Role Of MicroRNA-34a And ATG9A On Myocardial Hypertrophy

BackgroundMyoardial hypertrophy refers to a thickening myocardium, resulting in adecrease in size of the heart chamber. A common cause of myocardial hypertrophy ishypertension or heart valve stenosis. At the cell level, myocardial hypertrophy isgenerally characterized by an increase in the size of cardiomyocytes, without increasein cell numbers, and by cytoskeletal reorganization. At the molecular level,myocardial hypertrophy shows an increased expression of fetal-type genes.Physiologically, myocardial hypertrophy is initially an adaptive response to stressoverload. However, the continued presence of hypertrophic growth often carries apoor prognosis that may result in heart failure and sudden death of patients.To date, appropriate therapy and prevention methods of myocardial hypertrophyprogression have had limited success because the pathophysiological mechanismsresponsible for myocardial hypertrophy development remain to be defined. To thisend, a previous study showed that myocardial hypertrophy induced bypressure-overload stress triggers cardiomyocyte autophagy. Moreover, cardiomyocyteexcessive autophagy may lead to cardiomyocyte death although physiological levelsof autophagy are essential in eukaryotic cells to eliminate damaged proteins andorganelles as part of the maintenance of cell homeostasis. This excessive or deficientautophagy may therefore contribute to disease pathogenesis. As the only integralmembrane ATG protein, ATG9A is localized in the phagophore/pre-autophagosomal structure (PAS) and is an essential protein in the autophagic process.In addition, microRNAs (miRNAs) are a class of endogenous non-coding smallRNAs and modulate gene expression at the post-transcriptional level by binding to theseed-matched sequence of the3’-UTR region in their target mRNAs, which results ineither degradation or translational repression of target gene expression. Alteredexpression of miRNAs has been associated with development of myocardialhypertrophy. Recent studies have further indicated that miRNAs also plays a role incardiac development and physiology. For example, miR-34a is a multifunctionalregulator, which is involved in cell division, senescence, apoptosis and proliferationthrough regulating the expression of its target genes. Using microarray profiling,Cheng at al. demonstrated that miR-34a was aberrantly expressed in hypertrophicmouse hearts. However, the molecular mechanism regulating myocardial hypertrophyby miR-34a has been poorly understood. Yang et al. elucidated that miR-34amodulated Caenorhabditis elegans lifespan via the repression of ATG9A-mediatedautophagic activities. As known to us, Angiotensin II (AngII) is a critical growthfactor and mediates myocardial hypertrophy, and its receptors can regulatecardiomyocyte autophagy.However, it is unknown whether and how these factors work together to regulatemyocardial hypertrophy, whether ATG9A mediated autophagic activity is excessivelyactivated in AngII induced cardiomyocyte hypertrophy, and whether miR-34a canmodulate AngII-induced cardiomyocyte hypertrophy by targeting ATG9A expression.Thus, we hypothesized that during development of myocardial hypertrophy, thedecreased expression of miR-34a could accelarete the expression of ATG9A, whichcan mediate autophagic activity.In this study, in order to clarify the relation of miR-34a, ATG9A and autophagicactivity, we first construct a rat model of myocardial hypertrophy and detect thealternation of miR-34a, ATG9A expression and autophagic activity. In addition, inhypertrophic myocardiocytes induced by Ang II, we certify whether miR-34a canregulate ATG9A expression and the change of ATG9A expression can promoteautphagic activity. Through those, we go furtherly to demonstrate that in the process of cardiomyocyte hypertrophy, miR-34a modulates AngII-induced myocardialhypertrophy by inhibition of ATG9A-mediated autophagic activity. We took this novelapproach to help better understand the molecular mechanisms of myocardialhypertrophy development in order to develop a prospective therapeutic target forcontrol of cardiac hypertrophy in the future.Part oneChanges of autophagic activity, ATG9A and miR-34aexpression in rat tissue of myocardial hypertropyObjctiveTo explore the alternation of autophagic activity, ATG9A and miR-34aexpression in cardiac tissue from rats of myocardial hypertrophy induced by pressureover-load.MethodAnimal model produced a well-established rat myocardial hypertrophy bytransverse abdominal aortic banding and evaluated by echocardiography,histopathological analysis of heart size, the ratio of heart weight to body weight(HWI), and cardiomyocyte size in hematoxylin and eosin (H&E)-stained heartcross-sections. Relative expression level of miR-34a and ATG9A was analyzed byReal-time PCR in cardiac tissue. Two established marker protein for autophagicactivity (p62and LC3II/I) was analyzed by Western blot in cardiac tissue. In addition,autophagic vacuoles were detected by transmission electron microscopy in cardiactissue.Result1. Four weeks after the surgery, left ventricular well thickness and abdominal aorta was detected by Echocardiography. The data showed that the left ventricularend-diastolic and end-systolic dimension (LVIDd and LVIDs) and left ventricularposterior wall end-diastolic and end-systolic thickness (LVPWd and LVPWs) in ratsfrom the TAAC group were significantly higher than those in the Sham group.Spectral Doppler demonstrated that Filling defects of bloodstream, blood flowvelocity and pressure gradient increase where abdominal aortic constriction wasperformed.2. The whole heart tissue section was prepared by H&E staining. The wholeheart sections, cut at the papillary muscle level, showed that the heart volume wassignificantly expanded, while the papillary muscles and trabeculae carneae cordiswere much coarser in appearance in the TAAC group than in the Sham group.3. H&E stained tissue sections from the TAAC group displayed a lightly stainedcolor in the regions of myocardial hypertrophy with inhomogeneous staining andnumerous nuclear-free regions with an increased nucleolar density in regions ofmuscle fiber atrophy. Cardiac muscle fiber surface area measured by Image Pro Plussoft increased markedly in the TAAC group than in the Sham group.4. Compared to the Sham group of rats, the TAAC group showed up-regulationof ATG9A expression in cardiac tissue.5. Compared with those in the sham operation group, autophagic activity markerprotein of LC3II/I expression and autophagic vacuoles was enhanced in rats from theoperation group, but the p62protein expression was decreased.6. miR-34a expression was significantly down-regulated in the TAAC group ofrats compared to that of the Sham group.Brief summary1. The expression of autophagy-related protein9A and autophagic activity wereenhanced in rat tissue of cardiac hypertrophy.2. miR-34a expression was significantly down-regulated in rat hypertrophichearts. Part twoThe role of altered ATG9A and autophagic activity onAngII-induced myocardial hypertrophyObjectiveTo clarify the relation of ATG9A and autophagic activity, and the effect ofwhich had on AngII-induced myocardial hypertrophy.MethodAn in vitro cardiomyocytes hypertrophic medol was establised by teating with1μmol/L AngII in cardiomyocytes. The influence of RNA interference orover-expression of ATG9A gene on the autophagic activity was assessed. Incardiomyocytes, relative protein expression level of p62and LC3II/I detected bywestern blot, ratio of autophagic vacuoles analyzed by flow cytometry, the number ofautophagic vacuoles observed by transmission electron microscopy were evaluated onautophagic activity. In addition, cardiomyocytes hypertrophy was evaluated byqRT-PCR for the expression of hypertrophy-related gene (ANP and β-MHC) andconfocal microscopic detection of cardiomyocytes morphology.Result1. Compared to the control cardiomyocytes, AngII-treated cardiomyocytesshowed markedly increased expression of hypertrophy-related genes and cell area.2. Compared to the control cardiomyocytes, AngII-treated cardiomyocytesshowed markedly increased expression of autphagic activity marker protein LC3II/I,but decreased expression of the other autophagic activity marker protein p62.3. Compared with the controls, both the percentage of autophagic vacuoles andthe number of autophagic vacuoles were up-regulated in neonatal cardiomyocytesstimulated with AngII. 4. Compared with the control-vetor group, over-expression of ATG9A-treatedgroup demonstrated that the expression of autophagic activity marker protein LC3II/Iwas significantly enhanced, but the p62expression was markedly lowered.5. Compared to the negative control group, the expression of ATG9A and LC3II/Iwas up-regulated in neonatal cardiomyocytes stimulated with AngII plus negativecotrol, while the expression of p62was down-regulated.6. Compared to AngII plus negative group, the expression of ATG9A and LC3II/Iwas decreased in neonatal cardiomyocytes stimulated with AngII plus ATG9A-specificshRNA, but the p62expression was increased.7. Compared with the control-vector group, both the percentage of autophagicvacuoles and the number of autophagic activity were up-regulated in neonatalcardiomyocytes stimulated with overexpressed-ATG9A vector.8. Compared with the negative control group, autophagic vacuoles number andratio were increased in neonatal cardiomyocytes teated with AngII plus negativecontrol.9. Compare to AngII plus negative control group, cardiomyocytes treated withAngII plus ATG9A-specific shRNA showed that markedly decreased ratio ofautophagic vacuoles and the number of autophagic vacuoles.10. Compared with control-vector, cardiomyocytes treated with theoverexpressed-vector of ATG9A confirmed that significantly increased the expressionof hypertrophy-related gene and cell area.11. Compared with negative control, cardiomyocytes stimulated with AngII plusnegative control showed that increased both the hypertrophy-related gene expressionand cell area.12. Compared with AngII plus negative control, the expression ofhypertrophy-related gene and the cell area were lowered in neonatal cardiomyocytestreated with ATG9A-specific shRNA. Brief summary1. Myocardial hypertrophy, autophagic activity and the expression of ATG9Awere induced in cardiomyocytes stimulated with1μmol/L AngII.2. Overexpression of ATG9A could enhance myocardial hypertrophy andautophagic activity in cardiomyocytes.3. Specific-ATG9A shRNA in cardiomyocytes could mitigate myocardialhypertrophy and autophagic avtivity induced by AngII-treated cardiomyocytes.Part threeThe effect of miR-34a on ATG9A and its potential role inAngII-induced myocardial hypertrophyObjectiveTo explore whether miR-34a can modulate AngII-induced myocardialhypertrophy by binding to the3’UTR of ATG9A mRNA, which can alter autophagicactivity in cardiomyocytes.MethodAfter cardiomyocytes were cotransfected with Luciferase reporter vectorcontaining ATG9A3’UTR and internal control vector, miR-34a mimics or miR-34ainhibitors were transduced respectively, then performed the luciferase assay. Inaddition, after miR-34a expression was over-expressed or inhibited, the expression ofATG9A was analyzed by qRT-PCR and Western blot. After cardiomyocytes treatedwith AngII, the expression of miR-34a and hypertrophy-related gene was detected byqRT-PCR, while the alternation of cell morphology was observed by confocalmicroscopy. Finally, after over-expressed or inhibited miR-34a in cardiomyocytes, thechanges of hypertrophy-related gene expression and autophagic activity wereobserved. Result1. Compared to cells treated with a negative control, after cardiomyocytes weretransfected with pGL-3-ATG9A3’-UTR-Wild Type, transduction with miR-34amimics resulted in a45%reduction in the relative luciferase activity. In contrast,transduction with miR-34a inhibitors resulted in a1.38-fold increase in the relativeluciferase activity, compared to cells treated with a negative control.2. Compared to cells treated with a negative control, after in cardiomyocyteswere transfected with pGL-3-ATG9A3’-UTR-Mutant Type, neither transdution withmiR-34a mimics nor transduction with miR-34a inhibitors can alter luciferase activity.3. Compared with the negative control, after in cardiomyocytes were transducedwith miR-34a mimics or miR-34a inhibitors, the expression of ATG9A mRNA was notdifferent, while the ATG9A protein expression was down-regulated by0.48fold orup-regulated by0.7fold, respectively.4. Compared to cardiomyocytes treated with negative control, ratio of autophagicvacuoles was increased by11%in cells treated with AngII plus negative control.5. The percentage of autophagic vacuoles was markedly decreased incardiomyocytes treated with AngII plus miR-34a mimics, compared with AngII plusnegative control.6. Compared to cardiomyocytes treated with negative control, the number ofautophagic vacuoles was increased in cells treated with AngII plus negative control(0.9±0.1vs2.4±0.31，P<0.05).7. The percentage of autophagic vacuoles was markedly lowered incardiomyocytes treated with AngII plus miR-34a mimics, compared with AngII plusnegative control (2.4±0.31vs1.62±0.4，P<0.05).8. Compared to negative control, cardiomyocytes treated with AngII plusnegative control showed that markedly increased the expression of ANP and β-MHCand cell area.9. Compared to AngII plus negative control, the expression of ANP and β-MHCand cell area were mitigated in cardiomyocytes stimulated with AngII plus miR-34a mimics, whereas those was aggravated in cardiomyocytes treated with AngII plusmiR-34a inhibitors.Brief summary1. miR-34a can bind to ATG9A mRNA3’UTR, and regulate its proteinexpression.2. miR-34a was down-regulated in AngII-induced myocardial hypertrophy.3. Up-regulation of miR-34a can inhibit autophagic activity induced by AngII incardiomyocytes.4. miR-34a can modulate AngII-mediated myocardial hypertrophy. ConclusionThe present study shows that miR-34a can modulate AngII-inducedcardiomyocytes hypertrophy. This effect of regulation is implemented by directinhibition of ATG9A expression and autophagic activity.