The Impairment Of TRPV1 Regulating Mitochondria-Associated ER Membranes Formation In The Cardiac Hypertrophy

Background:Myocardial hypertrophy is the common and important pathological basis for hypertension,myocardial infarction,heart valvular disease,cardiomyopathy and other cardiovascular diseases to develop into heart failure.At the same time,it also increases the risk of arrhythmia,cardiac sudden death and other cardiovascular events.Therefore,preventing and delaying the occurrence and development of myocardial hypertrophy is the fundamental strategy for the treatment of heart failure.Due to the process of cardiac contraction requires a relatively high level of energy,the myocardial cells contain a large number of mitochondria.myocardial mitochondrial dysfunction affects the production of ATP,and causes oxidative stress and apoptosis,promoting the development of myocardial hypertrophy.However,the mechanism of myocardial mitochondrial dysfunction caused by pressure overload is unclear.The appropriate mitochondrial Ca²⁺levels maintain normal oxidative respiration and ATP production by regulating the activity of rate-limiting enzymes in the tricarboxylic acid cycle and ATP synthase,while disruption of mitochondrial Ca²⁺homeostasis leads to increased oxidative stress and inflammation and apoptosis.Mitochondria contacts with endoplasmic reticulum（ER）through mitochondria-associated endoplasmic reticulum membranes（MAMs）to maintain mitochondrial Ca²⁺homeostasis.However,whether the abnormal formation of MAMs is involved in the myocardial hypertrophic mitochondrial dysfunction remains unclear.Transient receptor potential vanilloid type 1（TRPV1）is a non-selective cationic channel protein that plays an important role in maintaining Ca²⁺homeostasis and mitochondrial function.Previous studies have shown that the activation of TRPV1 can effectively reduce the myocardial hypertrophy induced by pressure overload,but the specific mechanism is unclear.We speculated that TRPV1 may protect mitochondrial function by regulating the formation of MAMs,thus protecting myocardial hypertrophy.Objectives:1.To clarify the role of TRPV1 in myocardial hypertrophy.2.To study the role of TRPV1in regulating MAMs formation and maintaining mitochondrial function in myocardial hypertrophy.3.To elucidate the specific mechanism of TRPV1 regulating myocardial MAMs formation.Methods:1.Expression changes of TRPV1 in myocardial hypertrophy were detected.Western blotting,immunohistochemistry and real-time quantitative PCR were used to investigate the effects of phenylephrine（PE）-induced cardiomyocyte hypertrophy and transverse aortic constriction（TAC）-induced cardiac hypertrophy.The expression of TRPV1 protein and m RNA in myocardial hypertrophy model was detected.2.The effect of TRPV1 activation on myocardial hypertrophy was detected.In this study,male C57BL/6J mice aged 6-8 weeks（18-22g）were randomly divided into 4 groups:normal standard diet group,capsaicin diet group（0.01 g capsaicin per 100 g food）,TAC group and capsaicin diet+TAC group.At 8 weeks after TAC,cardiac function indexes of mice were detected by using animal ultrasonic instrument.Ejection fraction（EF）%,fractional shortening（FS）%,left ventricular internal diameter in end-diastole（LVIDd）,left Ventricular internal diameter in end-systole（LVIDs）.To observe the changes of the general shape of the heart,measure the weight of the heart and detect the pathological changes of the heart.In vitro,cardiomyocyte hypertrophy was induced by PE and TRPV1 was activated by capsaicin.To observe the effect of capsaicin on the area of cardiomyocytes,Meanwhile,m RNA and protein expression of atrial Natriuretic Peptide（ANP）、B-type Natriuretic Peptide（BNP）andβ-Myosin Heavy Chain（β-MHC）are detected.3.The effects of TRPV1 activation on mitochondria and MAMs were detected.The effects of TRPV1 activation on mitochondrial structure and function were observed by transmission electron microscopy,mitochondrial membrane potential and ATP production detection.Transmission electron microscopy,laser confocal microscopy and immunoprecipitation were used to analyze the regulation of TRPV1 on MAMs.In order to determine whether the protective effect of activation of TRPV1 on mitochondria through MAMs,the formation of MAMs was disrupted by transfection of MFN2 si RNA,and then the mitochondrial structure and function were detected to observe the changes in mitochondrial function.4.To explore the mechanism of TRPV1 regulating MAMs.To further investigate whether TRPV1 regulates MAMs through AMPK/MFN2 axis,the effect of AMPK inhibitor CC on MAMs was observed after activation of TRPV1 by capsaicin,and the expression of MFN2protein and the co-location of mitochondria and endoplasmic reticulum were detected.Results:1.Compared with the control group,protein and m RNA expression of TRPV1 were upregulated in both PE-induced hypertrophic myocardial cell model and TAC-induced hypertrophic myocardial tissue model.2.In vivo,capsaicin diet+TAC group showed better cardiac function compared with TAC group and lower heart/body weight ratio,smaller cell area.In vitro,the TRPV1 activation group showed a decrease in cell area compared with the PE group,and down-regulated the protein and m RNA expressions of ANP,BNP andβ-MHC genes.3.Primary cardiomyocytes treated with PE showed mitochondrial outer membrane rupture and crest dissolution,and the activation of TRPV1 by capsaicin could better protect mitochondrial structure.Compared with PE group,capsaicin-treated group showed better mitochondrial function.We also observed increased MAMs formation after TRPV1 activation.After transfection with si MFN2,the protective effect of TRPV1 activation on mitochondria decreased,the area of cardiomyocytes increased.These results suggest that the protective effect of TRPV1 activation on mitochondria is exerted by MAMs.4.TRPV1 activation increased the expression of MFN2 and promoted MAMs formation,and the effect was blocked by AMPK inhibitor compound C（CC）.Conclusions:our data described the role of TRPV1 in alleviating myocardial hypertrophy damage via regulating MAMs formation by affecting AMPK/MFN2 pathways,providing beneficial influence on mitochondrial homeostasis.Because TRPV1 is a key molecule in cardiovascular diseases,the domain and functional region of the protein may contribute to the development of new drugs for cardiovascular diseases.