Mitochondrial-Derived Vesicles Protect Cardiomyocytes Against Hypoxic Damage.
ABSTRACT: Myocardial ischemia is a condition with insufficient oxygen supporting the heart tissues, which may result from myocardial infarction or trauma-induced hemorrhagic shock. In order to develop better preventive and therapeutic strategies for myocardial ischemic damage, it is important that we understand the mechanisms underlying this type of injury. Mitochondrial-derived vesicles (MDVs) have been proposed as a novel player in maintaining mitochondrial quality control. This study aimed to investigate the role and possible mechanisms of MDVs in ischemia/hypoxia-induced myocardial apoptosis. H9C2 cardiomyocytes were used for the cellular experiments. A 40% fixed blood volume hemorrhagic shock rat model was used to construct the acute general ischemic models. MDVs were detected using immunofluorescence staining with PDH and TOM20. Exogenous MDVs were reconstituted in vitro from isolated mitochondria under different hypoxic conditions. The results demonstrate that MDV production was negatively correlated with cardiomyocyte apoptosis under hypoxic conditions; exogenous MDVs inhibited hypoxia-induced cardiomyocyte apoptosis; and MDV-mediated protection against hypoxia-induced cardiomyocyte apoptosis was accomplished via Bcl-2 interactions in the mitochondrial pathway. This study provides evidence that MDVs protect cardiomyocytes against hypoxic damage by inhibiting mitochondrial apoptosis. Our study used a novel approach that expands our understanding of MDVs and highlights that MDVs may be part of the endogenous response to hypoxia designed to mitigate damage. Strategies that stimulate cardiomyocytes to produce cargo-specific MDVs, including Bcl-2 containing MDVs, could theoretically be helpful in treating ischemic/hypoxic myocardial injury.
Project description: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 PPAR?-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.
Project description:Carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) is a tumor-associated factor that is known to be involved in apoptosis, but the role of CEACAM1 in cardiovascular disease is unclear. We aims to investigate whether CEACAM1 influences cardiac remodeling in mice with myocardial infarction (MI) and hypoxia-induced cardiomyocyte injury. Both serum in patients and myocardial CEACAM1 levels in mice were significantly increased in response to MI, while levels were elevated in neonatal rat cardiomyocytes (NRCs) exposed to hypoxia. Eight weeks after MI, a lower mortality rate, improved cardiac function, and less cardiac remodeling in CEACAM1 knock-out (KO) mice than in their wild-type (WT) littermates were observed. Moreover, myocardial expression of mitochondrial Bax, cytosolic cytochrome C, and cleaved caspase-3 was significantly lower in CEACAM1 KO mice than in WT mice. In cultured NRCs exposed to hypoxia, recombinant human CEACAM1 (rhCEACAM1) reduced mitochondrial membrane potential, upregulated mitochondrial Bax, increased cytosolic cytochrome C and cleaved caspase-3, and consequently increased apoptosis. RhCEACAM1 also increased the levels of GRP78 and CHOP in NRCs with hypoxia. All of these effects were abolished by silencing CEACAM1. Our study indicates that CEACAM1 exacerbates hypoxic cardiomyocyte injury and post-infarction cardiac remodeling by enhancing cardiomyocyte mitochondrial dysfunction and endoplasmic reticulum stress-induced apoptosis.
Project description:Cardiomyocytes are susceptible to apoptosis caused by hypoxia during the acute and subacute phases of myocardial infarction (MI). Angiogenesis can reduce MI-induced damage by mitigating hypoxia. It has been speculated that the ischemic border zone is a unique area rescued by angiogenic therapy. However, the mechanism and timing for new vessel formation in the mammalian heart following hypoxia are unclear. Identifying targets that benefit from angiogenesis treatment is indispensable for the development of revolutionary therapies. Here, we describe a novel circulatory system wherein new vessels develop from the endocardium of the left ventricle to perfuse the hypoxic area and salvage damaged cardiomyocytes at 3-14 days after MI by activating vascular endothelial growth factor signaling. Moreover, enhanced angiogenesis increased cardiomyocyte survival along the endocardium in the ischemic zone and suppressed ventricular remodeling in infarcted hearts. In contrast, cardiomyocytes in the border zone's hypoxic area underwent apoptosis within 12?h of MI, and the border area that was amenable to treatment disappeared. These data indicate that the non-perfused area along the endocardium is a site of active angiogenesis and a promising target for MI treatment.
Project description:<h4>Background</h4>Our previous studies suggested that brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) axis inhibited cardiomyocyte apoptosis in myocardial infarction (MI). However, the relationship between BDNF and microRNA (miRNA) in cardiomyocytes are unclear. The present study was performed to investigate the role of miR-195 and the interplay between BDNF and miR-195 in ischemic cardiomyocyte apoptosis.<h4>Methods</h4>Male Wistar rats were subjected to coronary artery ligation, and primary neonatal rat ventricular myocytes were treated with hypoxia or hydrogen peroxide (H2O2). BDNF level in rat ventricles was measured by enzyme linked immunosorbent assay (ELISA). miR-195 mimic, inhibitor or negative control was transfected into the cardiomyocytes. Cell viability and apoptosis were detected by MTT assay and TdT-mediated dUTP nick end labeling (TUNEL) staining, respectively. Cardiac function and apoptosis were detected in MI rats intravenously injected with antagomiR-195. Luciferase assay, Western blot and Real-time RT-PCR were employed to clarify the interplay between miR-195 and BDNF.<h4>Results</h4>miR-195 level was dynamically regulated in response to MI and significantly increased in ischemic regions 24 h post-MI as well as in hypoxic or H2O2-treated cardiomyocytes. Meanwhile, BDNF protein level was rapidly increased in MI rats and H2O2-treated cardiomyocytes. Apoptosis in both hypoxic and H2O2-treated cardiomyocytes were markedly reduced and cell viability was increased by miR-195 inhibitor. Moreover, inhibition of miR-195 significantly improved cardiac function of MI rats. Bcl-2 but not BDNF was validated as the direct target of miR-195. Furthermore, BDNF abolished the pro-apoptotic role of miR-195, which was reversed by its scavenger TrkB-Fc.<h4>Conclusion</h4>Up-regulation of miR-195 in ischemic cardiomyocytes promotes ischemic apoptosis by targeting Bcl-2. BDNF mitigated the pro-apoptotic effect of miR-195 in rat cardiomyocytes. These findings may provide better understanding of the pro-apoptotic role of miR-195 in MI and suggest that BDNF/miR-195/Bcl-2 axis may be beneficial for limiting myocardial ischemic injury.
Project description:Acute myocardial infarction (AMI) is an ischemic heart disease with high mortality worldwide. AMI triggers a hypoxic microenvironment and induces extensive myocardial injury, including autophagy and apoptosis. MiRNAs, which are a class of posttranscriptional regulators, have been shown to be involved in the development of ischemic heart diseases. We have previously reported that hypoxia significantly alters the miRNA transcriptome in rat cardiomyoblast cells (H9c2), including miR-27a-5p. In the present study, we further investigated the potential function of miR-27a-5p in the cardiomyocyte response to hypoxia, and showed that miR-27a-5p expression was downregulated in the H9c2 cells at different hypoxia-exposed timepoints and the myocardium of a rat AMI model. Follow-up experiments revealed that miR-27a-5p attenuated hypoxia-induced cardiomyocyte injury by regulating autophagy and apoptosis via <i>Atg7</i>, which partly elucidated the anti-hypoxic injury effects of miR-27a-5p. Taken together, this study shows that miR-27a-5p has a cardioprotective effect on hypoxia-induced H9c2 cell injury, suggesting it may be a novel target for the treatment of hypoxia-related heart diseases.
Project description:Cardiac shock wave therapy (SWT) has been described as a novel therapeutic strategy that is able to alleviate myocardial ischemic injury. microRNA (miRNA/miR)?210 plays a cytoprotective role in cardiomyocytes in response to hypoxia by regulating cell apoptosis. The aim of the present study was to investigate whether cardiac SWT could protect cardiomyocytes from hypoxia?induced injury by regulating miR?210 expression. The murine adult cardiomyocyte cell line HL?1 was incubated for 5 h in hypoxic conditions, followed by reoxygenation for 12 h and treatment with SWT immediately following hypoxia in the present study. The cell viability was determined using an MTS assay. Western blot analyses were performed in order to detect cell signaling changes. Reactive oxygen species production was detected using dihydroethidium staining, and malondialdehyde levels were measured using the thiobarbituric acid method. miRNA and mRNA expression levels were confirmed via reverse transcription?quantitative PCR. Apoptosis was evaluated by means of flow cytometry. HL?1 cells were then transfected with miR?210 mimics or inhibitors in order to alter miR?210 expression levels, and the effects on HL?1 cells were determined. Hypoxia led to elevated oxidative stress, enhanced cell apoptosis and upregulated miR?210 expression levels in HL?1 cells, while SWT could alleviate hypoxia?induced cell injury and further promote miR?210 expression. miR?210 overexpression decreased apoptosis and oxidative stress during hypoxic stress in HL?1 cells, whereas inhibition of miR?210 increased cell apoptosis and promoted oxidative stress. Furthermore, miR?210 inhibition could reverse the effects of SWT on HL?1 cells. Finally, the mRNA analysis revealed that SWT significantly attenuated apoptosis?inducing factor mitochondrion?associated 3 and caspase 8 associated protein 2 mRNA expression levels in cardiomyocytes exposed to hypoxia, which were two targets of miR?210. SWT could exert cardioprotective effects against hypoxia?induced cardiac injury by modulating miR?210.
Project description:Objective: Integrin ?3 is one of the main integrin heterodimer receptors on the surface of cardiac myocytes. Our previous studies showed that hypoxia induces apoptosis and increases integrin ?3 expression in cardiomyocytes. However, the exact mechanism by which integrin ?3 protects against apoptosis remains unclear. Hence, the present investigation aimed to explore the mechanism of integrin ?3 in cardiomyocyte proliferation and hypoxia-induced cardiomyocyte apoptosis. Methods: Stable cells and in vivo acute and chronic heart failure rat models were generated to reveal the essential role of integrin ?3 in cardiomyocyte proliferation and apoptosis. Western blotting and immunohistochemistry were employed to detect the expression of integrin ?3 in the stable cells and rat cardiac tissue. Flow cytometer was used to investigate the role of integrin ?3 in hypoxia-induced cardiomyocyte apoptosis. Confocal microscopy was used to detect the localization of integrin ?3 and integrin ?v in cardiomyocytes. Results: A cobaltous chloride-induced hypoxic microenvironment stimulated cardiomyocyte apoptosis and increased integrin ?3 expression in H9C2 cells, AC16 cells, and cardiac tissue from acute and chronic heart failure rats. The overexpression of integrin ?3 promoted cardiomyocyte proliferation, whereas silencing integrin ?3 expression resulted in decreased cell proliferation in vitro. Furthermore, knocking down integrin ?3 expression using shRNA or the integrin ?3 inhibitor cilengitide exacerbated cobaltous chloride-induced cardiomyocyte apoptosis, whereas overexpression of integrin ?3 weakened cobaltous chloride-induced cardiomyocytes apoptosis. We found that integrin ?3 promoted cardiomyocytes proliferation through the regulation of the PTEN/Akt/mTOR and ERK1/2 signaling pathways. In addition, we found that knockdown of integrin ?v or integrin ?1 weakened the effect of integrin ?3 in cardiomyocyte proliferation. Conclusion: Our findings revealed the molecular mechanism of the role of integrin ?3 in cardiomyocyte proliferation and hypoxia-induced cardiomyocyte apoptosis, providing new insights into the mechanisms underlying myocardial protection.
Project description:BACKGROUND:Qiliqiangxin (QLQX) is a preparation refined from a traditional Chinese medicine compound. It plays an important role in protecting cardiac function after myocardial infarction (MI). However, the underline mechanism of QLQX action is not clear. The purpose of this study was to detect the effects of QLQX on mitophagy after MI. METHODS:Male FVB/NJ mice aged 8-10?weeks were underwent left coronary artery ligation and were orally administered either QLQX (0.25?g/kg/d) or saline. Twenty-eight days after surgical operation, the cardiac function of mice was detected by echocardiography. Electron Microscopy was used to observe the microstructure of cardiomyocytes. Myocardial apoptosis was examined by TdT-mediated dUTP Nick-End Labeling (TUNEL) and western blot. H9c2 cells were cultured in a hypoxic incubator chamber (5% CO2, 1% O2, 94% N2) for 12?h and pretreated with or without QLQX (0.5?mg/mL). The cell apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential and mitophagy were detected. RESULTS:When compared to sham group, the cardiac function of MI mice decreased significantly, and their cardiomyocyte apoptosis and mitochondrial damage were more serious. These MI-induced cardiac changes could be reversed by QLQX treatment. In vitro experiments also confirmed that QLQX could protect cardiomyocytes from hypoxia-induced apoptosis and mitochondrial damage. Further study indicated that QLQX could increase the expression of Pink1 and Parkin in cardiomyocytes. CONCLUSION:Qiliqiangxin could reduce cardiomyocytes apotosis and improved heart function in infarcted heart through Pink1-mediated mitochondrial autophagy.
Project description:Programmed cell death of cardiomyocytes following myocardial ischemia increases biomechanical stress on the remaining myocardium, leading to myocardial dysfunction that may result in congestive heart failure or sudden death. Nogo-A is well characterized as a potent inhibitor of axonal regeneration and plasticity in the central nervous system, however, the role of Nogo-A in non-nervous tissues is essentially unknown. In this study, Nogo-A expression was shown to be significantly increased in cardiac tissue from patients with dilated cardiomyopathy and from patients who have experienced an ischemic event. Nogo-A expression was clearly associated with cardiomyocytes in culture and was localized predominantly in the endoplasmic reticulum. In agreement with the findings from human tissue, Nogo-A expression was significantly increased in cultured neonatal rat cardiomyocytes subjected to hypoxia/reoxygenation. Knockdown of Nogo-A in cardiomyocytes markedly attenuated hypoxia/reoxygenation-induced apoptosis, as indicated by the significant reduction of DNA fragmentation, phosphatidylserine translocation, and caspase-3 cleavage, by a mechanism involving the preservation of mitochondrial membrane potential, the inhibition of ROS accumulation, and the improvement of intracellular calcium regulation. Together, these data demonstrate that knockdown of Nogo-A may serve as a novel therapeutic strategy to prevent the loss of cardiomyocytes following ischemic/hypoxic injury.
Project description:Background. Nitric oxide can successfully compete with oxygen for sites of electron-transport chain in conditions of myocardial hypoxia. These features may prevent excessive oxidative stress occurring in cardiomyocytes during sudden hypoxia-reoxygenation. Aim. To study the action of the potent stable NO donor dinitrosyl iron complex with glutathione (Oxacom®) on the recovery of myocardial contractile function and Ca2+ transients in cardiomyocytes during hypoxia-reoxygenation. Results. The isolated rat hearts were subjected to 30?min hypoxia followed by 30?min reoxygenation. The presence of 30?nM Oxacom in hypoxic perfusate reduced myocardial contracture and improved recovery of left ventricular developed pressure partly due to elimination of cardiac arrhythmias. The same Oxacom concentration limited reactive oxygen species generation in hypoxic cardiomyocytes and increased the viability of isolated cardiomyocytes during hypoxia from 12 to 52% and after reoxygenation from 0 to 40%. Oxacom prevented hypoxia-induced elevation of diastolic Ca2+ level and eliminated Ca2+ transport alterations manifested by slow Ca2+ removal from the sarcoplasm and delay in cardiomyocyte relaxation. Conclusion. The potent stable NO donor preserved cardiomyocyte integrity and improved functional recovery at hypoxia-reoxygenation both in the isolated heart and in cardiomyocytes mainly due to preservation of Ca2+ transport. Oxacom demonstrates potential for cardioprotection during hypoxia-reoxygenation.