The Effect And Mechanisms Of TRPV1 On Autophagy In Hypoxic Mouse Cardiomyocytes

Background:The heart is a major target organ of hypoxic injury regardless of acute hypobaric hypoxia at high altitude,severe burn,traumatic hemorrhagic shock or other diseases.As the heart is a dynamic circulatory organ,severe heart hypoxic injury further reduces the oxygen supply to the body and aggravates other tissue and organ injury throughout the body.Therefore,exploring the mechanisms of myocardial injury under hypoxic stress helps to prevent and treat myocardial hypoxic diseases.Autophagy is a mechanism of survival and stress regulation widely existed in eukaryotic cells.Autophagy is a dynamic process,which involves the formation of double-membrane autophagosomes that engulf cytoplasmic materials and fuse with lysosomes,thus delivering the autophagosome into the lysosomal lumen for degradation.The dynamic process of all the above steps is called Autophagy flux.Autophagy promotes the degrading and recycling organelles and maintains cellular homeostasis by degrading long-lived and aging/damaged proteins,and plays an important role in the growth and development of various organisms and the development of various diseases.A large number of literatures have indicated that autophagy is closely related to myocardial hypoxia,heart failure and other cardiovascular diseases.Previous studies have reported that the fusion between autophagosomes and lysosomes was blocked in hypoxic cardiomyocytes or other cells,resulting in accumulation of autophagosomes.Then,a series of chain reactions takes place,such as impaired function of clearing damaged organelles,ROS aggregation,leading to cell necrosis,but its exact mechanism and role are still unclear.Previous studies have shown that many signaling pathways and cytokines participate in autophagy,Transient receptor potential vanilloid 1?TRPV1?is a nonselective cation channel that is widespread in cell and organelle membranes,and is activated by a variety of exogenous and endogenous physical and chemical stimuli.With the identification of TRPV1 localization in the left ventricle,the importance of this channel in the regulation of heart function has drawn much attention recently.Increased TRPV1 channel expression was observed in hypoxic hearts,exerting positive inotropic and positive chronotropic effects,thus protecting hypoxic cardiomyocytes.Most recently,activation of TRPV1 by capsaicin has been reported to induce autophagy in mouse thymocytes and hepatocytes,and this TRPV1-induced autophagy play a beneficial role to thymocytes and hepatocytes survival.Most of the TRP channels superfamily,including TRPV1 channel,are widespread on the lysosomal membrane,and participate in the regulation of lysosomal function and the maintenance of lysosomal homeostasis through the Ca²⁺signal.Adenosine 5'-monophosphate?AMP?-activated protein kinase?AMPK?a key molecule regulating bioenergy metabolism.AMPK signaling is closely related to autophagy and could be activated by the tumor suppressor factor hepatic kinase B1?LKB1?and Ca2+/calmodulin-dependent protein kinase-??CaMKK-??.TRPV1 is a nonselective cationic channel with good permeability to calcium ions.Whether TRPV1 induces autophagy in hypoxic cardiomyocyte by induced intracellular Ca²⁺influx to enhance function of lysosomal and phosphorylate of AMPK,promoting autophagic flux is not yet clear.Therefore,our study proposes a hypothesis that TRPV1 channel could improve the autophagic flux of cardiac myocytes to protect hypoxic cardiomyocytes and CPS-activated TRPV1 induces the AMPK signaling pathway by promoting Ca²⁺influx to activate autophagy under hypoxic stress.so as to provide a new theoretical basis for the prevention and treatment of myocardial hypoxic injury.Methods:1.The primary cardiomyocyte of C57BL/6 neonatal mice was used as the experimental object,The hypoxic environment was simulated in vitro to establish hypoxic cardiomyocytes model.we detected the changes in autophagy markers?LC3,Beclin-1,Atg5?by Western Blotting in cardiomyocytes under hypoxic stress for different time?0 h,3 h,6 h,9 h?.After blocking the autophagic flux of hypoxic cardiomyocytes with chloroquine,the activity of cardiomyocytes was detected by CCK8 assay and LDH assay.2.Expression of TRPV1 protein in primary mouse cardiomyocytes under hypoxia was detected by Western blotting and immunofluorescence.Capsaicin?CPS?and TRPV1 siRNA were used to promote or inhibit TRPV1 channel expression in cardiomyocytes respectively.Then,the expression of autophagy markers?LC3,Beclin-1,Atg5?in cardiomyocytes was detected by Western Blotting and the viability of cardiomyocytes was detected by CCK8assay and LDH assessment to determine the regulatory effect of TRPV1 on autophagy of hypoxic cardiomyocytes.3.To up-or down-regulation of TRPV1 expression by the above method.The expression of LAMP-1 was detected by Western blotting and the colocalization LAMP-1and LC3 was detected by immunofluorescence to clarify the regulatory effect of TRPV1 in promoting the fusion between autophagosome and lysosome to formation of autolysosomes.4.The expression of AMPK protein in primary mouse cardiomyocytes under hypoxic stress for different time?0 h,3 h,6 h,9 h?was determined by Western Blotting.Then,the expression of AMPK protein in primary mouse cardiomyocytes after regulation the expression of TRPV1 was determined by Western Blotting to explore the effect of TRPV1on the expression of AMPK protein.Next,A-179662?AMPK agonist?and Compound C?AMPK inhibitor?were used to regulate the expression of AMPK protein,and the autophagy marker proteins and LAMP-1 protein were detected to confirm the influence of AMPK signaling on autophagy of hypoxic cardiomyocytes.Next,we detected the expression of autophagy markers and LAMP-1 protein in cardiomyocytes after CPS activates TRPV1 protein expression while Compound C inhibits AMPK protein expression,Finally,we detected the change of Ca²⁺flow in cardiomyocytes after promoting TRPV1expression by CPS.and whether TRPV1 activates the autophagy level of cardiomyocytes through AMPK protein.These methods were used to determine whether TRPV1 activates autophagy in cardiomyocytes through AMPK signal pathway.Results:1.The ratio of LC3 II/LC3 I and the expression of Beclin-1,Atg5 protein were increased significantly in hypoxic mouse cardiomyocytes compared with the normoxic group.Chloroquine impaired autophagy flux of hypoxic cardiomyocytes and aggravated cell viability.It suggest that autophagosomes accumulate and autophagy flux is impaired in hypoxic cardiomyocytes.2.The expression of TRPV1 protein was increased in hypoxic cardiomyocytes.The ratio of LC3 II/LC3 I and the expression of Beclin-1,Atg5 protein in hypoxic cardiomyocytes were increased after inducing TRPV1 expression by CPS,while the viability of hypoxic cardiomyocytes was rescued after up-regulation of TRPV1 by CPS.After siRNA inhibits the expression of TRPV1 protein,the expression of autophagy markers were also decreased.3.The results of Western Blotting and immunofluorescence showed that the CPS significantly promoted the expression of LAMP-1 and the colocalization of LAMP-1 and LC3 in hypoxic cardiomyocytes.In contrast,transfection with TRPV1 siRNA combined with a hypoxic environment decreased the expression of LAMP-1 and the colocalization of LAMP-1 and LC3 in cardiomyocytes.These data indicated that activation of TRPV1protein could enhance the lysosomal activity and promote the fusion of autophagosomes and lysosomes in cardiomyocytes,promote autophagy-lysosomal pathways and improve autophagy activity of hypoxic cardiomyocytes.4.The expression of p-AMPK protein significantly increased in hypoxic cardiomyocytes compared with normoxic group.P-AMPK expression in hypoxic cardiomyocytes was further increased by CPS and decreased by TRPV1 siRNA,A-179662and Compound C were respectively used to stimulate or inhibit AMPK signaling in mouse cardiac myocytes.The two reagents were proved to be effective,as A-179662 increased and Compound C decreased the activation of AMPK signaling in hypoxic cardiomyocytes.In addition,the two reagents significantly promoted or inhibited the expression levels of LAMP-1,Beclin-1,Atg5 and the ratio of LC3 II/I in hypoxic cardiac myocytes.Moreover,we found that the expression levels of LAMP-1,Beclin-1,Atg5 and the ratio of LC3 II/I in hypoxic cardiomyocytes were remarkably increased by CPS,and this upregulation could be reversed by the inhibitor of AMPK signaling.Finally,we followed the CPS-induced Ca²⁺influx by monitoring intracellular cytosolic Ca²⁺fluorescence intensity using a Fluo-4AM stain.and the result showed that the Ca²⁺fluorescence intensity increased after 4 seconds or40 seconds of CPS addition in hypoxic group and normoxic group respectively,and peak of intracellular Ca²⁺fluorescence intensity rise in the hypoxic ischemic group were higher than those in the normal group.Conclusion:this study preliminarily verified that activation of TRPV1 protein could enhance the lysosomal activity and promote the fusion of autophagosomes and lysosomes in cardiomyocytes,promote autophagy-lysosomal pathways and improve autophagy activity of hypoxic cardiomyocytes to alleviate hypoxic cardiomyocytes injury.In addition,CPS-activated TRPV1 induces the AMPK signaling pathway by promoting Ca²⁺influx to activate autophagy under hypoxic stress.Therefore,this experiment further elaborated the role of autophagy in hypoxic cardiomyocytes and highlighted TRPV1 as a promising therapeutic target for hypoxic cardiomyopathy.