The Effect And Mechanisms Of Autophagy In Rat Cardiomyocyte Injury Induced By PH Downregualtion

BackgroundDue to burn shock/ischemia-hypoxia coursed by the loss of body fluid,stress,infection and other factors,severe burn will cause the accumulation of acidic metabolites such as lactic acid and keto acid,leading to acidosis in the body.In clinical diagnosis,the pH of arterial blood is one of the most intuitive indicators of acidosis.The decompensation of acid-base buffer system will cause a pH decline in arterial blood in varying degrees.Heart is an important circulating organ of the body.Previous studies have shown that cardiomyocytes are sensitive to the changes of extracellular pH,and a decrease in the pH of arterial blood can cause severe damage to heart function and increase the mortality rate of burn patients.Therefore,it has great significance to reserch the myocardial damage induced by acidosis and explore its possible mechanism.Autophagy is an important physiological process for cells to maintain metabolic balance and normal function.It provides necessary nutrients and substrates for cells by decomposing damaged organelles,misfolded proteins and the like.A large number of literatures indicate that autophagy plays an important role in physiological or pathological conditions.In hypoxic conditions,the damage of autophagy flux will cause a decrease of cardiomyocyte viability in rats,and the activation of autophagy induced by starvation can compensate the lack of nutrients in some degree and maintain the survival of cells.Our previous study found that acidic treatment can inhibit autophagosome generation.However,how autophagy participates in and regulates the pH downregulation-induced cardiomyocyte damage is not yet clear.Studies have shown that multiple signaling pathways are involved in the regulation of autophagy.Microtubules(MTs)are reticular frameworks in mammalian cells and play an important role in maintaining cell morphology,regulating intracellular transport,and cell migration.The performance of the normal function of microtubules requires the participation of various post-translational modifications,such as acetylation,methylation,glycosylation,etc.Microtubule acetylation is one of the most important modification methods.Previous studies have shown that the acetylation of microtubules is closely related to autophagy,and the absence of acetylation can hinder the degradation of autophagosomes.Therefore,we speculate that when the pH is lowered,acetylation of microtubules may be involved in the regulation of autophagy in cardiomyocytes.Histone deacetylase 6(HDAC6)is an important deacetylase that exists in the cytoplasm and plays a regulatory role in a variety of physiological or pathological processes.In mammalian cells,HDAC6 is an important upstream regulator of the acetylation of microtubules.However,whether HDAC6 participates in the regulation of microtubule acetylation induecd by pH decline in cardiomyocytes has not been reported.Therefore,our study proposes the hypothesis that pH decline regulates cardiomyocyte autophagy through microtubule acetylation which leads to cardiomyocyte injury.To prove this hypothesis an acidic treatment model in vitro was established to examine the effect of autophagy regulation on the acidic damage of cardiomyocytes when pH is lowered.And the detection and regulation of HDAC6 and microtubule acetylation were performed to elucidate the mechanism of autophagy induced by pH deline in cardiomyocytes.Methods1.Primary cultured neonatal rat cardiomyocytes were used as the research object,an acidic treatment model of rat cardiomyocytes in vitro by formulating acidic DMEM/F12 medium was established to observe the effects of different acidic pH(pH7.0,pH6.5,pH6.0)and different treatment times(1,3,and 6h)on autophagy and cardiomyocyte damage.The working point of declined pH on autophagy regulation of cardiomyocytes was confirmed by the inhibition test of Baf A1(100 nM).2.We construct ATG5 over-expression adenovirus to up-regulate the autophagy level of cardiomyocytes under low pH condition,and the effectiveness of adenovirus transfection was detected by Western Blot(WB).Lactate dehydrogenase(LDH)release and cell counting kit 8(cck-8)were used to observe the cardiomyocyte injury and viability to confirm the regulatory effect of autophagy on the cardiomyocyte damage caused by the decrease of pH.3.WB and Immunofluorescence(IF)assays were used to detect changes in acetylation levels of microtubules in cardiomyocytes after acidic treatment,and the cardiomyocytes were treated with Taxol(0.2?M)to upregulate the acetylation level of microtubules.The changes in the autophagy level and the damage of cardiomyocytes were observed to clarify the regulatory effect on the autophagy of cardiomyocytes by acetylation of microtubules under declined pH.4.The protein expression and activity of HDAC6 were detected by WB and HDAC activity kits when the pH was lowered.After selectively inhibition of the HDAC6 activity by Tubastatin A(TBSA,0.2?M),we detected the changes in the levels of microtubule acetylation and autophagy by WB and IF,and the damage of cardiomyocytes by LDH release and cck8 to confirm the regulatory effects of HDAC6 on microtubule acetylation and autophagy induced by pH downregulation.Results1.pH downregulation caused the damage of rat cardiomyocytes.At the same time,the expression of autophagy markers LC3 and p62 decreased significantly.Through Baf A1 inhibition experiments,the expression of LC3 II in the acidic treatment group was significantly reduced compared with the normal group after inhibiting the fusion of autophagosomes with lysosomes by Baf A1.It indicates that the reduction of the autophagosomes is due to the inhibition of the autophagosome generation rather than the excessive consumption of autophagosomes in cardiomyocytes.2.The ATG5 over-expression adenovirus was transfected into cardiomyocytes and its autophagy level was up-regulated under acidic treatment conditions.Through the LDH release and cck8 assays,we found that the up-regulation of autophagy level significantly reduced acidic damage and improved viability of cardiomyocytes,indicating that autophagy has a regulatory effect on the damage caused by pH downregulation in cardiomyocytes.3.The results of WB and IF showed that acidic treatment significantly down-regulated the acetylation of microtubules in cardiomyocytes.We treated the cardiomyocytes with Taxol(0.2?M),a microtubule stabilizer,to up-regulate the acetylation levels of microtubules and detected the level of autophagy after acidic treatment.Compared with the acidic treatment group,it was found that there was a significant increase in autophagy level in acidic treatment+Taxol group,at the same time,the damage of cardiomyocytes was significantly mitigated and the viability was improved,indicating that when the pH is decreased,the acetylation of microtubules is involved in the regulation of autophagy in cardiomyocytes.4.After acidic treatment,there was no significant change of the level of HDAC6 protein expression,but its activity of HDAC6 was remarkably increased.When Tubastatin A was used to selectively inhibit HDAC6 activity under acidic treatment conditions,the acetylation of microtubules in cardiomyocytes was significantly up-regulated.At the same time,through the detection of autophagy markers LC3 and p62 and IF experiment,the level of autophagy was also significantly rised.It indicates that HDAC6 plays an important regulatory role in the pH decline-induced downregulation of microtubule acetylation.Finally,by examining the changes in cardiomyocyte injury,it was found that the selective inhibition of HDAC6 activity reduced acidic damage and improve cell viability.ConclusionsWhen pH declined,the increase of HDAC6 activity down-regulates the acetylation level of microtubules,which resulting in dyspoiesis of autophagosomes generation,and the decreased viability and damage of cardiomyocytes.The results above suggest that autophagy is involved in the regulation of cardiomyocyte damage induced by pH downregulation,and that the regulation of microtubule acetylation mediated by HDAC6 is an important regulatory mechanism.This conclusion further enriches the theoretical basis of the cardiomyocyte damage after burns,and is helpful for us to explore new targets for the prevention and treatment of cardiomyocyte damage in burned patients.