Effects And Mechanisms Of Maslinic Acid On The Progress Of Cardiac Hypertrophy Via METTL3-mediated M~6A Methylation Modification

Objective:Cardiac Hypertrophy(CH)is a common cardiac physiological compensatory response to a variety of stressors to maintain its normal functions.However,cardiac enlargement due to myocardial injury,hypertensive stress,or excessive neurohumoral activation is associated with poor adaptive remodeling and cardiac dysfunction,and is classified as pathological hypertrophy.Pathological cardiac hypertrophy is a major risk factor for cardiomyopathy,heart failure,and sudden cardiac death.Although the understanding of the pathological regulators of cardiac hypertrophy has been improved,the molecular mechanism of cardiac hypertrophy is still unclear,and more effective therapies and intervention targets are urgently needed.Maslinic acid(MA)is a pentacyclic triterpenoid rich in olive pericarp and has a wide range of pharmacological properties.It has been reported that MA can play roles as a hypoglycemic agent,an oxidative damage inhibitor,and an inducer of human colon cancer cell apoptosis.MA inactivates the nuclear factor Kappa-B(NF-κB),which has anti-inflammatory and anti-arthritis effects.MA reduces the expression of hypoxia-inducible factor-1(HIF-1)in lung cancer cells and also provides prevention against many kinds of cardiovascular diseases.MA protects mice from diabetes-related cardiac injury by its anti-glucose and anti-coagulant activity.MA has antihyperlipidemic potential in rats,which inhibits isoproterenol-induced cardiotoxicity.However,the effect of MA on stress load-induced cardiac hypertrophy and its specific regulatory mechanism remains unclear.N6-methyladenosine(m~6A)is the most common post-transcriptional modification of m RNA in mammals.Recent studies have shown that it is important for the regulation of a variety of biological processes,such as embryo development,cell differentiation,regeneration,and tumorigenesis.However,there are still few studies related to m~6A in the cardiovascular field.It has been reported that the overall level of m~6A is increased in myocardial infarction and ischemia-reperfusion injury,thereby the decrease of m~6A can lead to autophagy flow enhancement and cardiac function improvement.However,how m~6A modifications affect cardiac functions and which underlying mechanisms mediating these changes are still unclear.The object of this study is to explore the role and mechanism of m~6A in inhibiting cardiac hypertrophy from the perspective of m~6A methylation modification,based on in vitro and in vivo experiments.Methods:The cytotoxicity of MA was evaluated in vitro using H9c2 cells and neonatal mouse cardiomyocytes.Cardiac hypertrophy was induced by stimulating neonatal mouse cardiomyocytes with type II angiotensin.In vivo,a mouse model of stress-induced cardiac hypertrophy was established using transverse aortic constriction(TAC).Cardiac ultrasound,H&E,WGA,and Masson staining were used to evaluate cardiac function and cardiac hypertrophy phenotype for transverse aortic constriction(TAC).Real-time PCR and Western Blot were used to detect the genes associated with hypertrophy(ANP,BNP,and b-MHC),fibrosis(Fibronectin Collagen I and a-SMA),and key enzymes for m~6A methylation modification(METTL3,METTL 14,WTAP,RBM15,VIRMA,FTO,and ALKBH5).The levels of m~6A RNA in myocardial cells and tissues were detected by chemiluminescence assay kit and Dot Blot.Finally,METTL3 overexpression was performed on the myocardium by adenovirus vector to verify the key regulatory role of METTL3-mediated m~6A in MA’s anti-cardiac hypertrophy.Results:In this study,we first found that the activity of H9c2 cells and Suckling mouse cardiomyocytes were not affected in vitro at a dose of MA less than 10~4μg/ml.However,Ang II-induced cardiomyocyte hypertrophy in Suckling mice was significantly inhibited at 10-10~3μg/ml of MA.In vivo,we found that 30 mg/kg injection dose significantly inhibited TAC-induced myocardial injury,cardiac hypertrophy phenotype and hypertrophy related gene expression,myocardial fibrosis phenotype,and fibrosis-related gene expression.Subsequently,we found that MA significantly inhibited the total level of m~6A RNA in cardiomyocytes,and after screening and verification,MA significantly inhibited the expression of METTL3 in cardiomyocytes.Finally,through adenovirus-mediated myocardial METTL3overexpression,we clarified that the inhibition of cardiac hypertrophy by MA required the involvement of METTL3-mediated m~6A.Conclusion:This study found that MA has a significant anti-cardiac hypertrophy effect,and its mechanism shows that the anti-cardiac hypertrophy effect of MA depends on the regulation of mettl3-mediated m~6A methylation modification.The findings of the present study provide a new target and strategy for the treatment of cardiac hypertrophy.