Sirt3-Bnip3 signaling axis alleviates myocardial ischemia/reperfusion-induced cardiac injury via regulating GSDME.

BackgroundMyocardial ischemia/reperfusion (MI/R) injury imposes devastating cardiovascular sequelae in particular cardiac dysfunction as a result of restored blood flow. However, the mechanism behind MI/R injury remains elusive. Mitochondrial ubiquitin ligase (MITOL/MARCH5) is localized at the mitochondria‐ER contact site and may be activated in response to a variety of pathophysiological processes, such as apoptosis, mitochondrial injury, ER stress, hypoxia, and reactive oxygen species (ROS) generation. Irisin as a cleaved product of fibronectin type III domain‐containing protein 5 (FNDC5) displays cardioprotection in diverse cardiac diseases.MethodsThis study was designed to examine the role of irisin and MITOL in MI/R injury. Male C57BL/6J mice (8‐10‐week‐old) were administered adenovirus MITOL shRNA through intracardiac injection followed by MI/R surgery through ligation and release the slipknot of cardiac left anterior descending coronary artery.ResultsOur results showed that irisin improved myocardial function in the face of MI/R injury as evidenced by reduced myocardial infarct size, apoptotic rate, serum lactate dehydrogenase (LDH), ROS generation, and malondialdehyde (MDA) levels as well as lessened ER stress injury. Moreover, our results indicated that protective role of irisin was mediated by upregulation of MITOL. Irisin also protected H9c2 cells against simulated I/R through negating ER stress, apoptosis, ROS and MDA levels, as well as facilitating superoxide dismutase (SOD) by way of elevated MITOL expression.ConclusionsTo this end, our data favored that irisin pretreatment protects against MI/R injury, ER stress, ROS production, and mitochondrial homeostasis through upregulation of MITOL. These findings depicted the therapeutic potential of irisin and MITOL in the management of MI/R injury in patients with ST‐segment elevation.

Background:Oxygen-glucose deprivation-nutrition resumption (OGD-NR) models on H9c2 cells are commonly used in vitro models of simulated myocardial ischemia-reperfusion injury (MIRI), but no study has assessed whether these methods for establishing in vitro models can effectively imitate the characteristics of MIRI in vivo. This experiment was designed to analyze the feasibility of six OGD-NR models of MIRI.Methods:By searching the PubMed database using the keywords “myocardial reperfusion injury H9c2 cells,” we obtained six commonly used OGD-NR in vitro models of MIRI performed on H9c2 cells from more than 400 published papers before January 30, 2017. For each model, control (C), simulated ischemia (SI), and simulated ischemia-reperfusion (SIR) groups were assigned, and cell morphology, lactate dehydrogenase (LDH) release, adenosine triphosphate (ATP) levels, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and inflammatory cytokines were examined to evaluate the characteristics of cell injury. Subsequently, a coculture system of cardiomyocyte-endothelial-macrophage was constructed. The coculture system was dealt with SI and SIR treatments to test the effect on cardiomyocytes survival.Results:For models 1, 2, 3, 4, 5, and 6, SI treatment caused morphological damage to cells, and subsequent SIR treatment did not cause further morphological damage. In the models 1, 2, 3, 4, 5 and 6, LDH release was significantly higher in the SI groups than that in the C group (P < 0.05), and was significantly lower in the SIR groups than that in the SI groups (P < 0.05), except for no significant differences in the LDH release between C, SI and SIR groups in model 6 receiving a 3-h SI treatment. In models 1, 2, 3, 4, 5, and 6, compared with the C group, ATP levels of the SI groups significantly decreased (P < 0.05), ROS levels increased (P < 0.05), and MMP levels decreased (P < 0.05). Compared with the SI group, ATP level of the SIR groups was significantly increased (P < 0.05), and there was no significant ROS production, MMP collapse, and over inflammatory response in the SIR groups. In a coculture system of H9c2 cells-endothelial cells-macrophages, the proportion of viable H9c2 cells in the SIR groups was not reduced compared with the SI groups.Conclusion:All the six OGD-NR models on H9c2 cells in this experiment can not imitate the characteristics of MIRI in vivo and are not suitable for MIRI-related study.

Oxidative stress and mitochondrial dysfunction are considered to be activators of apoptosis and serve a pivotal role in the pathogenesis of myocardial ischemia-reperfusion (MI/R) injury. Apurinic/apyrimidinic endonuclease/redox factor 1 (APE1) is a multifunctional protein that processes the cellular response to DNA damage and oxidative stress. Little is known about the role of APE1 in the pathogenesis of MI/R injury. The aim of the present study was to investigate the effects of APE1 on hypoxia-reoxygenation (H/R)-induced H9c2 cardiomyocyte injury and the underlying mechanism responsible. It was demonstrated that H/R decreased cell viability and increased lactic dehydrogenase (LDH) release, as well as reducing APE1 expression in H9c2 cells. However, APE1 overexpression induced by transfection with APE1-expressing lentivirus significantly increased H9c2 cell viability, decreased LDH release, decreased apoptosis and reduced caspase-3 activity in H/R-treated H9c2 cells. APE1 overexpression ameliorated the H/R-induced increases in reactive oxygen species and NAPDH oxidase expression, as well as the decreases in superoxide dismutase activity and glutathione expression. Furthermore, APE1 overexpression increased mitochondrial membrane potential and ATP production, stabilized electron transport chain activity (as illustrated by increased NADH-ubiquinone oxidoreductase, succinate dehydrogenase, coenzyme Q-cytochrome c oxidoreductase and cytochrome c oxidase activities) and decreased the ratio of B-cell lymphoma 2-associated X protein/B-cell lymphoma 2 in H/R, improving mitochondrial dysfunction. In conclusion, the results of the present study suggest that APE1 alleviates H/R-induced injury in H9c2 cells by attenuating oxidative stress and ameliorating mitochondrial dysfunction. APE1 may therefore be used as an effective treatment for MI/R injury.

Background Cardiomyocyte apoptosis is a crucial event underlying the development of cardiac abnormalities and dysfunction after myocardial ischemia reperfusion (MI/R) injury. A better understanding of the cell signaling pathways involved in cardiac remodeling may support the development of new therapeutic strategies for the treatment of heart failure (HF) after MI/R. Nicorandil, an ATP-sensitive potassium channel opener, could improve mitochondrial damage and reduce oxidative stress，which reduce cardiomyocyte apoptosis. Metallothionein (Mt), reactive oxygen species (ROS) scavenger, which can reduce oxidative stress, also attenuated MI/R-induced autophagy and cell apoptosis. Except as a potassium channel opener, nicorandil may play a cardioprotective role by regulating metallothionein, and its specific mechanism remains to be elucidated. Objective To clarify the protective effect of nicorandil in myocardial ischemia/reperfusion injury, and elucidate the specific mechanism which nicorandil regulate the expression of metallothiosin to reduce myocardial ischemia/reperfusion injury, so as to provide new drug targets or new measures for clinical prevention and treatment of MI/R. Methods A cardiac MI/R injury model was constructed by the ligation of the left anterior descending coronary artery to investigate the underlying molecular mechanisms. The apoptosis of myocardial tissue cells was detected by TUNEL staining. The ultrastructural changes of myocardial tissue were observed by transmission electron microscopy. The expressions of Mt family, mitochondrial dynamics and Glucose metabolism-associated genes were measured by Western blotting and qPCR. The content of ATP was determined by ELISA in heart tissue. AutodockVina was used to evaluate binding energy and interaction patterns between drug candidates and their targets. The protein-protein interaction model was predicted by ZDOCK software. Results Treatment with nicorandil mitigated left ventricular enlargement, improved cardiac dysfunction,reduced myocardial infarcation area and decreased cardiomyocyte apoptosis after I/R. Nicorandil up-regulated the expression of Mt2 and decreased the expressions of Drp-1,p-Drp-1(ser616), while enhanced the expression of Mfn1/2,GLUT4,HK II and MPC II in myocardium. Knockdown of Mt2 decreased the effects of nicorandil on apoptosis and mitochondrial dysfunction after I/R. Mechanistically, nicorandil significantly upregulated the expression of Mt2, which could bound to LKB1 and cause phosphorylation of LKB1, resulting in AMPK-dependent activation. Conclusions Nicorandil significantly upregulated the expression of Mt2, which interacted with LKB1 to promote LKB1 protein phosphorylation, resulting in AMPK-dependent activation and subsequently alleviating I/R-induced mitochondrial dysfunction and energy metabolism disorder, and thereby reducing cardiomyocyte apoptosis, eventually improving I/ R-induced cardiac remodeling and dysfunction.

Bergenin alleviates myocardial ischemia‐reperfusion injury via SIRT1 signaling

This study aimed to explore the role of dual specificity phosphatase 12 (DUSP12) in regulating myocardial ischemia-reperfusion (I/R) injury and the underlying mechanism. The expression of DUSP12 in myocardial tissuesand heat-shock protein beta-8(HSPB8) and mitophagy-related proteins in myocardial tissues and H9c2 cells were detected by western blot analysis. The serum creatine kinase isoenzymes (CK-MB)and lactate dehydrogenase (LDH), levels of reactive oxygen speciesand malondialdehyde,superoxide dismutase activity in myocardial tissuesand H9c2 cells, and caspase-3 activity in H9c2 cells were analyzed by corresponding assay kits. The infarct area in therat's heart was observed by triphenyl tetrazolium chloridestaining. The apoptosis of myocardial cells in myocardial tissues and H9c2 cells was detected by terminal-deoxynucleotidyl transferase dUTP-biotin nick-end labelingassay. The interaction between DUSP12 and HSPB8 was clarified by the coimmunoprecipitation assay. The transfection efficacy of si-HSPB8#1 and si-HSPB8#2 in H9c2 cells was confirmed by real-time quantitative-polymerasechain reactionand western blot analysis. As a result, DUSP12 expression was downregulated in I/R rats, which was promoted by lentivirus-expressing DUSP12. DUSP12 overexpression reduced the serumcreatine kinase isoenzymes (CK-MB) and LDH, decreased the infarct area in the rat's heart, and suppressed the apoptosis and oxidative stress in myocardial tissues. DUSP12 overexpression also upregulated the expression of HSPB8 to promote mitophagy. The coimmunoprecipitation assay indicated that DUSP12 could be combined with HSPB8. In addition, DUSP12 overexpression could inhibit hypoxia/reoxygenation-elicited apoptosis as well as oxidative stress in H9c2 cells by upregulating HSPB8 expression to promote mitophagy, which was countervailed by HSPB8 deficiency. In conclusion, DUSP12 overexpression decreased the apoptosis and oxidative stress in myocardial I/R injury through HSPB8-induced mitophagy.

SIRT3 and RORα are two prospective targets against mitophagy during simulated ischemia/reperfusion injury in H9c2 cells

Evidence suggests Ginsenoside Rd (GSRd), a biologically active extract from the medical plant Panax Ginseng, exerts antioxidant effect, decreasing reactive oxygen species (ROS) formation. Current study determined the effect of GSRd on myocardial ischemia/reperfusion (MI/R) injury (a pathological condition where ROS production is significantly increased) and investigated the underlying mechanisms. The current study utilized an in vivo rat model of MI/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of simulated ischemia/reperfusion (SI/R) injury. Infarct size was measured by Evans blue/TTC double staining. NRC injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. ROS accumulation and apoptosis were assessed by flow cytometry. Mitochondrial membrane potential (MMP) was determined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation of mitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2 family proteins, and phosphorylated Akt and GSK-3β were determined by western blot. Pretreatment with GSRd (50 mg/kg) significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ±dP/dt. GSRd reduced myocardial infarct size, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels after MI/R. In NRCs, GSRd (10 µM) inhibited SI/R-induced ROS generation (P<0.01), decreased cellular apoptosis, stabilized the mitochondrial membrane potential (MMP), and attenuated cytosolic translocation of mitochondrial cytochrome c. GSRd inhibited activation of caspase-9 and caspase-3, increased the phosphorylated Akt and GSK-3β, and increased the Bcl-2/Bax ratio. Together, these data demonstrate GSRd mediated cardioprotective effect against MI/R–induced apoptosis via a mitochondrial-dependent apoptotic pathway.

The prevention and management of myocardial ischemia/reperfusion (MI/R) injury is an essential part of coronary heart disease surgery and is becoming a major clinical problem in the treatment of ischemic heart disease. Previous studies by our group have demonstrated that pigment epithelium-derived factor (PEDF) improves cardiac function in rats with acute myocardial infarction and reduces hypoxia-induced cell injury. However, the protective function and mechanisms underlying the effect of PEDF in MI/R injury remain to be fully understood. In the present study, the positive effect of PEDF in MI/R injury was confirmed by construction of the adult Sprague-Dawley rat MI/R model. PEDF reduced myocardial infarct size and downregulated cardiomyocyte apoptosis in the I/R myocardium in this model. In addition, PEDF improved cardiac function and increased cardiac functional reserve in rats subjected to MI/R Injury. To further study the protective effect of PEDF and the underlying mechanisms in MI/R injury, a H9c2 cardiomyocyte hypoxia/reoxygenation (H/R) model was constructed. PEDF was confirmed to decrease H/R-induced apoptosis in H9c2 cells, and this anti-apoptotic function was abolished by pigment epithelium-derived factor-receptor (PEDF R) small interfering (si)RNA. Furthermore, administration of PEDF decreased the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in H/R H9c2 cells. Compared with the H/R group, PEDF decreased mitochondrial ROS, increased the mitochondrial DNA copy number, reduced xanthine oxidase and NADPH oxidase activity, as well as RAC family small GTPase 1 protein expression. However, these effects of PEDF were markedly attenuated by PEDF-R siRNA. To the best of our knowledge, the present study is the first to identify the protective effect of PEDF in MI/R injury, and confirm that the antioxidative effect PEDF occurred via inhibition of ROS generation via PEDF-R under MI/R conditions.

Cardioprotective effect of Danshensu against myocardial ischemia/reperfusion injury and inhibits apoptosis of H9c2 cardiomyocytes via Akt and ERK1/2 phosphorylation

Attenuation of Na/K-ATPase/Src/ROS amplification signal pathway with pNaktide ameliorates myocardial ischemia-reperfusion injury

Recent studies have revealed that exercise has myocardial protective effects, but the exact mechanism remains unclear. Studies have increasingly found that peptides play a protective role in myocardial ischaemia‐reperfusion (I/R) injury. However, little is known about the role of exercise‐induced peptides in myocardial I/R injury. To elucidate the effect of exercise‐induced peptide EIP‐22 in myocardial I/R injury, we first determined the effect of EIP‐22 on hypoxia/reperfusion (H/R)‐ or H2O2‐induced injury via assessing cell viability and lactate dehydrogenase (LDH) level. In addition, reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) was assessed by fluorescence microscope. Meanwhile, Western blot and TUNEL methods were used to detect apoptosis level. Then, we conducted mice I/R injury model and verified the effect of EIP‐22 by measuring cardiac function, evaluating heart pathology and detecting serum LDH, CK‐MB and cTnI level. Finally, the main signalling pathway was analysed by RNA‐seq. In vitro, EIP‐22 treatment significantly improved cells viabilities and MMP and attenuated the LDH, ROS and apoptosis level. In vivo, EIP‐22 distinctly improved cardiac function, ameliorated myocardial infarction area and fibrosis and decreased serum LDH, CK‐MB and cTnI level. Mechanistically, JAK/STAT signalling pathway was focussed by RNA‐seq and we confirmed that EIP‐22 up‐regulated the expression of p‐JAK2 and p‐STAT3. Moreover, AG490, a selective inhibitor of JAK2/STAT3, eliminated the protective roles of EIP‐22. The results uncovered that exercise‐induced peptide EIP‐22 protected cardiomyocytes from myocardial I/R injury via activating JAK2/STAT3 signalling pathway and might be a new candidate molecule for the treatment of myocardial I/R injury.

Over the past 10 to 15 years, a vast collection of studies have provided evidence indicating that reactive oxygen species (ROS), particularly superoxide (O2·−) and hydrogen peroxide (H2O2), contribute to the pathogenesis of cardiovascular diseases, such as heart failure and hypertension. Griendling et al1 first demonstrated that NADPH oxidase present in the vasculature is a primary source of the elevated ROS levels. Since these initial studies, NADPH oxidase-derived ROS in the kidney,2 heart,3 and brain4 have been linked to the development and progression of numerous cardiovascular-related diseases. More recently, however, mitochondria have also been identified as important sources of ROS in controlling cardiovascular function. Considering that mitochondria are the primary source of ROS in most cells during normal respiration because of the leaking of electrons from the electron transport chain (ETC), perhaps it should not be all that surprising that mitochondrial-produced ROS are involved in pathophysiological conditions of the cardiovascular system. To date, most of the evidence linking mitochondrial dysfunction and mitochondrial-produced ROS to the pathogenesis of cardiovascular diseases comes from studies on the peripheral renin-angiotensin system.5 For example, using a model of cardiac ischemic reperfusion injury, Kimura et al6 reported that angiotensin II (Ang II)-induced preconditioning is mediated by mitochondrial-produced ROS. The authors further demonstrated that Ang II-induced NADPH oxidase-derived ROS lie upstream of mitochondrial-produced ROS, thus, implicating a ROS-induced ROS mechanism. Similarly, it was demonstrated recently that, in aortic endothelial cells, Ang II-induced NADPH oxidase activation leads to an increase in mitochondrial ROS production, as well as mitochondrial dysfunction, as determined by a decrease in mitochondrial membrane potential and mitochondrial respiration.7 Together, these studies and others (detailed elsewhere5) clearly illustrate a role for mitochondrial-produced ROS and mitochondrial dysfunction in peripheral tissues in the pathogenesis of …

Myocardial ischemia/reperfusion (MI/R) leads to oxidative stress, which may in turn lead to myocardial injury. In the present study, we investigated the effects of exenatide, a glucagon-like peptide-1 (GLP-1) analogue, on oxidative stress-induced injury in vitro and in vivo. In in vitro experiments, H9c2 cells were incubated with exenatide to determine the direct cytoprotective effects of exenatide following exposure to hydrogen peroxide (H2O2). Pre-treatment with exenatide (1 nM), prior to H2O2 exposure, increased cell viability and inhibited H2O2-induced reactive oxygen species (ROS) production. Exenatide also decreased the levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in the cultured supernatants, as well as those of malondialdehyde (MDA) in the H9c2 cells and increased the total superoxide dismutase (T-SOD) levels in the H9c2 cells. In in vivo experiments, an animal model of MI/R was induced by coronary occlusion. Pre-treatment with exenatide (10 µg/kg/day) protected the rat hearts from MI/R-induced injury by decreasing the levels of LDH and CK-MB in plasma, increasing the levels of catalase, T-SOD and glutathione peroxidase (GSH-Px) in the heart and decreasing the MDA levels in the rats with MI/R-induced injury. Exenatide also reduced the infarct size and enhanced cardiac function in the rats with MI/R-induced injury. Moreover, pre-treatment with exenatide inhibited cardiomyocyte apoptosis, increased Aktserine473 and Badserine136 phosphorylation and decreased cleaved caspase-3 expression in vitro and in vivo; however, these effects were attenuated by the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002. Our results suggest that exenatide exerts significant cardioprotective effects against oxidative stress-induced injury in vitro and in vivo. The mechanisms involved may be attributed to the scavenging of oxidative stress products, such as ROS, the increase in the concentrations of antioxidant defense enzymes and the inhibition of cardiomyocyte apoptosis. The anti-apoptotic effects of exenatide were, at least in part, associated with the activation of the PI3K/Akt signaling pathway.

Necroptosis contribute to the pathogenesis of myocardial ischemia/reperfusion (MI/R) injury. Ginsenoside Rg2 has been reported to have cardioprotective effects against MI/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate the effect of ginsenoside Rg2 on necroptosis induced by MI/R and to explore the mechanism. In this study, hypoxia/reoxygenation (H/R) injury model was established in H9c2 cells. In vivo, male C57/BL6 mice were subjected to myocardial ischemia 30 min/reperfusion 4 h. Rg2 (50 mg/kg) or vehicle was intravenously infused 5 min before reperfusion. Cardiac function and the signaling pathway involved in necroptosis were investigated. Compared with H/R group, Rg2 significantly inhibited H/R-induced cardiomyocyte death. Rg2 treatment effectively inhibited the phosphorylation of RIP1, RIP3, and MLKL in H/R cardiomyocytes, and inhibited RIP1/RIP3 complex (necrosome) formation. In mice, Rg2 treatment manifested significantly lower ischemia/reperfusion (I/R)-induced myocardial necroptosis, as evidenced by decrease in phosphorylation of RIP1, RIP3, and MLKL, inhibited lactate dehydrogenase (LDH) release and Evans blue dye (EBD) penetration. Mechanically, an increased level of tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6, and MCP-1 were found in MI/R hearts, and Rg2 treatment significantly inhibit the expression of these factors. We found that TNFα-induced phosphorylation of RIP1, RIP3, and MLKL was negatively correlated with transforming growth factor-activated kinase 1 (TAK1) phosphorylation, and inhibition of TAK1 phosphorylation led to necroptosis enhancement. More importantly, Rg2 treatment significantly increased TAK1 phosphorylation, enhanced TAK1 binding to RIP1 while inhibiting RIP1/RIP3 complex, ultimately reducing MI/R-induced necroptosis. These findings highlight a new mechanism of Rg2-induced cardioprotection: reducing the formation of RIP1/RIP3 necrosome by regulating TAK1 phosphorylation to block necroptosis induced by MI/R.