Function And Mechanism Of Volume-sensitive Chloride Channel Regulates The Myocardial Ischemia/Reperfusion Injury Through Autophagy

Myocardial tissue reperfusion is essential for ischaemia recovery in heart stroke, however contribute to ischaemia-reperfusion injury due to the generation of excessive reactive oxygen species(ROS). It has generally been considered that I/R initiates ROS, calcium overload, neutrophile granulocyte and mitochondrial dysfunction, especially ion channels regulation of cytomembrane, but the paradoxical mechanism remains unclear. It is necessary to explore the relationship between ion channels and heart I/R injury, which should provide novel treatment strategies for I/R injury.Myocardial cell death in ischemia/reperfusion injury, including myocardial autophagy cell death, apoptosis and necrosis, is the primary reason of heart function failure. The myocardial necrosis and apoptosis in I/R injury have been reported frequently. In recent years, autophagy-- a novel programmed cell death mediated by lysosome, has become the focus of the research, which is an evolutionarily conserved intracellular lysosomal degradative process to eliminate protein aggregates and nonessential organelles, with double membrane-bound autophagosomes, and then fuse with lysosomes and degraded by lysosomal enzymes, providing energy to tissue in stress. Myocardial autophagy is increasing in ischemic insult, and further increased during reperfusion, which causes mitochondrial permeabilization leading to autophagic cell death in cardiac reperfusion injury. Reperfusion injury provokes ROS generation and autophagic myocardial death due to rapid decline in lysosome associated membrane protein-2(LAMP2) abundance, which results in autophagosome accumulation. Inhibiting excessive autophagy in reperfusion process can rescue myocardial ischemia-reperfusion injury. Foundational autophagy level is necessary for cell survival, obstacle of early stages of autophagy in Beclin-1 knockdown or impairment of late stages of autophagy in the absence of LAMP2 may result in cardiomyopathy.Recent studies find that many kinds of ion channel proteins on cytomembrane maintain the intracellular environment, which is associated with important cells function. In myocardial cell and vivo heart I/R model, we have proved the blocking VSOR Clchannels can inhibit myocardial apoptosis and myocardial injury. However, it is deceptive whether VSOR chlorine channel participates in autophagic cell death regulation. Our present experiment should undergo in myocardial vitro and vivo I/R model to reveal whether VSOR Cl- channel mediates autophagic cardiomyocyte fate.Aims:1. Establishment of ischemia/reperfusion(I/R) model in SD rats and simulation I/R injury model in neonatal SD rat cardiomyocytes.2. To study the change of VSOR Cl- channel proteins in myocardial I/R injury model, which influence cells survival, myocardial infarction area and the cardiac function, further explore the regulation role of autophagy in the I/R process.Method1. Establishment of I/R model: SD rat I/R model was subjected to ligation of left anterior descending coronary artery for ischemia 30 min, reperfusion 24-96 hours; I/R simulation in cardiac myocytes: neonatal SD rat myocardial cells, anoxia 12 h/reoxygenation 4 h;2. Grouping: The experiment randomly assigned to normal sham group(Sham group), ischemia/reperfusion group(I/R group); I/R+NAC pretreatment group(NAC group), I/R+DCPIB pretreatment group(DCPIB group) and I/R+3 MA pretreatment group(3 MA group). Those inhibitors were were administered to rat or cells at 10 min before reperfusion, respectively;3. Biochemical analytical instruments to detect serum CK、CK-MB and LDH enzyme;4. ELISA was performed to analyze NF-κB and TNF-α level;5. Cardiac uhrasonography for rat was used to detect index of cardiac function;6. TTC and Evans blue double staining to detect myocardial infarct size;7. The myocardial tissue LC3 and LAMP2 were detected by immunohistochemistry, immunofluorescence and western blot;8. Fluorescence spectrophotometer and fluorescence probe(DHE) were used to detect ROS levels in tissues and cells, respectively;9. TEM was performed to detect autophagosome and lysosomes;10. The whole-cell patch clamp recording technique was employed to test cardiomyocyteVSOR Cl- current.11. Real-time PCR was employed to detect the contents of Beclin 1 and liposomal transfection si Beclin-1 expression levels.ResultPlasma CK、CK-MB and LDH enzyme, NF-κB and LC3 stain in myocardial tissue were markedly increased and LAMP2 were dramaticlly reduced with I/R as compared with sham controls, whereas these may be reversed through DCPIB, NAC and 3MA, which increased heart function and reduced myocardial infarct. Moreover, neonatal rat cardiomyocytes subjected to H2O2 1μM siginificantly increased ROS expression leading to VSOR Cl- channel-activated, H2O2 1μM activated ICl,Vol with the similar peak amplitude, further increased LC3 but depleted of LAMP2 causing cell death. Blocking ROS and VSOR Cl- channel with NAC and DCPIB respectively, significantly inhibited ICl,Vol; 3MA could markedly inhibited autophagy but failed to thoroughly blocking ICl,Vol. Interesting, under blocking chloride channel, even increased ROS by adding H2O2, nevertheless ROS failed to increase autophagy strength; si Beclin-1 resulted in autophagy completely blocked, increased ROS and caspase 3 expression, cell survival activity were decreased. Our study provides strong evidence that I/R results in excessive generation of ROS, which rapidly deplete of LAMP2 abundance contribute to autophagosome accumulation and autophagic cell death. These indicate myocardial I/R injury influences heart function due to accelerate rapidly LAMP2 consumption, which contributes to autophagosome accumulation and inflammatory reaction. Inhibition of ROS and VSOR Cl- channel can continue to maintain LAMP2 expression, reducing inflammatory reaction. Overall, Blocking VSOR Cl- channel contributes a double benefit to rescue myocardial impairment through not only fractional restraint of Beclin1 but also restoration of LAMP2, the results indicate that VSOR Clchannel-activated directly modulates LAMP2 and mediates cardiomyocyte fate. Simultaneously, DCPIB eliminates inflammation to alleviate cardiomyocyte I/R damage.Conclusion:1. SD rat I/R model and neonatal rat cardiomyocytes I/R model were successfully established.2. VSOR Cl- channel blocker BCPIB could significantly inhibit the I/R-induced myocardial injury and myocardial cell autophagic death.3. VSOR Cl- channel blocker DCPIB protected myocardium from I/R injury, the main mechanisms due to inhibition of ROS, NF-k B level, partial inhibition of Beclin 1 expression and maintenance of LAMP2 expression, alleviate intracellular autophagosome accumulation.