji; Kajimura, Mayumi; Suematsu, Makoto

    2010-01-01

    Although cilostazol, an inhibitor of cyclic nucleotide phosphodiesterase 3 (PDE3), is known to exert a potent antiplatelet function by raising intracellular cAMP concentration, its effect on cerebral microcirculation upon an ischemic insult is not clearly understood. To examine effects of cilostazol on the global ischemic injury in the brain, we first measured the plasma leakage using modified Miles assay after mice had been subjected to 60 min of a bilateral common carotid artery (BCCA) occlusion followed by reperfusion for 4 h. Oral treatment with cilostazol (30 mg/kg) significantly increased plasma leakage. This result led us to examine if the treatment with cilostazol recruits more capillaries leading to an increase in surface area for exchange and oxygen transport to tissues. To do so, we simultaneously measured degrees of tissue hypoxia and vessel perfusion. Pimonidazol was injected intraperitoneally 1 h before sacrifice and capillary patency was assessed by fluorescein isothiocyanate-labeled Lycopersicon esculentum lectin bound to the endothelial surface. Treatment with cilostazol markedly increased the capillary patency which was accompanied by a reduction in the hypoxic area. Although the treatment with cilostazol caused an increase in the flux of plasma proteins across endothelial barrier that may imply an adverse role after a BCCA occlusion, this increase in protein leakage was attributable to the increased surface area for exchange which in turn brought about a reduction in tissue hypoxia. Taken together cilostazol appears to produce a protective effect against the ischemic-reperfusion injury.

  6. Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes.

    PubMed

    Alvarez, Julio L; Petzhold, Daria; Pankonien, Ines; Behlke, Joachim; Kouno, Michiyoshi; Vassort, Guy; Morano, Ingo; Haase, Hannelore

    2010-09-01

    Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.

  7. Cognitive Impairments Induced by Concussive Mild Traumatic Brain Injury in Mouse Are Ameliorated by Treatment with Phenserine via Multiple Non-Cholinergic and Cholinergic Mechanisms

    PubMed Central

    Li, Yazhou; Yu, Qian-sheng; Barak, Shani; Tamargo, Ian A.; Rubovitch, Vardit; Holloway, Harold W.; Lehrmann, Elin; Wood, William H.; Zhang, Yongqing; Becker, Kevin G.; Perez, Evelyn; Van Praag, Henriette; Luo, Yu; Hoffer, Barry J.; Becker, Robert E.; Pick, Chaim G.; Greig, Nigel H.

    2016-01-01

    Traumatic brain injury (TBI), often caused by a concussive impact to the head, affects an estimated 1.7 million Americans annually. With no approved drugs, its pharmacological treatment represents a significant and currently unmet medical need. In our prior development of the anti-cholinesterase compound phenserine for the treatment of neurodegenerative disorders, we recognized that it also possesses non-cholinergic actions with clinical potential. Here, we demonstrate neuroprotective actions of phenserine in neuronal cultures challenged with oxidative stress and glutamate excitotoxicity, two insults of relevance to TBI. These actions translated into amelioration of spatial and visual memory impairments in a mouse model of closed head mild TBI (mTBI) two days following cessation of clinically translatable dosing with phenserine (2.5 and 5.0 mg/kg BID x 5 days initiated post mTBI) in the absence of anti-cholinesterase activity. mTBI elevated levels of thiobarbituric acid reactive substances (TBARS), a marker of oxidative stress. Phenserine counteracted this by augmenting homeostatic mechanisms to mitigate oxidative stress, including superoxide dismutase [SOD] 1 and 2, and glutathione peroxidase [GPx], the activity and protein levels of which were measured by specific assays. Microarray analysis of hippocampal gene expression established that large numbers of genes were exclusively regulated by each individual treatment with a substantial number of them co-regulated between groups. Molecular pathways associated with lipid peroxidation were found to be regulated by mTBI, and treatment of mTBI animals with phenserine effectively reversed injury-induced regulations in the ‘Blalock Alzheimer’s Disease Up’ pathway. Together these data suggest that multiple phenserine-associated actions underpin this compound’s ability to ameliorate cognitive deficits caused by mTBI, and support the further evaluation of the compound as a therapeutic for TBI. PMID:27254111

  8. Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy.

    PubMed

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

    2013-10-22

    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.

  9. Micropost arrays for measuring stem cell-derived cardiomyocyte contractility

    PubMed Central

    Beussman, Kevin M.; Rodriguez, Marita L.; Leonard, Andrea; Taparia, Nikita; Thompson, Curtis R.; Sniadecki, Nathan J.

    2015-01-01

    Stem cell-derived cardiomyocytes have the potential to be used to study heart disease and maturation, screen drug treatments, and restore heart function. Here, we discuss the procedures involved in using micropost arrays to measure the contractile forces generated by stem cell-derived cardiomyocytes. Cardiomyocyte contractility is needed for the heart to pump blood, so measuring the contractile forces of cardiomyocytes is a straightforward way to assess their function. Microfabrication and soft lithography techniques are utilized to create identical arrays of flexible, silicone microposts from a common master. Micropost arrays are functionalized with extracellular matrix protein to allow cardiomyocytes to adhere to the tips of the microposts. Live imaging is used to capture videos of the deflection of microposts caused by the contraction of the cardiomyocytes. Image analysis code provides an accurate means to quantify these deflections. The contractile forces produced by a beating cardiomyocyte are calculated by modeling the microposts as cantilever beams. We have used this assay to assess techniques for improving the maturation and contractile function of stem cell-derived cardiomyocytes. PMID:26344757

  10. Acoustical sensing of cardiomyocyte cluster beating

    SciTech Connect

    Tymchenko, Nina; Kunze, Angelika; Dahlenborg, Kerstin; Svedhem, Sofia; Steel, Daniella

    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 cell 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.

  11. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease

    PubMed Central

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A.; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K.; Schübeler, Dirk; Hein, Lutz

    2014-01-01

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity. PMID:25335909

  12. Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease.

    PubMed

    Gilsbach, Ralf; Preissl, Sebastian; Grüning, Björn A; Schnick, Tilman; Burger, Lukas; Benes, Vladimir; Würch, Andreas; Bönisch, Ulrike; Günther, Stefan; Backofen, Rolf; Fleischmann, Bernd K; Schübeler, Dirk; Hein, Lutz

    2014-10-22

    The heart is a highly specialized organ with essential function for the organism throughout life. The significance of DNA methylation in shaping the phenotype of the heart remains only partially known. Here we generate and analyse DNA methylomes from highly purified cardiomyocytes of neonatal, adult healthy and adult failing hearts. We identify large genomic regions that are differentially methylated during cardiomyocyte development and maturation. Demethylation of cardiomyocyte gene bodies correlates strongly with increased gene expression. Silencing of demethylated genes is characterized by the polycomb mark H3K27me3 or by DNA methylation. De novo methylation by DNA methyltransferases 3A/B causes repression of fetal cardiac genes, including essential components of the cardiac sarcomere. Failing cardiomyocytes partially resemble neonatal methylation patterns. This study establishes DNA methylation as a highly dynamic process during postnatal growth of cardiomyocytes and their adaptation to pathological stress in a process tightly linked to gene regulation and activity.

  13. Renal progenitors derived from human iPSCs engraft and restore function in a mouse model of acute kidney injury

    PubMed Central

    Imberti, Barbara; Tomasoni, Susanna; Ciampi, Osele; Pezzotta, Anna; Derosas, Manuela; Xinaris, Christodoulos; Rizzo, Paola; Papadimou, Evangelia; Novelli, Rubina; Benigni, Ariela; Remuzzi, Giuseppe; Morigi, Marina

    2015-01-01

    Acute kidney injury (AKI) is one of the most relevant health issues, leading to millions of deaths. The magnitude of the phenomenon remarks the urgent need for innovative and effective therapeutic approaches. Cell-based therapy with renal progenitor cells (RPCs) has been proposed as a possible strategy. Studies have shown the feasibility of directing embryonic stem cells or induced Pluripotent Stem Cells (iPSCs) towards nephrogenic intermediate mesoderm and metanephric mesenchyme (MM). However, the functional activity of iPSC-derived RPCs has not been tested in animal models of kidney disease. Here, through an efficient inductive protocol, we directed human iPSCs towards RPCs that robustly engrafted into damaged tubuli and restored renal function and structure in cisplatin-mice with AKI. These results demonstrate that iPSCs are a valuable source of engraftable cells with regenerative activity for kidney disease and create the basis for future applications in stem cell-based therapy. PMID:25744951

  14. Metabolomic profiling of a modified alcohol liquid diet model for liver injury in the mouse uncovers new markers of disease

    SciTech Connect

    Bradford, Blair U.; O'Connell, Thomas M.; Han, Jun; Kosyk, Oksana; Shymonyak, Svitlana; Ross, Pamela K.; Winnike, Jason; Kono, Hiroshi; Rusyn, Ivan

    2008-10-15

    Metabolomic evaluation of urine and liver was conducted to assess the biochemical changes that occur as a result of alcohol-induced liver injury. Male C57BL/6J mice were fed an isocaloric control- or alcohol-containing liquid diet with 35% of calories from corn oil, 18% protein and 47% carbohydrate/alcohol for up to 36 days ad libitum. Alcohol treatment was initiated at 7 g/kg/day and gradually reached a final dose of 21 g/kg/day. Urine samples were collected at 22, 30 and 36 days and, in additional treatment groups, liver and serum samples were harvested at 28 days. Steatohepatitis was induced in the alcohol-fed group since a 5-fold increase in serum alanine aminotransferase activity, a 6-fold increase in liver injury score (necrosis, inflammation and steatosis) and an increase in lipid peroxidation in liver were observed. Liver and urine samples were analyzed by nuclear magnetic resonance spectroscopy and electrospray infusion/Fourier transform ion cyclotron resonance-mass spectrometry. In livers of alcohol-treated mice the following changes were noted. Hypoxia and glycolysis were activated as evidenced by elevated levels of alanine and lactate. Tyrosine, which is required for L-DOPA and dopamine as well as thyroid hormones, was elevated possibly reflecting alterations of basal metabolism by alcohol. A 4-fold increase in the prostacyclin inhibitor 7,10,13,16-docosatetraenoic acid, a molecule important for regulation of platelet formation and blood clotting, may explain why chronic drinking causes serious bleeding problems. Metabolomic analysis of the urine revealed that alcohol treatment leads to decreased excretion of taurine, a metabolite of glutathione, and an increase in lactate, n-acetylglutamine and n-acetylglycine. Changes in the latter two metabolites suggest an inhibition of the kidney enzyme aminoacylase I and may be useful as markers for alcohol consumption.

  15. Metabolomic profiling of a modified alcohol liquid diet model for liver injury in the mouse uncovers new markers of disease

    PubMed Central

    Bradford, Blair U.; O’Connell, Thomas M.; Han, Jun; Kosyk, Oksana; Shymonyak, Svitlana; Ross, Pamela K.; Winnike, Jason; Kono, Hiroshi; Rusyn, Ivan

    2008-01-01

    Metabolomic evaluation of urine and liver was conducted to assess the biochemical changes that occur as a result of alcohol-induced liver injury. Male C57BL/6J mice were fed an isocaloric control-or alcohol-containing liquid diet with 35% of calories from corn oil, 18% protein and 47% carbohydrate/alcohol for up to 36 days ad libitum. Alcohol treatment was initiated at 7 g/kg/day and gradually reached a final dose of 21 g/kg/day. Urine samples were collected at 22, 30 and 36 days and in additional treatment groups, liver and serum samples were harvested at 28 days. Steatohepatitis was induced in the alcohol-fed group since a 5-fold increase in serum alanine aminotransferase activity, a 6-fold increase in liver injury score (necrosis, inflammation and steatosis) and an increase in lipid peroxidation in liver were observed. Liver and urine samples were analyzed by nuclear magnetic resonance spectroscopy and electrospray infusion/Fourier transform ion cyclotron resonance-mass spectrometry. In livers of alcohol-treated mice the following changes were noted. Hypoxia and glycolysis were activated as evidenced by elevated levels of alanine and lactate. Tyrosine, which is required for L-DOPA and dopamine as well as thyroid hormones, was elevated possibly reflecting alterations of basal metabolism by alcohol. A 4-fold increase in the prostacyclin inhibitor 7,10,13,16-docosatetraenoic acid, a molecule important for regulation of platelet formation and blood clotting, may explain why chronic drinking causes serious bleeding problems. Metabolomic analysis of the urine revealed that alcohol treatment leads to decreased excretion of taurine, a metabolite of glutathione, and an increase in lactate, n-acetylglutamine and n-acetylglycine. Changes in the latter two metabolites suggest an inhibition of the kidney enzyme aminoacylase I and may be useful as markers for alcohol consumption. PMID:18674555

  16. DIETARY FLAXSEED PREVENTS RADIATION-INDUCED OXIDATIVE LUNG DAMAGE, INFLAMMATION AND FIBROSIS IN A MOUSE MODEL OF THORACIC RADIATION INJURY

    PubMed Central

    Lee, James C.; Krochak, Ryan; Blouin, Aaron; Kanterakis, Stathis; Chatterjee, Shampa; Arguiri, Evguenia; Vachani, Anil; Solomides, Charalambos C.; Cengel, Keith A.; Christofidou-Solomidou, Melpo

    2009-01-01

    Flaxseed (FS) has high contents of omega-3 fatty acids and lignans with antioxidant properties. Its use in preventing thoracic X-ray radiation therapy (XRT)-induced pneumonopathy has never been evaluated. We evaluated FS supplementation given to mice given before and post-XRT. FS-derived lignans, known for their direct antioxidant properties, were evaluated in abrogating ROS generation in cultured endothelial cells following gamma radiation exposure. Mice were fed 10% FS or isocaloric control diet for three weeks and given 13.5 Gy thoracic XRT. Lungs were evaluated at 24 hours for markers of radiation-induced injury, three weeks for acute lung damage (lipid peroxidation, lung edema and inflammation), and at four months for late lung damage (inflammation and fibrosis). FS-Lignans blunted ROS generation in vitro, resulting from radiation in a dose-dependent manner. FS-fed mice had reduced expression of lung injury biomarkers (Bax, p21, and TGF-beta1) at 24 hours following XRT and reduced oxidative lung damage as measured by malondialdehyde (MDA) levels at 3 weeks following XRT. In addition, FS-fed mice had decreased lung fibrosis as determined by hydroxyproline content and decreased inflammatory cell influx into lungs at 4 months post XRT. Importantly, when Lewis Lung carcinoma cells were injected systemically in mice, FS dietary supplementation did not appear to protect lung tumors from responding to thoracic XRT. Dietary FS is protective against pulmonary fibrosis, inflammation and oxidative lung damage in a murine model. Moreover, in this model, tumor radioprotection was not observed. FS lignans exhibited potent radiation-induced ROS scavenging action. Taken together, these data suggest that dietary flaxseed may be clinically useful as an agent to increase the therapeutic index of thoracic XRT by increasing the radiation tolerance of lung tissues. PMID:18981722

  17. A Soluble Activin Receptor IIB Fails to Prevent Muscle Atrophy in a Mouse Model of Spinal Cord Injury.

    PubMed

    Graham, Zachary A; Collier, Lauren; Peng, Yuanzhen; Saéz, Juan C; Bauman, William A; Qin, Weiping; Cardozo, Christopher P

    2016-06-15

    Myostatin (MST) is a potent regulator of muscle growth and size. Spinal cord injury (SCI) results in marked atrophy of muscle below the level of injury. Currently, there is no effective pharmaceutical treatment available to prevent sublesional muscle atrophy post-SCI. To determine whether inhibition of MST with a soluble activin IIB receptor (RAP-031) prevents sublesional SCI-induced muscle atrophy, mice were randomly assigned to the following groups: Sham-SCI; SCI+Vehicle group (SCI-VEH); and SCI+RAP-031 (SCI-RAP-031). SCI was induced by complete transection at thoracic level 10. Animals were euthanized at 56 days post-surgery. RAP-031 reduced, but did not prevent, body weight loss post-SCI. RAP-031 increased total lean tissue mass compared to SCI-VEH (14.8%). RAP-031 increased forelimb muscle mass post-SCI by 38% and 19% for biceps and triceps, respectively (p < 0.001). There were no differences in hindlimb muscle weights between the RAP-031 and SCI-VEH groups. In the gastrocnemius, messenger RNA (mRNA) expression was elevated for interleukin (IL)-6 (8-fold), IL-1β (3-fold), and tumor necrosis factor alpha (8-fold) in the SCI-VEH, compared to the Sham group. Muscle RING finger protein 1 mRNA was 2-fold greater in the RAP-031 group, compared to Sham-SCI. RAP-031 did not influence cytokine expression. Bone mineral density of the distal femur and proximal tibia were decreased post-SCI (-26% and -28%, respectively) and were not altered by RAP-031. In conclusion, MST inhibition increased supralesional muscle mass, but did not prevent sublesional muscle or bone loss, or the inflammation in paralyzed muscle.

  18. Exercise Training after Spinal Cord Injury Selectively Alters Synaptic Properties in Neurons in Adult Mouse Spinal Cord

    PubMed Central

    Flynn, Jamie R.; Dunn, Lynda R.; Galea, Mary P.; Callister, Robin; Rank, Michelle M.

    2013-01-01

    Abstract Following spinal cord injury (SCI), anatomical changes such as axonal sprouting occur within weeks in the vicinity of the injury. Exercise training enhances axon sprouting; however, the exact mechanisms that mediate exercised-induced plasticity are unknown. We studied the effects of exercise training after SCI on the intrinsic and synaptic properties of spinal neurons in the immediate vicinity (<2 segments) of the SCI. Male mice (C57BL/6, 9–10 weeks old) received a spinal hemisection (T10) and after 1 week of recovery, they were randomized to trained (treadmill exercise for 3 weeks) and untrained (no exercise) groups. After 3 weeks, mice were killed and horizontal spinal cord slices (T6–L1, 250 μm thick) were prepared for visually guided whole cell patch clamp recording. Intrinsic properties, including resting membrane potential, input resistance, rheobase current, action potential (AP) threshold and after-hyperpolarization (AHP) amplitude were similar in neurons from trained and untrained mice (n=67 and 70 neurons, respectively). Neurons could be grouped into four categories based on their AP discharge during depolarizing current injection; the proportions of tonic firing, initial bursting, single spiking, and delayed firing neurons were similar in trained and untrained mice. The properties of spontaneous excitatory synaptic currents (sEPSCs) did not differ in trained and untrained animals. In contrast, evoked excitatory synaptic currents recorded after dorsal column stimulation were markedly increased in trained animals (peak amplitude 78.9±17.5 vs. 42.2±6.8 pA; charge 1054±376 vs. 348±75 pA·ms). These data suggest that 3 weeks of treadmill exercise does not affect the intrinsic properties of spinal neurons after SCI; however, excitatory synaptic drive from dorsal column pathways, such as the corticospinal tract, is enhanced. PMID:23320512

  19. Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury

    PubMed Central

    Avetisyan, Marina; Wang, Hongtao; Schill, Ellen Merrick; Bery, Saya; Grider, John R.; Hassell, John A.; Stappenbeck, Thaddeus

    2015-01-01

    Factors providing trophic support to diverse enteric neuron subtypes remain poorly understood. We tested the hypothesis that hepatocyte growth factor (HGF) and the HGF receptor MET might support some types of enteric neurons. HGF and MET are expressed in fetal and adult enteric nervous system. In vitro, HGF increased enteric neuron differentiation and neurite length, but only if vanishingly small amounts (1 pg/ml) of glial cell line-derived neurotrophic factor were included in culture media. HGF effects were blocked by phosphatidylinositol-3 kinase inhibitor and by MET-blocking antibody. Both of these inhibitors and MEK inhibition reduced neurite length. In adult mice, MET was restricted to a subset of calcitonin gene-related peptide-immunoreactive (IR) myenteric plexus neurons thought to be intrinsic primary afferent neurons (IPANs). Conditional MET kinase domain inactivation (Metfl/fl; Wnt1Cre+) caused a dramatic loss of myenteric plexus MET-IR neurites and 1–1′-dioctodecyl-3,3,3′,3′-tetramethylindocarbocyamine perchlorate (DiI) labeling suggested reduced MET-IR neurite length. In vitro, Metfl/fl; Wnt1Cre+ mouse bowel had markedly reduced peristalsis in response to mucosal deformation, but normal response to radial muscle stretch. However, whole-bowel transit, small-bowel transit, and colonic-bead expulsion were normal in Metfl/fl; Wnt1Cre+ mice. Finally, Metfl/fl; Wnt1Cre+ mice had more bowel injury and reduced epithelial cell proliferation compared with WT animals after dextran sodium sulfate treatment. These results suggest that HGF/MET signaling is important for development and function of a subset IPANs and that these cells regulate intestinal motility and epithelial cell proliferation in response to bowel injury. SIGNIFICANCE STATEMENT The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well

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

    PubMed

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

    2014-11-01

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

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

    PubMed Central

    Welsh, David K.

    2016-01-01

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

  2. The Role of Reactive Oxygen Species in β-Adrenergic Signaling in Cardiomyocytes from Mice with the Metabolic Syndrome

    PubMed Central

    Llano-Diez, Monica; Sinclair, Jon; Yamada, Takashi; Zong, Mei; Fauconnier, Jeremy; Zhang, Shi-Jin; Katz, Abram; Jardemark, Kent; Westerblad, Håkan; Andersson, Daniel C.

    2016-01-01

    The metabolic syndrome is associated with prolonged stress and hyperactivity of the sympathetic nervous system and afflicted subjects are prone to develop cardiovascular disease. Under normal conditions, the cardiomyocyte response to acute β-adrenergic stimulation partly depends on increased production of reactive oxygen species (ROS). Here we investigated the interplay between beta-adrenergic signaling, ROS and cardiac contractility using freshly isolated cardiomyocytes and whole hearts from two mouse models with the metabolic syndrome (high-fat diet and ob/ob mice). We hypothesized that cardiomyocytes of mice with the metabolic syndrome would experience excessive ROS levels that trigger cellular dysfunctions. Fluorescent dyes and confocal microscopy were used to assess mitochondrial ROS production, cellular Ca2+ handling and contractile function in freshly isolated adult cardiomyocytes. Immunofluorescence, western blot and enzyme assay were used to study protein biochemistry. Unexpectedly, our results point towards decreased cardiac ROS signaling in a stable, chronic phase of the metabolic syndrome because: β-adrenergic-induced increases in the amplitude of intracellular Ca2+ signals were insensitive to antioxidant treatment; mitochondrial ROS production showed decreased basal rate and smaller response to β-adrenergic stimulation. Moreover, control hearts and hearts with the metabolic syndrome showed similar basal levels of ROS-mediated protein modification, but only control hearts showed increases after β-adrenergic stimulation. In conclusion, in contrast to the situation in control hearts, the cardiomyocyte response to acute β-adrenergic stimulation does not involve increased mitochondrial ROS production in a stable, chronic phase of the metabolic syndrome. This can be seen as a beneficial adaptation to prevent excessive ROS levels. PMID:27907040

  3. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury.

    PubMed Central

    Marber, M S; Mestril, R; Chi, S H; Sayen, M R; Yellon, D M; Dillmann, W H

    1995-01-01

    Myocardial protection and changes in gene expression follow whole body heat stress. Circumstantial evidence suggests that an inducible 70-kD heat shock protein (hsp70i), increased markedly by whole body heat stress, contributes to the protection. Transgenic mouse lines were constructed with a cytomegalovirus enhancer and beta-actin promoter driving rat hsp70i expression in heterozygote animals. Unstressed, transgene positive mice expressed higher levels of myocardial hsp70i than transgene negative mice after whole body heat stress. This high level of expression occurred without apparent detrimental effect. The hearts harvested from transgene positive mice and transgene negative littermates were Langendorff perfused and subjected to 20 min of warm (37 degrees C) zero-flow ischemia and up to 120 min of reflow while contractile recovery and creatine kinase efflux were measured. Myocardial infarction was demarcated by triphenyltetrazolium. In transgene positive compared with transgene negative hearts, the zone of infarction was reduced by 40%, contractile function at 30 min of reflow was doubled, and efflux of creatine kinase was reduced by approximately 50%. Our findings suggest for the first time that increased myocardial hsp70i expression results in protection of the heart against ischemic injury and that the antiischemic properties of hsp70i have possible therapeutic relevance. Images PMID:7706448

  4. Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

    PubMed Central

    Harder, Yves; Schmauss, Daniel; Wettstein, Reto; Egaña, José T.; Weiss, Fabian; Weinzierl, Andrea; Schuldt, Anna; Machens, Hans-Günther; Menger, Michael D.; Rezaeian, Farid

    2014-01-01

    Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue. PMID:25489743

  5. Temporal Characterization of Microglia/Macrophage Phenotypes in a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

    PubMed Central

    Hellström Erkenstam, Nina; Smith, Peter L. P.; Fleiss, Bobbi; Nair, Syam; Svedin, Pernilla; Wang, Wei; Boström, Martina; Gressens, Pierre; Hagberg, Henrik; Brown, Kelly L.; Sävman, Karin; Mallard, Carina

    2016-01-01

    Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 days post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activation) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6 h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11b+CD86+ cells at 24 h after HI (+3667%), which was coupled with a relative suppression of CD11b+CD206+ cells and cells that did not express neither CD86 nor CD206. The CD11b+CD206+ population was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured gray and white matter. Protein concentration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI. In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also contained a subset of cells expressing both CD86 and CD206 and a large population that

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

    PubMed

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

    2005-08-01

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

  7. Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling

    PubMed Central

    Urlep, Žiga; Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Matz-Soja, Madlen; Gebhardt, Rolf; Björkhem, Ingemar; Hall, Jason A.; Bonneau, Richard; Littman, Dan R.; Rozman, Damjana

    2017-01-01

    Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51−/−) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51−/− and Rorc−/− expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51−/− females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity. PMID:28098217

  8. Intranasal C3a treatment ameliorates cognitive impairment in a mouse model of neonatal hypoxic-ischemic brain injury.

    PubMed

    Morán, Javier; Stokowska, Anna; Walker, Frederik R; Mallard, Carina; Hagberg, Henrik; Pekna, Marcela

    2017-04-01

    Perinatal asphyxia-induced brain injury is often associated with irreversible neurological complications such as intellectual disability and cerebral palsy but available therapies are limited. Novel neuroprotective therapies as well as approaches stimulating neural plasticity mechanism that can compensate for cell death after hypoxia-ischemia (HI) are urgently needed. We previously reported that single i.c.v. injection of complement-derived peptide C3a 1h after HI induction prevented HI-induced cognitive impairment when mice were tested as adults. Here, we tested the effects of intranasal treatment with C3a on HI-induced cognitive deficit. Using the object recognition test, we found that intranasal C3a treated mice were protected from HI-induced impairment of memory function assessed 6weeks after HI induction. C3a treatment ameliorated HI-induced reactive gliosis in the hippocampus, while it did not affect the extent of hippocampal tissue loss, neuronal cell density, expression of the pan-synaptic marker synapsin I or the expression of growth associated protein 43. In conclusion, our results reveal that brief pharmacological treatment with C3a using a clinically feasible non-invasive mode of administration ameliorates HI-induced cognitive impairment. Intranasal administration is a plausible route to deliver C3a into the brain of asphyxiated infants at high risk of developing hypoxic-ischemic encephalopathy.

  9. Disrupting Hepatocyte Cyp51 from Cholesterol Synthesis Leads to Progressive Liver Injury in the Developing Mouse and Decreases RORC Signalling

    NASA Astrophysics Data System (ADS)

    Urlep, Žiga; Lorbek, Gregor; Perše, Martina; Jeruc, Jera; Juvan, Peter; Matz-Soja, Madlen; Gebhardt, Rolf; Björkhem, Ingemar; Hall, Jason A.; Bonneau, Richard; Littman, Dan R.; Rozman, Damjana

    2017-01-01

    Development of mice with hepatocyte knockout of lanosterol 14α-demethylase (HCyp51‑/‑) from cholesterol synthesis is characterized by the progressive onset of liver injury with ductular reaction and fibrosis. These changes begin during puberty and are generally more aggravated in the knockout females. However, a subgroup of (pre)pubertal knockout mice (runts) exhibits a pronounced male prevalent liver dysfunction characterized by downregulated amino acid metabolism and elevated Casp12. RORC transcriptional activity is diminished in livers of all runt mice, in correlation with the depletion of potential RORC ligands subsequent to CYP51 disruption. Further evidence for this comes from the global analysis that identified a crucial overlap between hepatic Cyp51‑/‑ and Rorc‑/‑ expression profiles. Additionally, the reduction in RORA and RORC transcriptional activity was greater in adult HCyp51‑/‑ females than males, which correlates well with their downregulated amino and fatty acid metabolism. Overall, we identify a global and sex-dependent transcriptional de-regulation due to the block in cholesterol synthesis during development of the Cyp51 knockout mice and provide in vivo evidence that sterol intermediates downstream of lanosterol may regulate the hepatic RORC activity.

  10. Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism.

    PubMed

    Chaturvedi, Pankaj; Kalani, Anuradha; Givvimani, Srikanth; Kamat, Pradip Kumar; Familtseva, Anastasia; Tyagi, Suresh C

    2014-04-01

    The mechanisms of homocysteine-mediated cardiac threats are poorly understood. Homocysteine, being the precursor to S-adenosyl methionine (a methyl donor) through methionine, is indirectly involved in methylation phenomena for DNA, RNA, and protein. We reported previously that cardiac-specific deletion of N-methyl-d-aspartate receptor-1 (NMDAR1) ameliorates homocysteine-posed cardiac threats, and in this study, we aim to explore the role of NMDAR1 in epigenetic mechanisms of heart failure, using cardiomyocytes during hyperhomocysteinemia (HHcy). High homocysteine levels activate NMDAR1, which consequently leads to abnormal DNA methylation vs. histone acetylation through modulation of DNA methyltransferase 1 (DNMT1), HDAC1, miRNAs, and MMP9 in cardiomyocytes. HL-1 cardiomyocytes cultured in Claycomb media were treated with 100 μM homocysteine in a dose-dependent manner. NMDAR1 antagonist (MK801) was added in the absence and presence of homocysteine at 10 μM in a dose-dependent manner. The expression of DNMT1, histone deacetylase 1 (HDAC1), NMDAR1, microRNA (miR)-133a, and miR-499 was assessed by real-time PCR as well as Western blotting. Methylation and acetylation levels were determined by checking 5'-methylcytosine DNA methylation and chromatin immunoprecipitation. Hyperhomocysteinemic mouse models (CBS+/-) were used to confirm the results in vivo. In HHcy, the expression of NMDAR1, DNMT1, and matrix metalloproteinase 9 increased with increase in H3K9 acetylation, while HDAC1, miR-133a, and miR-499 decreased in cardiomyocytes. Similar results were obtained in heart tissue of CBS+/- mouse. High homocysteine levels instigate cardiovascular remodeling through NMDAR1, miR-133a, miR-499, and DNMT1. A decrease in HDAC1 and an increase in H3K9 acetylation and DNA methylation are suggestive of chromatin remodeling in HHcy.

  11. Maresin 1 Mitigates High Glucose-Induced Mouse Glomerular Mesangial Cell Injury by Inhibiting Inflammation and Fibrosis

    PubMed Central

    Tang, Shi; Long, Yang; Huang, Wei; Chen, Jiao; Fan, Fang; Jiang, Chunxia

    2017-01-01

    Background. Inflammation and fibrosis are the important pathophysiologic processes in diabetic nephropathy (DN). Maresin 1 is a potential anti-inflammatory lipid mediator, which has displayed powerful proresolving activities. Aim. We determine whether maresin 1 has protective effect on mouse glomerular mesangial cells (GMCs) induced by high glucose. Methods. We cultured GMCs stimulated by high glucose and categorized as follows: normal glucose group (5.6 mmol/L), high glucose group (30 mmol/L), mannitol group, maresin 1 intervention group (1, 10, and 100 nmol/L), maresin 1 and normal glucose group, and the N-acetylcysteine (NAC) intervention group (10 μmol/L NAC). After 24 h, the expression of ROS, NLRP3, caspase-1, procaspase-1, IL-1β, and pro-IL-1β was detected by western-blot, RT-PCR, and immunofluorescence. After 48 h, the expression of TGF-β1 and FN was detected by RT-PCR and ELISA. Results. Compared with normal glucose group, the expression of ROS, NLRP3, caspase-1, IL-1β, TGF-β1, and FN increased in high glucose group (P < 0.05), but it decreased after the treatment of maresin 1 in different concentrations. On the contrary, the expression of procaspase-1 and pro-IL-1β protein was restrained by high glucose and enhanced by maresin 1 in a dose-dependent manner (P < 0.05). Conclusion. Maresin 1 can inhibit NLRP3 inflammasome, TGF-β1, and FN in GMCs; it may have protective effect on DN by mitigating the inflammation and early fibrosis. PMID:28182085

  12. Repair of sublethal radiation injury after multiple small doses in mouse kidney: an estimate of flexure dose

    SciTech Connect

    Stewart, F.A.; Oussoren, Y.; Luts, A.; Begg, A.C.; Dewit, L.; Lebesque, J.; Bartelink, H.

    1987-05-01

    Functional kidney damage in mice was measured after a series of fractionated X-irradiations. Doses per fraction of 0.75-12.5 Gy were given as 2, 5, 10, 30, 40, 60, or 80 equal doses in a total treatment time of 4 weeks. Renal function (measured by clearance of /sup 51/CrEDTA or hematocrit levels) deteriorated progressively, in a dose related manner, from 20 to 46 weeks after the start of treatment. The changes in renal function versus time were fitted by a polynomial regression through all data and interpolated values for /sup 51/CrEDTA clearance were then calculated at 30 and 40 weeks after treatment. Steep dose response curves were obtained and these were used to calculate isoeffective doses for the different fractionation schedules. There was a marked increase in total isoeffective doses from 2-30 fractions and these data were well described by a linear quadratic (L.Q.) expression for damage with an alpha/beta ratio of 2.3 +/- 0.2 Gy. There was only a slight increase in the total isoeffect dose as the size of the dose per fraction was decreased below 2 Gy and the measured isoeffect doses after 40 to 80 fractions were lower than predicted on the basis of an L.Q. model assuming complete repair between successive irradiations. The flexure dose for mouse kidneys irradiated 3 times per day was, effectively, 1 to 2 Gy and hyperfractionation using lower doses per fraction did not lead to significant, additional repair.

  13. Sinomenine activation of Nrf2 signaling prevents hyperactive inflammation and kidney injury in a mouse model of obstructive nephropathy.

    PubMed

    Qin, Tian; Du, Ronghui; Huang, Fengjie; Yin, Shasha; Yang, Jun; Qin, Siyuan; Cao, Wangsen

    2016-03-01

    Sinomenine is originally derived from medicinal herb and used preferentially in treatment of rheumatoid diseases in Far East regions. SIN has strong anti-inflammatory and immune-regulatory properties, acting mainly through inhibiting NF-kB signaling. Although the upstream target through which SIN affects NF-kB activity is unknown, evidence suggests that SIN might regulate inflammation through Nrf2 signaling. In this study we explored the role of Nrf2 in mediating SIN's anti-inflammation and kidney protection in a mouse model of obstructive nephropathy. We found that SIN is an activator of Nrf2 signaling. It markedly increased Nrf2 protein level, Nrf2 nuclear translocation, Nef2 transcription capacity, and the downstream protein expression. We further demonstrated that SIN activation of Nrf2 is likely due to its repression of the Nrf2 inhibitor Keap1 since it drastically reduced Keap1 protein through the PKC-sensitive ubiquitination-proteasomal degradation. SIN treatment of nephropathy mice effectively reduced the kidney damage and inflammatory responses, balanced renal oxidative stress, and improved the pathological protein expression in an Nrf2 dependent manner. In addition, SIN also Nrf2-dependently modulated macrophage M1/M2 polarization and inhibited the IkBα phosphorylation and NF-kB nuclear translocation, hence revealing an important upstream event that contributed to its anti-inflammation and tissue protection. Taken together our study has identified a novel pathway through which SIN exerts its anti-inflammation and renal protective functions, and provided a molecular basis for SIN potential applications in the treatment of kidney and other inflammatory disorders.

  14. Cardiomyocyte proliferation in zebrafish and mammals: lessons for human disease.

    PubMed

    Matrone, Gianfranco; Tucker, Carl S; Denvir, Martin A

    2017-04-01

    Cardiomyocytes proliferate profusely during early development and for a brief period after birth in mammals. Within a month after birth, this proliferative capability is dramatically reduced in mammals unlike lower vertebrates where it persists into adult life. The zebrafish, for example, retains the ability to regenerate the apex of the heart following resection by a mechanism predominantly driven by cardiomyocyte proliferation. Differences in proliferative capacity of cardiomyocytes in adulthood between mammals and lower vertebrates are closely liked to ontogenetic or phylogenetic factors. Elucidation of these factors has the potential to provide enormous benefits if they lead to the development of therapeutic strategies that facilitate cardiomyocyte proliferation. In this review, we highlight the differences between Mammalian and Zebrafish cardiomyocytes, which could explain at least in part the different proliferative capacities in these two species. We discuss the advantages of the zebrafish as a model of cardiomyocyte proliferation, particularly at the embryonic stage. We also identify a number of key molecular pathways with potential to reveal key steps in switching cardiomyocytes from a quiescent to a proliferative phenotype.

  15. Microscopic heat pulses induce contraction of cardiomyocytes without calcium transients

    SciTech Connect

    Oyama, Kotaro; Mizuno, Akari; Shintani, Seine A.; Itoh, Hideki; Serizawa, Takahiro; Fukuda, Norio; Suzuki, Madoka

    2012-01-06

    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 heat 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.

  16. Antibody induced injury to podocytes with proteinuria and foot process swelling in a transgenic (T16) mouse

    PubMed Central

    Ebrahim, H; Evans, D J

    1999-01-01

    T16 mice contain a human 3′ untranslated sequence of the Thy 1.1 gene. Unlike normal mice they express Thy 1.1 protein on the podocytes which was immuno-localized to podocyte apical and basal plasma membranes and filtration slit. When monoclonal anti-Thy 1.1 antibody (OX7) was injected in nonproteinuric heterozygous mice there was rapid podocyte foot process swelling and proteinuria. Immunofluorescence showed granular glomerular OX7 binding at one hour. Progressive loss of pedicels occurred with 17.9 ± 2.5, 14.4 ± 1.1 and 10.5 ± 3.5 per 10 nm glomerular basement membrane (GBM) remaining 1, 6 and 24 hours, respectively, after 1 mg OX7, vs 32.2 ± 2.0 in T16 mice given saline. Twenty-four hour proteinuria was OX7 dose-dependent, peaked at 1–3 days and reduced to near basal levels 9–11 days thereafter. Proteinuria was nonselective except at very low doses (0.1 mg OX7) where microalbuminuria was seen. F(ab′)2 OX7 administration also caused proteinuria in T16 mice. One milligram F(ab′)1 OX7 caused diffuse foot process swelling without manifest proteinuria in T16 mice. Anti-Thy 1.1 IgM monoclonal antibody did not produce the effects of OX7 in T16 mice. Foot process swelling was not modified by histamine or 5-hydroxytryptamine antagonists. OX7 did not cause complement activation or leucocyte infiltration, hence glomerular injury appeared to be mediated directly by the antibody. PMID:10469262

  17. Neuroprotective and Anti-Inflammatory Effects of the Flavonoid-Enriched Fraction AF4 in a Mouse Model of Hypoxic-Ischemic Brain Injury

    PubMed Central

    Keddy, Paul G. W.; Dunlop, Kate; Warford, Jordan; Samson, Michel L.; Jones, Quinton R. D.; Rupasinghe, H. P. Vasantha; Robertson, George S.

    2012-01-01

    We report here neuroprotective and anti-inflammatory effects of a flavonoid-enriched fraction isolated from the peel of Northern Spy apples (AF4) in a mouse of model of hypoxic-ischemic (HI) brain damage. Oral administration of AF4 (50 mg/kg, once daily for 3 days) prior to 50 min of HI completely prevented motor performance deficits assessed 14 days later that were associated with marked reductions in neuronal cell loss in the dorsal hippocampus and striatum. Pre-treatment with AF4 (5, 10, 25 or 50 mg/kg, p.o.; once daily for 3 days) produced a dose-dependent reduction in HI-induced hippocampal and striatal neuron cell loss, with 25 mg/kg being the lowest dose that achieved maximal neuroprotection. Comparison of the effects of 1, 3 or 7 doses of AF4 (25 mg/kg; p.o.) prior to HI revealed that at least 3 doses of AF4 were required before HI to reduce neuronal cell loss in both the dorsal hippocampus and striatum. Quantitative RT-PCR measurements revealed that the neuroprotective effects of AF4 (25 mg/kg; p.o.; once daily for 3 days) in the dorsal hippocampus were associated with a suppression of HI-induced increases in the expression of IL-1β, TNF-α and IL-6. AF4 pre-treatment enhanced mRNA levels for pro-survival proteins such as X-linked inhibitor of apoptosis and erythropoietin following HI in the dorsal hippocampus and striatum, respectively. Primary cultures of mouse cortical neurons incubated with AF4 (1 µg/ml), but not the same concentrations of either quercetin or quercetin-3-O-glucose or its metabolites, were resistant to cell death induced by oxygen glucose deprivation. These findings suggest that the inhibition of HI-induced brain injury produced by AF4 likely involves a transcriptional mechanism resulting from the co-operative actions of various phenolics in this fraction which not only reduce the expression of pro-inflammatory mediators but also enhance pro-survival gene signalling. PMID:23251498

  18. Neuroprotective and anti-inflammatory effects of the flavonoid-enriched fraction AF4 in a mouse model of hypoxic-ischemic brain injury.

    PubMed

    Keddy, Paul G W; Dunlop, Kate; Warford, Jordan; Samson, Michel L; Jones, Quinton R D; Rupasinghe, H P Vasantha; Robertson, George S

    2012-01-01

    We report here neuroprotective and anti-inflammatory effects of a flavonoid-enriched fraction isolated from the peel of Northern Spy apples (AF4) in a mouse of model of hypoxic-ischemic (HI) brain damage. Oral administration of AF4 (50 mg/kg, once daily for 3 days) prior to 50 min of HI completely prevented motor performance deficits assessed 14 days later that were associated with marked reductions in neuronal cell loss in the dorsal hippocampus and striatum. Pre-treatment with AF4 (5, 10, 25 or 50 mg/kg, p.o.; once daily for 3 days) produced a dose-dependent reduction in HI-induced hippocampal and striatal neuron cell loss, with 25 mg/kg being the lowest dose that achieved maximal neuroprotection. Comparison of the effects of 1, 3 or 7 doses of AF4 (25 mg/kg; p.o.) prior to HI revealed that at least 3 doses of AF4 were required before HI to reduce neuronal cell loss in both the dorsal hippocampus and striatum. Quantitative RT-PCR measurements revealed that the neuroprotective effects of AF4 (25 mg/kg; p.o.; once daily for 3 days) in the dorsal hippocampus were associated with a suppression of HI-induced increases in the expression of IL-1β, TNF-α and IL-6. AF4 pre-treatment enhanced mRNA levels for pro-survival proteins such as X-linked inhibitor of apoptosis and erythropoietin following HI in the dorsal hippocampus and striatum, respectively. Primary cultures of mouse cortical neurons incubated with AF4 (1 µg/ml), but not the same concentrations of either quercetin or quercetin-3-O-glucose or its metabolites, were resistant to cell death induced by oxygen glucose deprivation. These findings suggest that the inhibition of HI-induced brain injury produced by AF4 likely involves a transcriptional mechanism resulting from the co-operative actions of various phenolics in this fraction which not only reduce the expression of pro-inflammatory mediators but also enhance pro-survival gene signalling.

  19. Recipient T-Cell TIM-3 and Hepatocyte Galectin-9 Signaling Protects Mouse Liver Transplants Against Ischemia-Reperfusion Injury

    PubMed Central

    Liu, Yuanxing; Ji, Haofeng; Zhang, Yu; Shen, Xiuda; Gao, Feng; He, Xiangyi; Li, Gabriella A.; Busuttil, Ronald W.; Kuchroo, Vijay K.; Kupiec-Weglinski, Jerzy W.

    2015-01-01

    Background & Aims By binding to T-cell immunoglobulin mucin-3 (TIM-3) on activated Th1 cells, Galectin-9 (Gal-9) negatively regulates Th1-type alloimmunity. Although T cells contribute to hepatic ischemia-reperfusion injury (IRI), it is unknown whether negative T cell-dependent TIM-3 costimulation may rescue IR-stressed orthotopic liver transplants (OLT) from innate immunity-driven inflammation. Methods We used WT and TIM-3Tg mice (C57BL6) as liver donors and recipients in a clinically-relevant model of hepatic cold storage (20h at 4°C in UW solution) and syngeneic OLT. Results OLTs in WT or TIM-3Tg->TIM-3Tg groups were resistant against IR- stress, evidenced by preserved hepatocellular function (sALT levels) and liver architecture (Suzuki’s score). In contrast, OLTs in WT or TIM-3Tg->WT groups were susceptible to IRI. TIM-3 induction in recipient circulating CD4+ T cells: 1/ depressed Tbet/IFN-γ, while amplifying GATA3 and IL-4/IL-10 expression in OLTs; 2/ promoted T cell exhaustion (PD-1, LAG-3) phenotype; and 3/ depressed neutrophil and macrophage infiltration/function in OLTs. In parallel studies, we have documented, for the first time that Gal-9, a natural TIM-3 ligand, was produced primarily by and released from IR-stressed hepatocytes, both in-vivo and in-vitro. Moreover, exogenous rGal-9 potentiated liver resistance against IRI by depressing T cell activation and promoting apoptosis of CD4+ T cells. Conclusion Harnessing TIM-3–Gal-9 signaling at T cell–hepatocyte interface facilitates homeostasis in IR-stressed OLTs. Enhancing anti-oxidant hepatocyte Gal-9 potentiates liver IR-resistance. Negative regulation by recipient TIM-3+CD4+ cells provides evidence for cytoprotective functions of a discrete T cell subset, which should be spared when applying T cell-targeted immunosuppression in transplant recipients. PMID:25450716

  20. Evans blue staining of cardiomyocytes induced by myocardial contrast echocardiography in rats: evidence for necrosis instead of apoptosis.

    PubMed

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

    2007-12-01

    High mechanical index (MI) echocardiography with contrast agent has been shown to induce Evans blue staining of cardiomyocytes, seen 1 d after exposure, in addition to contraction band necrosis, seen immediately after exposure. This research examined the roles of necrosis vs. apoptosis in these bioeffects. Myocardial contrast echocardiography at high MI with 1:4 electrocardiogram triggering was performed in anesthetized rats at 1.5 MHz. Histologically observable cell injury was accumulated by infusing a high dose of 50 microL/kg ultrasound contrast media via tail vein for 5 min at the start of 10 min of scanning. Evans blue dye or propidium iodide was injected as an indicator of cardiomyocyte plasma membrane integrity. Histologic sections were stained using the terminal dUTP nick-end labeling (TUNEL) method for labeling nuclei with DNA degradation (e.g., apoptosis). Evans blue fluorescent cells were counted on frozen sections or on hematoxylin-stained and TUNEL-labeled paraffin sections. In addition, transmission electron microscopy was used to assess potential apoptotic nuclei. Hypercontraction and propidium iodide staining were observed immediately after imaging exposure. Although TUNEL-positive cells were evident after 4 h, these also had indications of contraction band necrosis, and features of apoptosis were not confirmed by electron microscopy. Inflammatory cell infiltration was evident after 24 h. A second, more subtle injury was recognized by Evans blue staining, with minimal inflammatory cell infiltration at the morphologically intact stained cells after 24 h. Apoptosis was not detected by the TUNEL method in the cardiomyocytes stained with Evans blue at 24 h. However, Evans blue-stained cell numbers declined after 48 h, with continued inflammatory cell infiltration. The initial insult for Evans blue-stained cardiomyocytes apparently induced partial permeability of the plasma membrane, which led to gradual degeneration (but not apoptosis) and necrosis

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

    PubMed Central

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

    2016-01-01

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

  2. Transplanted Glial Restricted Precursor Cells Improve Neurobehavioral and Neuropathological Outcomes in a Mouse Model of Neonatal White Matter Injury Despite Limited Cell Survival

    PubMed Central

    Porambo, Michael; Phillips, Andre W.; Marx, Joel; Ternes, Kylie; Arauz, Edwin; Pletnikov, Mikhail; Wilson, Mary Ann; Rothstein, Jeffery D.; Johnston, Michael V.; Fatemi, Ali

    2014-01-01

    Objective Neonatal White Matter Injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI. Methods Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-day 5 (P5). At P22, intracallosal injections of either eGFP+ GRPs or saline were performed in control and ligated mice. Neurobehavioral and postmortem studies were performed at four and eight weeks post-transplantation. Results GRP survival was comparable at one month but significantly lower at two months post-transplantation in NWMI mice compared to unligated controls. Surviving cells showed better migration capability in controls; however, the differentiation capacity of transplanted cells was similar in control and NWMI. Saline-treated NWMI mice showed significantly altered response in startle amplitude and pre-pulse inhibition paradigms compared to unligated controls, while these behavioral tests were completely normal in GRP-transplanted animals. Similarly, there was significant increase in hemispheric myelin basic protein density, along with significant decrease in pathologic axonal staining in cell-treated NWMI mice compared to saline-treated NWMI animals. Interpretation The Reduced long-term survival and migration of transplanted GRPs in an ischemia-induced NWMI model suggests that neonatal ischemia leads to long-lasting detrimental effects on oligodendroglia even months after the initial insult. Despite limited GRP-survival, behavioral and neuropathological outcomes were improved after GRP-transplantation. Our results suggest that exogenous GRPs improve myelination through trophic effects in addition to differentiation into mature oligodendrocytes. PMID:25377280

  3. Detection of age-dependent brain injury in a mouse model of brain amyloidosis associated with Alzheimer's disease using magnetic resonance diffusion tensor imaging.

    PubMed

    Sun, Shu-Wei; Song, Sheng-Kwei; Harms, Michael P; Lin, Shiow-Jiuan; Holtzman, David M; Merchant, Kalpana M; Kotyk, John J

    2005-01-01

    Using magnetic resonance diffusion tensor imaging (DTI), the present study investigates changes in both gray and white matter in the APPsw transgenic mouse (Tg2576), a model of beta-amyloid plaque deposition associated with Alzheimer's disease (AD). DTI analyses were performed in cross-sectional groups of transgene-positive and -negative mice at 8, 12, 16, and 18 months of age to assess the magnitude of water diffusion in gray matter (i.e., Tr(D)) and changes in diffusion in white matter that may be indicative of axonal degeneration (i.e., reduced water diffusion parallel to axonal tracts, lambda(||)) and myelin degradation (i.e., increased water diffusion perpendicular to axonal tracts, lambda(perpendicular)). No appreciable changes in gray or white matter were observed between the APPsw and the age-matched control mice at 8 months of age. Reduced Tr(D) and lambda(||) were observed in gray and white matter, respectively, for the APPsw mice at ages greater than 8 months, which coincides with the time period when appreciable amyloid plaque accumulation was confirmed by ex vivo histopathological studies. The decreases in lambda(||) suggest the presence of axonal injury in multiple white matter tracts of APPsw mice. Unlike lambda(||), lambda(perpendicular) was unaltered between control and APPsw mice in most white matter tracts. However, in the corpus collosum (CC), lambda(perpendicular) increased at 16 and 18 months of age, suggesting the possibility of myelin damage in the CC at these later ages. This work demonstrates the potential for DTI as a noninvasive modality to detect evolving pathology associated with changes in tissue water diffusion properties in brain tissues.

  4. Uniform Action Potential Repolarization within the Sarcolemma of In Situ Ventricular Cardiomyocytes

    PubMed Central

    Bu, Guixue; Adams, Heather; Berbari, Edward J.; Rubart, Michael

    2009-01-01

    Previous studies have speculated, based on indirect evidence, that the action potential at the transverse (t)-tubules is longer than at the surface membrane in mammalian ventricular cardiomyocytes. To date, no technique has enabled recording of electrical activity selectively at the t-tubules to directly examine this hypothesis. We used confocal line-scan imaging in conjunction with the fast response voltage-sensitive dyes ANNINE-6 and ANNINE-6plus to resolve action potential-related changes in fractional dye fluorescence (ΔF/F) at the t-tubule and surface membranes of in situ mouse ventricular cardiomyocytes. Peak ΔF/F during action potential phase 0 depolarization averaged −21% for both dyes. The shape and time course of optical action potentials measured with the water-soluble ANNINE-6plus were indistinguishable from those of action potentials recorded with intracellular microelectrodes in the absence of the dye. In contrast, optical action potentials measured with the water-insoluble ANNINE-6 were significantly prolonged compared to the electrical recordings obtained from dye-free hearts, suggesting electrophysiological effects of ANNINE-6 and/or its solvents. With either dye, the kinetics of action potential-dependent changes in ΔF/F during repolarization were found to be similar at the t-tubular and surface membranes. This study provides what to our knowledge are the first direct measurements of t-tubule electrical activity in ventricular cardiomyocytes, which support the concept that action potential duration is uniform throughout the sarcolemma of individual cells. PMID:19289075

  5. 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

  6. 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.

  7. Endocrine and other physiologic modulators of perinatal cardiomyocyte endowment

    PubMed Central

    Jonker, S S; Louey, S

    2015-01-01

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

  8. 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.

  9. Amelioration of Ischemia/Reperfusion Injury During Resuscitation from Hemorrhage by Induction of Heme Oxygenase-1 (HO-1) in a Conscious Mouse Model of Uncontrolled Hemorrhage

    DTIC Science & Technology

    2012-10-01

    September 2009 – 30 August 2012 4. TITLE AND SUBTITLE Amelioration of Ischemia/ Reperfusion Injury During Resuscitation from Hemorrhage by Induction of Heme...initial insult is followed by further injury that occurs during the reintroduction of oxygen with the restoration of blood flow. This injury occurs...following hemorrhage. 15. SUBJECT TERMS- Hemorrhage shock, cytoprotection, ischemia/ reperfusion injury , drugs 16. SECURITY CLASSIFICATION OF: 17

  10. NOD1 Activation Induces Cardiac Dysfunction and Modulates Cardiac Fibrosis and Cardiomyocyte Apoptosis

    PubMed Central

    Fernández-Velasco, María; Prieto, Patricia; Terrón, Verónica; Benito, Gemma; Flores, Juana M.; Delgado, Carmen; Zaragoza, Carlos; Lavin, Begoña; Gómez-Parrizas, Mónica; López-Collazo, Eduardo; Martín-Sanz, Paloma; Boscá, Lisardo

    2012-01-01

    The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure. PMID:23028889

  11. Analysis of cardiomyocyte movement in the developing murine heart.

    PubMed

    Hashimoto, Hisayuki; Yuasa, Shinsuke; Tabata, Hidenori; Tohyama, Shugo; Seki, Tomohisa; Egashira, Toru; Hayashiji, Nozomi; Hattori, Fumiyuki; Kusumoto, Dai; Kunitomi, Akira; Takei, Makoto; Kashimura, Shin; Yozu, Gakuto; Shimojima, Masaya; Motoda, Chikaaki; Muraoka, Naoto; Nakajima, Kazunori; Sakaue-Sawano, Asako; Miyawaki, Atsushi; Fukuda, Keiichi

    2015-09-04

    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 cell 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.

  12. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

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

    PubMed Central

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

    2013-01-01

    While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell–cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes. PMID:24311969

  15. Cyclic stretch of Embryonic Cardiomyocytes Increases Proliferation, Growth, and Expression While Repressing Tgf-β Signaling

    PubMed Central

    Banerjee, Indroneal; Carrion, Katrina; Serrano, Ricardo; Dyo, Jeffrey; Sasik, Roman; Lund, Sean; Willems, Erik; Aceves, Seema; Meili, Rudolph; Mercola, Mark; Chen, Ju; Zambon, Alexander; Hardiman, Gary; Doherty, Taylor A; Lange, Stephan; del Álamo, Juan C.; Nigam, Vishal

    2014-01-01

    Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS. PMID:25446186

  16. Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons.

    PubMed

    Chuang, Wenpo; Sharma, Arun; Shukla, Praveen; Li, Guang; Mall, Moritz; Rajarajan, Kuppusamy; Abilez, Oscar J; Hamaguchi, Ryoko; Wu, Joseph C; Wernig, Marius; Wu, Sean M

    2017-03-22

    Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

  17. Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons

    PubMed Central

    Chuang, Wenpo; Sharma, Arun; Shukla, Praveen; Li, Guang; Mall, Moritz; Rajarajan, Kuppusamy; Abilez, Oscar J.; Hamaguchi, Ryoko; Wu, Joseph C.; Wernig, Marius; Wu, Sean M.

    2017-01-01

    Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs. PMID:28327614

  18. Platelet-derived growth factor (PDGF) signaling directs cardiomyocyte movement toward the midline during heart tube assembly

    PubMed Central

    Bloomekatz, Joshua; Singh, Reena; Prall, Owen WJ; Dunn, Ariel C; Vaughan, Megan; Loo, Chin-San; Harvey, Richard P; Yelon, Deborah

    2017-01-01

    Communication between neighboring tissues plays a central role in guiding organ morphogenesis. During heart tube assembly, interactions with the adjacent endoderm control the medial movement of cardiomyocytes, a process referred to as cardiac fusion. However, the molecular underpinnings of this endodermal-myocardial relationship remain unclear. Here, we show an essential role for platelet-derived growth factor receptor alpha (Pdgfra) in directing cardiac fusion. Mutation of pdgfra disrupts heart tube assembly in both zebrafish and mouse. Timelapse analysis of individual cardiomyocyte trajectories reveals misdirected cells in zebrafish pdgfra mutants, suggesting that PDGF signaling steers cardiomyocytes toward the midline during cardiac fusion. Intriguingly, the ligand pdgfaa is expressed in the endoderm medial to the pdgfra-expressing myocardial precursors. Ectopic expression of pdgfaa interferes with cardiac fusion, consistent with an instructive role for PDGF signaling. Together, these data uncover a novel mechanism through which endodermal-myocardial communication can guide the cell movements that initiate cardiac morphogenesis. DOI: http://dx.doi.org/10.7554/eLife.21172.001 PMID:28098558

  19. Plakophilin-2 loss promotes TGF-β1/p38 MAPK-dependent fibrotic gene expression in cardiomyocytes

    PubMed Central

    Dubash, Adi D.; Kam, Chen Y.; Aguado, Brian A.; Patel, Dipal M.; Delmar, Mario; Shea, Lonnie D.

    2016-01-01

    Members of the desmosome protein family are integral components of the cardiac area composita, a mixed junctional complex responsible for electromechanical coupling between cardiomyocytes. In this study, we provide evidence that loss of the desmosomal armadillo protein Plakophilin-2 (PKP2) in cardiomyocytes elevates transforming growth factor β1 (TGF-β1) and p38 mitogen-activated protein kinase (MAPK) signaling, which together coordinate a transcriptional program that results in increased expression of profibrotic genes. Importantly, we demonstrate that expression of Desmoplakin (DP) is lost upon PKP2 knockdown and that restoration of DP expression rescues the activation of this TGF-β1/p38 MAPK transcriptional cascade. Tissues from PKP2 heterozygous and DP conditional knockout mouse models also exhibit elevated TGF-β1/p38 MAPK signaling and induction of fibrotic gene expression in vivo. These data therefore identify PKP2 and DP as central players in coordination of desmosome-dependent TGF-β1/p38 MAPK signaling in cardiomyocytes, pathways known to play a role in different types of cardiac disease, such as arrhythmogenic or hypertrophic cardiomyopathy. PMID:26858265

  20. Purinergic inhibition of glucose transport in cardiomyocytes.

    PubMed

    Fischer, Y; Becker, C; Löken, C

    1999-01-08

    ATP is known to act as an extracellular signal in many organs. In the heart, extracellular ATP modulates ionic processes and contractile function. This study describes a novel, metabolic effect of exogenous ATP in isolated rat cardiomyocytes. In these quiescent (i.e. noncontracting) cells, micromolar concentrations of ATP depressed the rate of basal, catecholamine-stimulated, or insulin-stimulated glucose transport by up to 60% (IC50 for inhibition of insulin-dependent glucose transport, 4 microM). ATP decreased the amount of glucose transporters (GLUT1 and GLUT4) in the plasma membrane, with a concomitant increase in intracellular microsomal membranes. A similar glucose transport inhibition was produced by P2 purinergic agonists with the following rank of potencies: ATP approximately ATPgammaS approximately 2-methylthio-ATP (P2Y-selective) > ADP > alpha,betameATP (P2X-selective), whereas the P1 purinoceptor agonist adenosine was ineffective. The effect of ATP was suppressed by the poorly subtype-selective P2 antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonic acid but, surprisingly, not by the nonselective antagonist suramin nor by the P2Y-specific Reactive Blue 2. Glucose transport inhibition by ATP was not affected by a drastic reduction of the extracellular concentrations of calcium (down to 10(-9) M) or sodium (down to 0 mM), and it was not mimicked by a potassium-induced depolarization, indicating that purinoceptors of the P2X family (which are nonselective cation channels whose activation leads to a depolarizing sodium and calcium influx) are not involved. Inhibition was specific for the transmembrane transport of glucose because ATP did not inhibit (i) the rate of glycolysis under conditions where the transport step is no longer rate-limiting nor (ii) the rate of [1-14C]pyruvate decarboxylation. In conclusion, extracellular ATP markedly inhibits glucose transport in rat cardiomyocytes by promoting a redistribution of glucose transporters from the

  1. Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells.

    PubMed

    Garcia, Nahuel A; Ontoria-Oviedo, Imelda; González-King, Hernán; Diez-Juan, Antonio; Sepúlveda, Pilar

    2015-01-01

    Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship that is essential for maintaining normal development and function in the heart. Little is known about the mechanisms that regulate cardiac and endothelial crosstalk, particularly in situations of acute stress when local active processes are required to regulate endothelial function. We examined whether CM-derived exosomes could modulate endothelial function. Under conditions of glucose deprivation, immortalized H9C2 cardiomyocytes increase their secretion of exosomes. CM-derived exosomes are loaded with a broad repertoire of miRNA and proteins in a glucose availability-dependent manner. Gene Ontology (GO) analysis of exosome cargo molecules identified an enrichment of biological process that could alter EC activity. We observed that addition of CM-derived exosomes to ECs induced changes in transcriptional activity of pro-angiogenic genes. Finally, we demonstrated that incubation of H9C2-derived exosomes with ECs induced proliferation and angiogenesis in the latter. Thus, exosome-mediated communication between CM and EC establishes a functional relationship that could have potential implications for the induction of local neovascularization during acute situations such as cardiac injury.

  2. Oxidant stress during simulated ischemia primes cardiomyocytes for cell death during reperfusion.

    PubMed

    Robin, Emmanuel; Guzy, Robert D; Loor, Gabriel; Iwase, Hirotaro; Waypa, Gregory B; Marks, Jeremy D; Hoek, Terry L Vanden; Schumacker, Paul T

    2007-06-29

    Ischemia-reperfusion injury induces oxidant stress, and the burst of reactive oxygen species (ROS) production after reperfusion of ischemic myocardium is sufficient to induce cell death. Mitochondrial oxidant production may begin during ischemia prior to reperfusion because reducing equivalents accumulate and promote superoxide production. We utilized a ratiometric redox-sensitive protein sensor (heat shock protein 33 fluorescence resonance energy transfer (HSP-FRET)) to assess oxidant stress in cardiomyocytes during simulated ischemia. HSP-FRET consists of the cyan and yellow fluorescent protein fluorophores linked by the cysteine-containing regulatory domain from bacterial HSP-33. During ischemia, ROS-mediated oxidation of HSP-FRET was observed, along with a decrease in cellular reduced glutathione levels. These findings were corroborated by measurements using redox-sensitive green fluorescent protein, another protein thiol ratiometric sensor, which became 93% oxidized by the end of simulated ischemia. However, cell death did not occur during ischemia, indicating that this oxidant stress is not sufficient to induce death before reperfusion. However, interventions that attenuate ischemic oxidant stress, including antioxidants or scavengers of residual O(2) that attenuate/prevent ROS generation during ischemia, abrogated cell death during simulated reperfusion. These findings reveal that, in isolated cardiomyocytes, sublethal H(2)O(2) generation during simulated ischemia regulates cell death during simulated reperfusion, which is mediated by the reperfusion oxidant burst.

  3. Glucose Starvation in Cardiomyocytes Enhances Exosome Secretion and Promotes Angiogenesis in Endothelial Cells

    PubMed Central

    Garcia, Nahuel A.; Ontoria-Oviedo, Imelda; González-King, Hernán; Diez-Juan, Antonio; Sepúlveda, Pilar

    2015-01-01

    Cardiomyocytes (CMs) and endothelial cells (ECs) have an intimate anatomical relationship that is essential for maintaining normal development and function in the heart. Little is known about the mechanisms that regulate cardiac and endothelial crosstalk, particularly in situations of acute stress when local active processes are required to regulate endothelial function. We examined whether CM-derived exosomes could modulate endothelial function. Under conditions of glucose deprivation, immortalized H9C2 cardiomyocytes increase their secretion of exosomes. CM-derived exosomes are loaded with a broad repertoire of miRNA and proteins in a glucose availability-dependent manner. Gene Ontology (GO) analysis of exosome cargo molecules identified an enrichment of biological process that could alter EC activity. We observed that addition of CM-derived exosomes to ECs induced changes in transcriptional activity of pro-angiogenic genes. Finally, we demonstrated that incubation of H9C2-derived exosomes with ECs induced proliferation and angiogenesis in the latter. Thus, exosome-mediated communication between CM and EC establishes a functional relationship that could have potential implications for the induction of local neovascularization during acute situations such as cardiac injury. PMID:26393803

  4. ERK5 knock down aggravates detrimental effects of hypothermal stimulation on cardiomyocytes via Bim upregulation.

    PubMed

    Wang, Yao-Sheng; Zhou, Jing; Liang, Chun; Hong, Kui; Cheng, Xiao-Shu; Wu, Zong-Gui

    2013-09-01

    Mechanism of cold induced myocardial injury remained unclear. Our study investigated the role of ERK5/Bim pathway in hypothermal stimulation-induced apoptosis or damage of cardiomyocytes (CMs). Results showed that in CMs which under hypothermal stimulation, ERK5 siRNA promoted expression of Bim protein. Bim siRNA did not influence ERK5 expression but attenuated production of p-ERK5. ERK5 siRNA induced higher apoptosis rate; intracellular Ca(2+) overload; ROS activity; ΔΨm damage in hypothermia stimulated CMs, when compared with hypothermal stimulation solely treated group, while Bim siRNA effected oppositely and canceled pro-apoptotic effect of ERK5 siRNA. In conclusion, ERK5 knock down releases inhibition to Bim expression, induces aggravated apoptosis in CMs under hypothermal stimulation, which related to higher intracellular Ca(2+) overload, ROS activity, and more severe ΔΨm damage. Results revealed regulative role of ERK5/Bim pathway in hypothermal stimulation-induced injure or apoptosis of cardiomyocytes.

  5. The nuclear melatonin receptor RORα is a novel endogenous defender against myocardial ischemia/reperfusion injury.

    PubMed

    He, Ben; Zhao, Yichao; Xu, Longwei; Gao, Lingchen; Su, Yuanyuan; Lin, Nan; Pu, Jun

    2016-04-01

    Circadian rhythm disruption or decrease in levels of circadian hormones such as melatonin increases ischemic heart disease risk. The nuclear melatonin receptors RORs are pivotally involved in circadian rhythm regulation and melatonin effects mediation. However, the functional roles of RORs in the heart have never been investigated and were therefore the subject of this study on myocardial ischemia/reperfusion (MI/R) injury pathogenesis. RORα and RORγ subtypes were detected in the adult mouse heart, and RORα but not RORγ was downregulated after MI/R. To determine the pathological consequence of MI/R-induced reduction of RORα, we subjected RORα-deficient staggerer mice and wild-type (WT) littermates to MI/R injury, resulting in significantly increased myocardial infarct size, myocardial apoptosis and exacerbated contractile dysfunction in the former. Mechanistically, RORα deficiency promoted MI/R-induced endoplasmic reticulum stress, mitochondrial impairments, and autophagy dysfunction. Moreover, RORα deficiency augmented MI/R-induced oxidative/nitrative stress. Given the emerging evidence of RORα as an essential melatonin effects mediator, we further investigated the RORα roles in melatonin-exerted cardioprotection, in particular against MI/R injury, which was significantly attenuated in RORα-deficient mice, but negligibly affected by cardiac-specific silencing of RORγ. Finally, to determine cell type-specific effects of RORα, we generated mice with cardiomyocyte-specific RORα overexpression and they were less vulnerable to MI/R injury. In summary, our study provides the first direct evidence that the nuclear melatonin receptor RORα is a novel endogenous protective receptor against MI/R injury and an important mediator of melatonin-exerted cardioprotection; melatonin-RORα axis signaling thus appears important in protection against ischemic heart injury.

  6. 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.

  7. miR-613 suppresses ischemia-reperfusion-induced cardiomyocyte apoptosis by targeting the programmed cell death 10 gene.

    PubMed

    Wu, Zhenhua; Qi, Yujuan; Guo, Zhigang; Li, Peijun; Zhou, Ding

    2016-09-05

    MicroRNAs (miRNAs) are important gene regulators in both biological and pathological processes, including myocardial ischemia/reperfusion (I/R) injury. This study investigated the effect of miR-613 on I/R-induced cardiomyocyte apoptosis and its molecular mechanism of action. Hypoxia/reoxygenation (H/R) significantly increased the release of lactate dehydrogenase (LDH), levels of malondialdehyde (MDA), and cardiomyocyte apoptosis, but these effects were attenuated by an miR-613 mimic. Programmed cell death 10 (PDCD10) was identified as a target gene of miR-613. miR-613 significantly increased the phosphorylation of Akt (p-Akt). An miR-613 mimic lowered the level of expression of pro-apoptotic proteins, C/EBP homologous protein (CHOP), and phosphorylated c-Jun N-terminal kinase (p-JNK), and it up-regulated the expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2). All of these effects were reversed by restoration of PDCD10. Taken together, the current findings indicate that miR-613 inhibits I/R-induced cardiomyocyte apoptosis by targeting PDCD10 by regulating the PI3K/AKT signaling pathway.

  8. Clonal differentiation of skeletal muscle-derived CD34(-)/45(-) stem cells into cardiomyocytes in vivo.

    PubMed

    Tamaki, Tetsuro; Uchiyama, Yoshiyasu; Okada, Yoshinori; Tono, Kayoko; Masuda, Maki; Nitta, Masahiro; Hoshi, Akio; Akatsuka, Akira

    2010-04-01

    The differentiation and/or therapeutic potential of skeletal muscle-derived stem cells for cardiac infarction have been studied extensively for use in cellular cardiomyoplasty, as injured cardiomyocytes exhibit limited regenerative capacity. We previously reported cardio-myogenic differentiation of skeletal muscle-derived CD34+/45(-) (Sk-34) stem cells after therapeutic transplantation. However, the clonal differentiation potential of these cells remains unknown. Here, we show that skeletal muscle-derived CD34(-)/45(-) (Sk-DN) stem cells, which are situated upstream of Sk-34 cells in the same lineage, exhibit clonal differentiation into cardiomyocytes after single cell-derived single-sphere implantation into myocardium. Sk-DN cells were enzymatically isolated from green fluorescent protein (GFP) transgenic mice and purified by flow cytometry, and were then clonally cultured in collagen-based medium with bFGF and EGF after clonal cell sorting. Single cell-derived single-sphere colonies of Sk-DN cells were directly implanted into the wild-type mouse myocardium. At 4 weeks after implantation, donor cells exhibited typical cardiomyocyte structure with the formation of gap-junctions between donor and recipient cells. Expression of specific mRNAs for cardiomyocytes, such as cardiac actin and GATA-4, Nkx2-5, Isl-1, Mef2, and Hand2, were also seen in clonal cell cultures of Sk-DN cells. Cell fusion-independent differentiation was also confirmed by bulk cell transplantation using Cre- and loxP (enhanced GFP)-mice. We conclude that Sk-DN cells can give rise to cardiac muscle cells clonally, and that skeletal muscle includes a practical cell source for cellular cardiomyoplasty.

  9. Cardiomyocyte Formation by Skeletal Muscle-Derived Multi-Myogenic Stem Cells after Transplantation into Infarcted Myocardium

    PubMed Central

    Tamaki, Tetsuro; Akatsuka, Akira; Okada, Yoshinori; Uchiyama, Yoshiyasu; Tono, Kayoko; Wada, Mika; Hoshi, Akio; Iwaguro, Hideki; Iwasaki, Hiroto; Oyamada, Akira; Asahara, Takayuki

    2008-01-01

    Background Cellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported. Methods and Results Skeletal muscle interstitium-derived CD34+/CD45− (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed. Conclusions and Significance Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for

  10. 14-3-3ε Plays a Role in Cardiac Ventricular Compaction by Regulating the Cardiomyocyte Cell Cycle

    PubMed Central

    Kosaka, Yasuhiro; Cieslik, Katarzyna A.; Li, Ling; Lezin, George; Maguire, Colin T.; Saijoh, Yukio; Toyo-oka, Kazuhito; Gambello, Michael J.; Vatta, Matteo; Wynshaw-Boris, Anthony; Baldini, Antonio; Yost, H. Joseph

    2012-01-01

    Trabecular myocardium accounts for the majority of the ventricles during early cardiogenesis, but compact myocardium is the primary component at later developmental stages. Elucidation of the genes regulating compact myocardium development is essential to increase our understanding of left ventricular noncompaction (LVNC), a cardiomyopathy characterized by increased ratios of trabecular to compact myocardium. 14-3-3ε is an adapter protein expressed in the lateral plate mesoderm, but its in vivo cardiac functions remain to be defined. Here we show that 14-3-3ε is expressed in the developing mouse heart as well as in cardiomyocytes. 14-3-3ε deletion did not appear to induce compensation by other 14-3-3 isoforms but led to ventricular noncompaction, with features similar to LVNC, resulting from a selective reduction in compact myocardium thickness. Abnormal compaction derived from a 50% decrease in cardiac proliferation as a result of a reduced number of cardiomyocytes in G2/M and the accumulation of cardiomyocytes in the G0/G1 phase of the cell cycle. These defects originated from downregulation of cyclin E1 and upregulation of p27Kip1, possibly through both transcriptional and posttranslational mechanisms. Our work shows that 14-3-3ε regulates cardiogenesis and growth of the compact ventricular myocardium by modulating the cardiomyocyte cell cycle via both cyclin E1 and p27Kip1. These data are consistent with the long-held view that human LVNC may result from compaction arrest, and they implicate 14-3-3ε as a new candidate gene in congenital human cardiomyopathies. PMID:23071090

  11. Cox-2 Inhibition Protects against Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Akt-Dependent Enhancement of iNOS Expression

    PubMed Central

    Cai, Yin; Tang, Eva Hoi Ching; Yan, Dan; Kosuru, Ramoji

    2016-01-01

    The present study explored the potential causal link between ischemia-driven cyclooxygenase-2 (COX-2) expression and enhanced apoptosis during myocardial ischemia/reperfusion (I/R) by using H9C2 cardiomyocytes and primary rat cardiomyocytes subjected to hypoxia/reoxygenation (H/R). The results showed that H/R resulted in higher COX-2 expression than that of controls, which was prevented by pretreatment with Helenalin (NFκB specific inhibitor). Furthermore, pretreatment with NS398 (COX-2 specific inhibitor) significantly attenuated H/R-induced cell injury [lower lactate dehydrogenase (LDH) leakage and enhanced cell viability] and apoptosis (higher Bcl2 expression and lower level of cleaved caspases-3 and TUNEL-positive cells) in cardiomyocytes. The amelioration of posthypoxic apoptotic cell death was paralleled by significant attenuation of H/R-induced increases in proinflammatory cytokines [interleukin 6 (IL6) and tumor necrosis factor (TNFα)] and reactive oxygen species (ROS) production and by higher protein expression of phosphorylated Akt and inducible nitric oxide synthase (iNOS) and enhanced nitric oxide production. Moreover, the application of LY294002 (Akt-specific inhibitor) or 1400W (iNOS-selective inhibitor) cancelled the cellular protective effects of NS398. Findings from the current study suggest that activation of NFκB during cardiomyocyte H/R induces the expression of COX-2 and that higher COX-2 expression during H/R exacerbates cardiomyocyte H/R injury via mechanisms that involve cross talks among inflammation, ROS, and Akt/iNOS/NO signaling. PMID:27795807

  12. Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor

    PubMed Central

    Deng, Shengli; Yao, Gang

    2016-01-01

    Ischemic postconditioning (IPC) and ATP sensitive potassium channel (KATP) agonists (e.g. pinacidil and diazoxide) postconditioning are effective methods to defeat myocardial ischemia-reperfusion (I/R) injury, but their specific mechanisms of reducing I/R injury are not fully understood. We observed an intracellular free calcium ([Ca2+]i) overload in Anoxia/reoxygenation (A/R) cardiomyocytes, which can be reversed by KATP agonists diazoxide or pinacidil. The calcium-sensing receptor (CaSR) regulates intracellular calcium homeostasis. CaSR was reported to be involved in the I/R-induced apoptosis in rat cardiomyocytes. We therefore hypothesize that IPC and pinacidil postconditioning (PPC) reduce calcium overload in I/R cardiomyocytes by the down-regulation of CaSR. A/R model was established with adult rat caridomyocyte. mRNA and protein expression of CaSR were detected, IPC, PPC and KATP’s effects on [Ca2+]i concentration was assayed too. IPC and PPC ameliorated A/R insult induced [Ca2+]i overload in cardiomyocytes. In addition, they down-regulated the mRNA and protein level of CaSR as we expected. CaSR agonist spermine and KATP blocker glibenclamide offset IPC’s effects on CaSR expression and [Ca2+]i modulation. Our data indicate that CaSR down-regulation contributes to the mitigation of calcium overload in A/R cardiomyocytes, which may partially represents IPC and KATP’s myocardial protective mechanism under I/R circumstances. PMID:27833799

  13. Current recordings at the single channel level in adult mammalian isolated cardiomyocytes.

    PubMed

    Guinamard, Romain; Hof, Thomas; Sallé, Laurent

    2014-01-01

    This chapter describes appropriate methods to investigate mammalian cardiac channels properties at the single channel level. Cell isolation is performed from new born or adult heart by enzymatic digestion on minced tissue or using the Langendorff apparatus. Isolation proceeding is suitable for rabbit, rat, and mouse hearts. In addition, isolation of human atrial cardiomyocytes is described. Such freshly isolated cells or cells maintained in primary culture are suitable for patch-clamp studies. Here we describe the single channel variants of the patch-clamp technique (cell-attached, inside-out, outside-out) used to investigate channel properties. Proceedings for the evaluation of biophysical properties such as conductance, ionic selectivity, regulations by extracellular and intracellular mechanisms are described. To illustrate the study, we provide an example by the characterization of a calcium-activated non-selective cation channel (TRPM4).

  14. Human Stem Cell Derived Cardiomyocytes: An Alternative ...

    EPA Pesticide Factsheets

    Chemical spills and associated deaths in the US has increased 2.6-fold and 16-fold from 1983 to 2012, respectfully. In addition, the number of chemicals to which humans are exposed to in the environment has increased almost 10-fold from 2001 to 2013 within the US. Internationally, a WHO report on the global composite impact of chemicals on health reported that 16% of the total burden of cardiovascular disease was attributed to environmental chemical exposure with 2.5 million deaths per year. Clearly, the cardiovascular system, at all its various developmental and life stages, represents a critical target organ system that can be adversely affected by existing and emerging chemicals (e.g., engineered nanomaterials) in a variety of environmental media. The ability to assess chemical cardiac risk and safety is critically needed but extremely challenging due to the number and categories of chemicals in commerce, as indicated. This presentation\\session will evaluate the use of adult human stem cell derived cardiomyocytes, and existing platforms, as an alternative model to evaluate environmental chemical cardiac toxicity as well as provide key information for the development of predictive adverse outcomes pathways associated with environmental chemical exposures. (This abstract does not represent EPA policy) Rapid and translatable chemical safety screening models for cardiotoxicity current status for informing regulatory decisions, a workshop sponsored by the Society

  15. Xenotransplantation of Human Cardiomyocyte Progenitor Cells Does Not Improve Cardiac Function in a Porcine Model of Chronic Ischemic Heart Failure. Results from a Randomized, Blinded, Placebo Controlled Trial

    PubMed Central

    Jansen of Lorkeers, Sanne J.; Gho, Johannes M. I. H.; Koudstaal, Stefan; van Hout, Gerardus P. J.; Zwetsloot, Peter Paul M.; van Oorschot, Joep W. M.; van Eeuwijk, Esther C. M.; Leiner, Tim; Hoefer, Imo E.; Goumans, Marie-José; Doevendans, Pieter A.; Sluijter, Joost P. G.; Chamuleau, Steven A. J.

    2015-01-01

    Background Recently cardiomyocyte progenitor cells (CMPCs) were successfully isolated from fetal and adult human hearts. Direct intramyocardial injection of human CMPCs (hCMPCs) in experimental mouse models of acute myocardial infarction significantly improved cardiac function compared to controls. Aim Here, our aim was to investigate whether xenotransplantation via intracoronary infusion of fetal hCMPCs in a pig model of chronic myocardial infarction is safe and efficacious, in view of translation purposes. Methods & Results We performed a randomized, blinded, placebo controlled trial. Four weeks after ischemia/reperfusion injury by 90 minutes of percutaneous left anterior descending artery occlusion, pigs (n = 16, 68.5 ± 5.4 kg) received intracoronary infusion of 10 million fetal hCMPCs or placebo. All animals were immunosuppressed by cyclosporin (CsA). Four weeks after infusion, endpoint analysis by MRI displayed no difference in left ventricular ejection fraction, left ventricular end diastolic and left ventricular end systolic volumes between both groups. Serial pressure volume (PV-)loop and echocardiography showed no differences in functional parameters between groups at any timepoint. Infarct size at follow-up, measured by late gadolinium enhancement MRI showed no difference between groups. Intracoronary pressure and flow measurements showed no signs of coronary obstruction 30 minutes after cell infusion. No premature death occurred in cell treated animals. Conclusion Xenotransplantation via intracoronary infusion of hCMPCs is feasible and safe, but not associated with improved left ventricular performance and infarct size compared to placebo in a porcine model of chronic myocardial infarction. PMID:26678993

  16. MicroRNA-145 suppresses ROS-induced Ca{sup 2+} overload of cardiomyocytes by targeting CaMKIIδ

    SciTech Connect

    Cha, Min-Ji; Jang, Jin-Kyung; Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon; Lee, Chang Yeon; Park, Jun-Hee; Lee, Jiyun; Seo, Hyang-Hee; Choi, Eunhyun; Jeon, Woo-min; Hwang, Hye Jin; Shin, Hyun-Taek; and others

    2013-06-14

    Highlights: •CaMKIIδ mediates H{sub 2}O{sub 2}-induced Ca{sup 2+} overload in cardiomyocytes. •miR-145 can inhibit Ca{sup 2+} overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca{sup 2+}) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca{sup 2+} signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca{sup 2+}-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H{sub 2}O{sub 2}-mediated Ca{sup 2+} overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca{sup 2+} overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca{sup 2+}-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca{sup 2+} overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

  17. 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

  18. The role of glucagon-like peptide-2 on apoptosis, cell proliferation, and oxidant-antioxidant system at a mouse model of intestinal injury induced by tumor necrosis factor-alpha/actinomycin D.

    PubMed

    Arda-Pirincci, Pelin; Bolkent, Sehnaz

    2011-04-01

    Tumor necrosis factor-alpha (TNF-α) is a multifunctional cytokine, which has the ability to produce cytotoxicity via induction of cell death and cell cycle arrest. Blocking the synthesis of protective proteins through a transcriptional inhibitor such as actinomycin D (Act D) sensitizes many cell types to TNF-α toxicity. Teduglutide, h[Gly(2)]GLP-2, is a protease-resistant synthetic analog of glucagon-like peptide-2 (GLP-2) which is an intestinotrophic peptide. In this study, we evaluated this potential of GLP-2 on apoptosis, cell proliferation, and oxidant-antioxidant system on a mouse model of intestinal injury induced by TNF-α/Act D. The intestinal injury was induced by intraperitoneal administration of 15 μg/kg TNF-α and 800 μg/kg Act D per mouse. Animals were injected subcutaneously 200 μg/kg h[Gly(2)]GLP-2 every 12 h for 10 consecutive days prior to the administration of TNF-α and Act D. The model of intestinal injury induced by TNF-α/Act D, which is the new animal model for the intestinal disorders, was characterized by the degeneration of intestinal mucosa, an increase in apoptotic index, expression of active caspase-3, lipid peroxidation and glutathione (GSH) levels, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities; a decrease in cell proliferation and catalase (CAT) activity. h[Gly(2)]GLP-2 pretreatment prevented the TNF-α/Act D-induced oxidative injury by a significant reduction in the intestinal injury, apoptotic index, expression of active caspase-3, lipid peroxidation and GSH levels, GPx and SOD activities; a markedly increase in cell proliferation, and CAT activity. These results demonstrate that GLP-2 has a protective, antiapoptotic, proliferative, and antioxidant effects against to TNF-α/Act D-induced intestinal injury. It is suggested that GLP-2 may potentially be useful as a therapeutic agent in TNF-α-mediated intestinal disorders.

  19. AMPK and substrate availability regulate creatine transport in cultured cardiomyocytes.

    PubMed

    Darrabie, Marcus D; Arciniegas, Antonio Jose Luis; Mishra, Rajashree; Bowles, Dawn E; Jacobs, Danny O; Santacruz, Lucia

    2011-05-01

    Profound alterations in myocellular creatine and phosphocreatine levels are observed during human heart failure. To maintain its intracellular creatine stores, cardiomyocytes depend upon a cell membrane creatine transporter whose regulation is not clearly understood. Creatine transport capacity in the intact heart is modulated by substrate availability, and it is reduced in the failing myocardium, likely adding to the energy imbalance that characterizes heart failure. AMPK, a key regulator of cellular energy homeostasis, acts by switching off energy-consuming pathways in favor of processes that generate energy. Our objective was to determine the effects of substrate availability and AMPK activation on creatine transport in cardiomyocytes. We studied creatine transport in rat neonatal cardiomyocytes and HL-1 cardiac cells expressing the human creatine transporter cultured in the presence of varying creatine concentrations and the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Transport was enhanced in cardiomyocytes following incubation in creatine-depleted medium or AICAR. The changes in transport were due to alterations in V(max) that correlated with changes in total and cell surface creatine transporter protein content. Our results suggest a positive role for AMPK in creatine transport modulation for cardiomyocytes in culture.

  20. Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

    SciTech Connect

    Aguilar, David; Strom, Joshua; Chen, Qin M.

    2014-04-01

    Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA.

  1. Depressed phosphatidic acid-induced contractile activity of failing cardiomyocytes.

    PubMed

    Tappia, Paramjit S; Maddaford, Thane G; Hurtado, Cecilia; Panagia, Vincenzo; Pierce, Grant N

    2003-01-10

    The effects of phosphatidic acid (PA), a known inotropic agent, on Ca(2+) transients and contractile activity of cardiomyocytes in congestive heart failure (CHF) due to myocardial infarction were examined. In control cells, PA induced a significant increase (25%) in active cell shortening and Ca(2+) transients. The phospholipase C (PLC) inhibitor, 2-nitro-4-carboxyphenyl N,N-diphenylcarbonate, blocked the positive inotropic action induced by PA, indicating that PA induces an increase in contractile activity and Ca(2+) transients through stimulation of PLC. Conversely, in failing cardiomyocytes there was a loss of PA-induced increase in active cell shortening and Ca(2+) transients. PA did not alter resting cell length. Both diastolic and systolic [Ca(2+)] were significantly elevated in the failing cardiomyocytes. In vitro assessment of the cardiac sarcolemmal (SL) PLC activity revealed that the impaired failing cardiomyocyte response to PA was associated with a diminished stimulation of SL PLC activity by PA. Our results identify an important defect in the PA-PLC signaling pathway in failing cardiomyocytes, which may have significant implications for the depressed contractile function during CHF.

  2. Notch signaling regulates cardiomyocyte proliferation during zebrafish heart regeneration.

    PubMed

    Zhao, Long; Borikova, Asya L; Ben-Yair, Raz; Guner-Ataman, Burcu; MacRae, Calum A; Lee, Richard T; Burns, C Geoffrey; Burns, Caroline E

    2014-01-28

    The human heart's failure to replace ischemia-damaged myocardium with regenerated muscle contributes significantly to the worldwide morbidity and mortality associated with coronary artery disease. Remarkably, certain vertebrate species, including the zebrafish, achieve complete regeneration of amputated or injured myocardium through the proliferation of spared cardiomyocytes. Nonetheless, the genetic and cellular determinants of natural cardiac regeneration remain incompletely characterized. Here, we report that cardiac regeneration in zebrafish relies on Notch signaling. Following amputation of the zebrafish ventricular apex, Notch receptor expression becomes activated specifically in the endocardium and epicardium, but not the myocardium. Using a dominant negative approach, we discovered that suppression of Notch signaling profoundly impairs cardiac regeneration and induces scar formation at the amputation site. We ruled out defects in endocardial activation, epicardial activation, and dedifferentiation of compact myocardial cells as causative for the regenerative failure. Furthermore, coronary endothelial tubes, which we lineage traced from preexisting endothelium in wild-type hearts, formed in the wound despite the myocardial regenerative failure. Quantification of myocardial proliferation in Notch-suppressed hearts revealed a significant decrease in cycling cardiomyocytes, an observation consistent with a noncell autonomous requirement for Notch signaling in cardiomyocyte proliferation. Unexpectedly, hyperactivation of Notch signaling also suppressed cardiomyocyte proliferation and heart regeneration. Taken together, our data uncover the exquisite sensitivity of regenerative cardiomyocyte proliferation to perturbations in Notch signaling.

  3. Timing of cardiomyocyte growth, maturation, and attrition in perinatal sheep

    PubMed Central

    Jonker, Sonnet S.; Louey, Samantha; Giraud, George D.; Thornburg, Kent L.; Faber, J. Job

    2015-01-01

    Studies in altricial rodents attribute dramatic changes in perinatal cardiomyocyte growth, maturation, and attrition to stimuli associated with birth. Our purpose was to determine whether birth is a critical trigger controlling perinatal cardiomyocyte growth, maturation and attrition in a precocial large mammal, sheep (Ovis aries). Hearts from 0–61 d postnatal lambs were dissected or enzymatically dissociated. Cardiomyocytes were measured by micromorphometry, cell cycle activity assessed by immunohistochemistry, and nuclear number counted after DNA staining. Integration of this new data with published fetal data from our laboratory demonstrate that a newly appreciated >30% decrease in myocyte number occurred in the last 10 d of gestation (P < 0.0005) concomitant with an increase in cleaved poly (ADP-ribose) polymerase 1 (P < 0.05), indicative of apoptosis. Bisegmental linear regressions show that most changes in myocyte growth kinetics occur before birth (median = 15.2 d; P < 0.05). Right ventricular but not left ventricular cell number increases in the neonate, by 68% between birth and 60 d postnatal (P = 0.028). We conclude that in sheep few developmental changes in cardiomyocytes result from birth, excepting the different postnatal degrees of free wall hypertrophy between the ventricles. Furthermore, myocyte number is reduced in both ventricles immediately before term, but proliferation increases myocyte number in the neonatal right ventricle.—Jonker, S. S., Louey, S., Giraud, G. D., Thornburg, K. L., Faber, J. J. Timing of cardiomyocyte growth, maturation, and attrition in perinatal sheep. PMID:26139099

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. 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

  7. Human Placenta-Derived Multipotent Cells (hPDMCs) Modulate Cardiac Injury: From Bench to Small and Large Animal Myocardial Ischemia Studies.

    PubMed

    Liu, Yuan-Hung; Peng, Kai-Yen; Chiu, Yu-Wei; Ho, Yi-Lwun; Wang, Yao-Horng; Shun, Chia-Tung; Huang, Shih-Yun; Lin, Yi-Shuan; de Vries, Antoine A F; Pijnappels, Daniël A; Lee, Nan-Ting; Yen, B Linju; Yen, Men-Luh

    2015-01-01

    Cardiovascular disease is the leading cause of death globally, and stem cell therapy remains one of the most promising strategies for regeneration or repair of the damaged heart. We report that human placenta-derived multipotent cells (hPDMCs) can modulate cardiac injury in small and large animal models of myocardial ischemia (MI) and elucidate the mechanisms involved. We found that hPDMCs can undergo in vitro cardiomyogenic differentiation when cocultured with mouse neonatal cardiomyocytes. Moreover, hPDMCs exert strong proangiogenic responses in vitro toward human endothelial cells mediated by secretion of hepatocyte growth factor, growth-regulated oncogene-α, and interleukin-8. To test the in vivo relevance of these results, small and large animal models of acute MI were induced in mice and minipigs, respectively, by permanent left anterior descending (LAD) artery ligation, followed by hPDMC or culture medium-only implantation with follow-up for up to 8 weeks. Transplantation of hPDMCs into mouse heart post-acute MI induction improved left ventricular function, with significantly enhanced vascularity in the cell-treated group. Furthermore, in minipigs post-acute MI induction, hPDMC transplantation significantly improved myocardial contractility compared to the control group (p = 0.016) at 8 weeks postinjury. In addition, tissue analysis confirmed that hPDMC transplantation induced increased vascularity, cardiomyogenic differentiation, and antiapoptotic effects. Our findings offer evidence that hPDMCs can modulate cardiac injury in both small and large animal models, possibly through proangiogenesis, cardiomyogenesis, and suppression of cardiomyocyte apoptosis. Our study offers mechanistic insights and preclinical evidence on using hPDMCs as a therapeutic strategy to treat severe cardiovascular diseases.

  8. Downregulation of miR-181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1.

    PubMed

    Peng, Zhifeng; Li, Jiefei; Li, Yun; Yang, Xuan; Feng, Sujuan; Han, Song; Li, Junfa

    2013-10-01

    Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)-induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR-181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform-specific interacting proteins in HPC mouse brain. In this study, the role of miR-181b in oxygen-glucose deprivation (OGD)-induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR-181b expression levels significantly decreased in mouse brain following MCAO and in OGD-treated N2A cells. Up- and downregulation of miR-181b by transfection of pre- or anti-miR-181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD-treated N2A cells. By using a T7 promoter-driven control dual luciferase assay, we confirmed that miR-181b could bind to the 3'-untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR-181b antagomir reduced caspase-3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti-miR-181b-mediated neuroprotection against OGD-induced N2A cell injury in vitro. These results suggest that the downregulated miR-181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke.

  9. Dietary levels of acrylamide affect rat cardiomyocyte properties.

    PubMed

    Walters, Brandan; Hariharan, Venkatesh; Huang, Hayden

    2014-09-01

    The toxic effects of acrylamide on cytoskeletal integrity and ion channel balance is well-established in many cell types, but there has been little examination regarding the effects of acrylamide on primary cardiomyocytes, despite the importance of such components in their function. Furthermore, acrylamide toxicity is generally examined using concentrations higher than those found in vivo under starch-rich diets. Accordingly, we sought to characterize the dose-dependent effects of acrylamide on various properties, including cell morphology, contraction patterns, and junctional connexin 43 staining, in primary cardiomyocytes. We show that several days exposure to 1-100 μM acrylamide resulted in altered morphology, irregular contraction patterns, and an increase in the amount of immunoreactive signal for connexin 43 at cell junctions. We conclude that dietary levels of acrylamide may alter cellular function with prolonged exposure, in primary cardiomyocytes.

  10. An Animal Model with a Cardiomyocyte-Specific Deletion of Estrogen Receptor Alpha: Functional, Metabolic, and Differential Network Analysis

    PubMed Central

    Devanathan, Sriram; Whitehead, Timothy; Schweitzer, George G.; Fettig, Nicole; Kovacs, Attila; Korach, Kenneth S.; Finck, Brian N.; Shoghi, Kooresh I.

    2014-01-01

    Estrogen exerts diverse biological effects in multiple tissues in both animals and humans. Much of the accumulated knowledge on the role of estrogen receptor (ER) in the heart has been obtained from studies using ovariectomized mice, whole body ER gene knock-out animal models, ex vivo heart studies, or from isolated cardiac myocytes. In light of the wide systemic influence of ER signaling in regulating a host of biological functions in multiple tissues, it is difficult to infer the direct role of ER on the heart. Therefore, we developed a mouse model with a cardiomyocyte-specific deletion of the ERα allele (cs-ERα−/−). Male and female cs-ERα−/− mice with age/sex-matched wild type controls were examined for differences in cardiac structure and function by echocardiogram and differential gene expression microarray analysis. Our study revealed sex-differences in structural parameters in the hearts of cs-ERα−/− mice, with minimal functional differences. Analysis of microarray data revealed differential variations in the expression of 208 genes affecting multiple transcriptional networks. Furthermore, we report sex-specific differences in the expression of 56 genes. Overall, we developed a mouse model with cardiac-specific deletion of ERα to characterize the role of ERα in the heart independent of systemic effects. Our results suggest that ERα is involved in controlling the expression of diverse genes and networks in the cardiomyocyte in a sex-dependent manner. PMID:25000186

  11. Structural Phenotyping of Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-01-01

    Summary 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