The Mechanism Whereby MiR-29a Targets PPAR? In Cardiac Hypertrophy Induced By Isoproterenol

BackgroundCardiac hypertrophy is the adaptive response of heart to external stimuli.It manifests as an increase in the size of myocardial cells,an increase in the synthesis of contractile proteins,and reactivation of the embryonic development marker gene atrial natriuretic peptide(ANF).Although cardiac hypertrophy can initially compensate for the heart demand in face of cardiac overload,long-term increase in the size of myocardial cells often leads to cell death and the proliferation of myocardial interstitial cells,such as fibroblasts.As a result,myocardial structure is in disorder and cardiac contractility is decreased,which will consequently lead to heart dysfunction,heart failure and sudden death.Therefore,cardiac hypertrophy is an independent risk factor for many cardiovascular diseases.However,neither method to cure cardiac hypertrophy nor effective means of prevention or treatment have been found.There is still an urgent need for better clinically therapeutic drugs.Therefore,it is an important subject in the field of cardiovascular biology to explore the molecular mechanism of cardiac hypertrophy in order to develop safe and effective molecular drugs against cardiac hypertrophy in the future.Myocardial energy metabolism disorder is also a key factor in inducing cardiac hypertrophy,which is actually caused by the expression disorder of key genes involved in the regulation of cardiac energy metabolism.As one of the key genes in myocardial energy metabolism,the nuclear receptor peroxisome proliferator-activated receptor ?(PPAR?)plays an important role in the regulation of myocardial energy metabolism.Therefore,the study about the expression and regulation of PPAR? will be conducive for us to deeply explore the molecular mechanisms of cardiac hypertrophy.According to the miRNA target prediction tools Target Scan and PicTar,we found that PPAR? was the target protein of miR-29 a.In addition,other studies have shown that miR-29 a was highly expressed in mouse embryonic hearts and played an important role in ventricular remodeling.In view of this,we infered that miR-29 a played an important role in the development of cardiac hypertrophy,which was closely related to its target protein PPAR?.Based on the above analysis,we carried out our research work.ObjectiveIn this study,we aim to explore the mechanism of miR-29 a in the development of cardiac hypertrophy and demonstrate whether this effect is achieved by targeting PPAR?.Our research will elucidate the molecular mechanism and pathological basis of cardiac hypertrophy at the molecular level,and provide more clues for exploring ideal drugs against cardiac hypertrophy.Methods1.Hearts from 60-day-old mice were infused with isoproterenol and/or miR-29 a for 14 days.Echocardiography were used to test fractional shortening(FS),left ventricular mass(LV mass),diastolic left ventricular thickness(LVPWd),end-diastolic left ventricular internal diameter(LVIDd)and end-systolic left ventricular internal diameter(LVIDs).2.Immunohistochemistry analysis were used to analyze the levels of ANF,GATA-4 and PPAR?.3.Real-time PCR analysis were used to analyze the transcript levels of miR-29 a,U6,S16,ANF,BNF,GATA4,PPAR?,GAPDH in the ventricles of mice or neonatal cardiomyocytes.4.Luciferase reporter gene were used to analyze the match between the seed region of miR-29 a and the 3'UTR of PPAR? and the activation of the ANF promoter.Results1.mi R-29 a attenuates the isoproterenol-induced cardiac hypertrophic response.1.1 Gross morphometric and histology analysis results showed that miR-29 a agomir could reverse cardiac morphological changes induced by isoproterenol.1.2 Echocardiography results showed that mi R-29 a efficiently protected cardiac function from isoproterenol-induced dysfunction.1.3 Real-time PCR and immunohistochemistry analysis results revealed that miR-29 a could down-regulate the expression of genes related to cardiac hypertrophy.2.mi R-29 a can maintain cardiac function and down-regulate ANF expression.2.1 Gross morphometric and histology analysis results showed that miR-29 a could maintain the proper heart structure.2.2 Echocardiography results showed that normal heart function was maintained by miR-29 a.2.3 Real-time PCR and immunohistochemistry analysis results revealed that miR-29 a could down-regulate ANF expression.3.mi R-29 a targets PPAR? to inhibit ANF expression.3.1 Real-time PCR and immunohistochemistry analysis results revealed that miR-29 a could down-regulate PPAR? expression.3.2 Luciferase analysis showed that there was a perfect match between theseed region of miR-29 a and the 3'UTR of PPAR?,and then the activation of the PPAR? 3'-UTR-Luc was reduced.3.3 Luciferase analysis showed that the activation of the ANF promoter was activated by PPAR? in a dose-dependent manner.ConclusionmiR-29 a can attenuate isoproterenol(ISO)-induced cardiac hypertrophy,because it can bind to the 3' UTR of PPAR? mRNA and thereby down-regulate the expression of PPAR?,which eventually decreases the expression of cardiac hypertrophy marker gene ANF and attenuates isoproterenol(ISO)-induced cardiac hypertrophy.