Shock Wave Therapy Promotes H9c2 Myoblast Autophagy And Survival During Hypoxia

Background:Cardiovascular diseases are the leading cause of death worldwide.Despite robust successes of drug,percutaneous coronary intervention(PCI),and surgical therapies,the morbidity and mortality of coronary heart disease(CHD)are still high.Indeed,PCI and coronary artery bypass grafting(CABG)treatments are not suitable for some patients with severe and complicated coronary lesions,and some patients still suffer from angina pectoris even after PCI and/or CABG treatment.Chronic ischemia/hypoxia may cause repeated episodes of angina pectoris,myocardial infarction,or heart failure and seriously affect a patient's quality of life.The development of a new therapeutic strategy for the treatment of refractory and symptomatic CHD is urgently needed.Extracorporeal shock waves(SWs)were first introduced for clinical use for urolithiasis more than 20 years ago.Recently,an increasing number of studies have indicated that cardiac shock wave therapy(CSWT)is an efficient method for treating ischemic heart disease in animal models and human patients.The mechanisms of heart protection after CSWT are still unclear but may include promotion of the recruitment of endothelial progenitor cells,stimulation of neovascularization,and reduction of oxidative stress,inflammation,and fibrosis.Our recent in vitro and in vivo studies(unpublished data)demonstrated that CSWT could inhibit apoptosis in ischemic/hypoxic myocardium and consequently enhance cell vitality and heart function.However,the mechanism of CSWT appears to be multifactorial and needs to be further elucidated.Autophagy is an evolutionarily conserved energy-dependent process that occurs during a series of pathological stresses and plays an essential role in diseases such as cancer,infection,neurodegeneration,diabetes,and cardiac diseases.It is a unique biological process used to cope with stress and maintain cellular metabolism,homeostasis,and survival.It has been well established for several years that autophagy is a key regulator of the cardiovascular system and is essential for normal maintenance,repair,and adaptation of the heart over the course of a lifetime.Several studies have demonstrated that autophagy is obviously induced during myocardial ischemia,but the functional role of autophagy is still unclear and is currently under intense investigation.Despite the key beneficial effect of autophagic activity,excessive or insufficient autophagy may contribute to cell death.Furthermore,whether SW treatment can protect cardiac myocytes against hypoxic injury by influencing the process of autophagy is still unknown.Objectives:In the present study,we established an ischemia/hypoxia model in vitro using H9c2 myoblasts to explore whether SW could protect cardiomyocyte function by regulating cell autophagy and its possible signal transduction pathways.Methods:The rat cardiomyocyte cell line H9c2 derived from embryonic heart tissue of BDIX rat was cultured under an atmosphere of 5%C02 and 95%air(v/v)at 37?,After synchronization,H9c2 cells were cultured in an anoxia chamber saturated with 92%N2,5%CO2 and 3%O2(v/v/v)at 37? for 0,6,12,24,36,48 to 72 hours respectively.The cells were digested and re-suspended into a density of 5×106 cell/mL.Every centrifuge tube containing 1mL cell suspension was treated with the device of SW.Two-hundred shocks with the energy of 0.02,0.05 or 0.10 mJ/mm2 were given to different tubes.After SW,the cells were cultured in the incubator with 5%CO2 and 95%air for another 24 hours before being harvested.The cell viability and intracellular ATP level were examined.Western blot analysis was used to assess the expression of LC3B,AMPK,mTOR,Beclin-1,Sirtl and HIF-la.Autophagic vacuoles were visualized by monodansylcadaverine staining.Results:1.The viability of H9c2 cells was detected by the MTS method after incubation in low-oxygen conditions for 0,6,12,24,and 36 h.It was found that with the increase in hypoxic time,the number of viable cells gradually decreased.At 12,24,and 36 h,the cellular viability decreased by 30.31%,34.90%,and 49.63%,respectively.Hypoxia significantly decreased the intracellular ATP levels in a time-dependent manner.ATP level was reduced by 19.26%in H9c2 cells after hypoxic incubation for 24 h.It was found that LC3B-?/? increased over time and that the ratio reached a peak at 24 h of hypoxia,whereas it exhibited a decreasing trend from 36 to 72 h.2.After incubation in a normal or hypoxic chamber for 24 h,H9c2 cells were digested,re-suspended,and prepared for SW treatment.The number of viable cells was significantly increased,especially those treated with an energy of 0.05 mJ/mm2 in the hypoxic group.However,none of the three energies of SW treatment affected cell viability in the NC group.Furthermore,the expression of LC3B-? was significantly increased following treated with 0.05 mJ/mm2 SWs after hypoxia(24 h),whereas no significant changes were found following SW treatment at the milder(0.02 mJ/mm2)or higher(0.10 mJ/mm2)energy.Similarly,there was no obvious autophagy level change in the NC group following SW treatment at different energies.3.To further confirm the occurrence of autophagy,we used MDC staining to visualize autophagic vacuoles in the H9c2 cells and found that the number of autophagic vacuoles significantly increased after hypoxia.Furthermore,the intensity was enhanced by SW treatment.The quantification of fluorescence was consistent with the results of LC3B-II expression determined by Western blot analysis.Simultaneously,intracellular ATP was detected before and after the SW treatment.It was found that the ATP level was significantly increased by SWs after hypoxia induction.4.The results showed that hypoxia significantly attenuated mTOR phosphorylation(Ser2448)but increased the phosphorylation of AMPK(Thr172)and the expression of Beclin-1 in H9c2 cells.The SW treatment further weakened mTOR phosphorylation and enhanced AMPK phosphorylation.However,SWs did not significantly influence the expression of Beclin-1.5.We found that the expression of Sirt-1 was markedly decreased during hypoxia but that SW treatment increased its expression.Conversely,the upregulation of HIF-la after hypoxia was significantly attenuated by SW treatment.Conclusions:1.After hypoxia,cardiomyocyte viability and ATP level were decreased,and autophagy was increased obviously in H9c2 cells.2.SW with an energy of 0.05 mJ/mm2 significantly increased the ratio of LC3B-II/I,autophagic vacuoles,cellular viability and ATP level.3.Ischemia/hypoxia induced cardiomyocyte death and function loss.The treatment of SW potentially promoted cell autophagy through AMPK/mTOR pathway and improved cell viability and function during hypoxia through Sirtland HIF-la signals.4.SW may serve as an effective alternative therapeutic option for the treatment of ischemic heart disease.