MicroRNA And Autophagy Regulation Of Myocardial Remodeling

BACKGROUNDHeart failure is the ultimate outcome of various cardiovascular diseases and is a leading cause of morbidity and mortality worldwide. Although drugs and other therapies have been developed for the management of heart failure, its five-year mortality rate remains high. The process of heart failure involves the dysregulation of many coding and non-coding genes; however, not all of these genes have been well characterized. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that post-transcriptionally regulate the degradation and/or translation of their target genes. A large body of evidence indicates that miRNA-mediated gene regulation plays important roles in the control of cardiac homeostasis and pathological remodeling. We previously found that miR-221is significantly up-regulated in patients with hypertrophic cardiomyopathy and in a mouse model of cardiac hypertrophy and heart failure induced by pressure overload. The in vitro overexpression of miR-221alone is sufficient to increase the size of cardiomyocyte. However, the in vivo roles and molecular mechanisms of miR-221in the regulation of cardiac remodeling remain unclear.METHODS AND RESULTSTo investigate whether miR-221regulates cardiac remodeling in vivo, we generated transgenic mice (Tg-miR-221) with cardiac-specific overexpression of miR-221. Compared with their non-transgenic (NTG) littermates, the hearts of the Tg-miR-221mice were significantly enlarged at4weeks of age. The heart-to-body weight ratios and the expression levels of ANP and BNP were significantly higher in Tg-miR-221mice than in their NTG littermates. Increased interstitial fibrosis and apoptosis were observed in the myocardia from Tg-miR-221mice by histological examination. Cardiac function was evaluated by using high-resolution echocardiography at4and16weeks of age. Compared with their age-matched NTG controls, Tg-miR-221mice exhibited a progressive thickening of the end-diastolic left ventricular posterior wall, an increased internal dimension of the left ventricle and left ventricular mass/body weight ratio, and decreased fractional shortening. Taken together, these results indicated that the overexpression of miR-221interrupted cardiac homeostasis and induced cardiac dysfunction and heart failure in mice.We further analyzed the ultrastructure of the myocardia by using transmission electron microscopy. In cardiomyocytes from Tg-miR-221mice, the mitochondria were disorganized and dispersed, with degraded cristae. In addition, a large amount of vacuoles with low electron densities were found in the cardiomyocytes from Tg-miR-221mice. The lipidated form of microtubule-associated protein1light chain3(LC3-Ⅱ), a marker of autophagy, were dramatically decreased in Tg-miR-221hearts. Consistently, the levels of sequestosome1(p62), an indicator of cytosolic protein clearance, were significantly increased. H9c2cells were forced to overexpress miR-221by the transient transfection of miR-221mimics. An autophagy-reporter plasmid encoding the EGFP-LC3recombinant protein was also co-transfected into H9c2cells, and the formation of autophagosomes was monitored based on the appearance of EGFP-LC3puncta. H9c2cells overexpressing miR-221had significantly lower levels of punctate EGFP-LC3compared with those of the control cells, indicating that autophagy was directly inhibited by miR-221.As the mammalian target of rapamycin (mTOR) is a well-known negative regulator of autophagy. We first measured the activity of mTOR by detecting the phosphorylation levels of mTOR and its substrates. At4weeks of age, the mTOR signaling pathway was significantly activated in’transgenic hearts compared with those in the NTG controls. Likewise, overexpression of miR-221in cultured neonatal rat cardiomyocytes resulted in the hyper-activation of mTOR. To assess whether the cardiac remodeling induced by miR-221overexpression is dependent on the inhibition of autophagy, we used rapamycin to inhibit the activity of mTOR and re-activate autophagy in cultured neonatal rat cardiomyocytes through the transfection of miR-221mimics. Rapamycin (20nM) significantly antagonized the hypertrophic effect of miR-221overexpression. Our results indicated that the inhibition of autophagy induced by miR-221overexpression was dependent on mTOR activation and was necessary for miR-221-induced pathogenic cardiac remodeling.Previously, we determined that p27was a direct target of miR-221in cardiomyocytes in vitro. Consistently, in the present study, we observed that the in vivo expression levels of p27were significantly down-regulated by the transgenic overexpression of miR-221in mouse heart. To determine whether the suppression of p27was responsible for the inhibition of autophagy induced by miR-221overexpression, we knocked down p27by using two different siRNAs in H9c2cells. As expected, p27knockdown showed similar effects of miR-221overexpression on autophagy and mTOR pathway. P27is a cyclin dependent kinase (CDK) inhibitor that interacts with CDK2to inhibit the cell cycle. We employed SU9516, a selective CDK2inhibitor, to suppress CDK2activity. In neonatal rat cardiomyocytes overexpressing miR-221, SU9516treatment significantly activated autophagy and attenuated cardiac hypertrophy. Thus, our results indicated that CDK2acted as a downstream mediator of p27and was required for the autophagy inhibition and pathological cardiac remodeling induced by miR-221overexpression in cardiomyocytes.CONCLUSIONSWe found that miR-221promoted heart failure through mTOR-mediated autophagy inhibition. A previously unknown signaling pathway, the p27/CDK2/mTOR, governed the regulation of autophagy by miR-221in cardiomyocytes. Our study provides insights into the regulation of cardiomyocyte autophagy by miRNAs and implicates miR-221as a potential therapeutic target for heart failure. BACKGROUNDHypertrophic cardiomyopathy (HCM) is a common monogenetic disease of myocardium. HCM is characterized by unexplained cardiac hypertrophy, myocyte disarray and interstitial fibrosis, and is an important cause of sudden cardiac death and heart failure. This disease is mostly caused by mutations in genes encoding sarcomere proteins. However, the molecular mechanisms that drive the expression of HCM phenotypes are still poorly understood.MicroRNAs (miRs) are endogenous non-coding small RNAs that usually act as repressors of target genes by either inhibiting translation and/or by promoting degradation of the target mRNAs. Previous studies have shown that a number of microRNAs play critical roles in controlling cardiac homeostasis and pathological remodeling, and suggest that microRNAs may serve as potential therapeutic targets for cardiac disease.METHODS AND RESULTSTo identify differently expressed microRNAs between NCM and HCM hearts, a microarray assay was performed on specimens from5NCM donors and7HCM patients. In total,3microRNAs were significantly up-regulated (>2fold, P<0.01), whereas10down-regulated (≤0.5fold, P<0.05) in hearts from HCM patients. Among these differently expressed microRNAs, miR-451ranked as the most down-regulated. To validate the under-expression of miR-451, a qRT-PCR assay was performed with expanded tissue samples (NCM, n=8and HCM, n=16). As expected, the expression of miR-451had decreased more than four fold P<0.01).To analyze the effects of miR-451on cardiac hypertrophy, we transfected primary neonatal rat cardiomyocytes with miR-451mimics or miR-451antagomir for48h after which the cell surface areas were analyzed. The surface area of miR-451overexpressed cardiomyocytes was significantly decreased. Conversely, knockdown of miR-451with antagomir increased surface area. The results from dual luciferase assays suggested that TSC1was a direct target of miR-451. Overexpression of miR-451in Primary neonatal rat cardiomyocytes and HeLa cells induced the down-regulation of TSC1expression. As the expression of miR-451was down-regulated in the hearts from HCM patients, we observed that the expression of TSC1was consistently up-regulated in HCM hearts.As TSC1positively regulates autophagy, we assessed whether autophagy was regulated by miR-451. An autophagy-reporter plasmid encoding EGFP-LC3recombinant protein was co-transfected with miR-451mimics or antagomir into HeLa cells. The formation of autophagosomes was observed based on the appearance of EGFP-LC3punctae. We found that HeLa cells transfected with miR-451mimics had a significantly lower number of EGFP-LC3punctae compared with those transfected with scrambled microRNAs. Conversely, the suppression of miR-451in HeLa cells resulted in larger amounts of EGFP-LC3punctae.We transfected miR-451mimics or miR-451antagomir into primary neonatal rat cardiomyocytes and HeLa cells. We observed that the LC3-Ⅱ in cells with miR-451overexpression was significantly decreased, whereas cells transfected with miR-451antagomir showed increased LC3-Ⅱ.Transmission electron microscopy showed that large quantities of early and late autophagic vacuoles with cytoplasmic remnants and mitochondria had accumulated in the cytosol of cardiomyocytes from HCM hearts. The levels of LC3-Ⅱ were dramatically increased in hearts from HCM patients. The expression of Beclin-1, a required protein in membrane nucleation during the early stage of autophagy, was also significantly up-regulated in HCM hearts, whereas Bcl-2protein, an inhibitor of autophagy, is down-regulated. Taken together, these results indicated that autophagy was increased in HCM hearts.CONCLUSIONSIn summary, our study reveals that miR-451is one of the most down-regulated microRNAs in HCM and regulates cardiac hypertrophy and cardiac autophagy by targeting TSC1. The down-regulation of miR-451may contribute to the development of HCM and may be a potential therapeutic target for the disease. BACKGROUNDModifier genes contribute to the diverse clinical manifestations of hypertrophic cardiomyopathy (HCM), but are still largely unknown. Muscle ring finger (MuRF) proteins are a class of muscle-specific ubiquitin E3-ligases that appear to modulate cardiac mass and function by regulating the ubiquitin-proteasome system. MuRF1,2and3comprise a subfamily of the RING-finger E3ubiquitin ligases that are specifically expressed in striated muscles. Recently, mutations in the gene encoding MuRF1were reported to cause HCM by impairing protein degradation in cardiomyocytes, suggesting that genetic variants in genes encoding MuRF proteins might be involved in the pathogenesis of HCM. In present study, we evaluated the association between genetic variants of MuRF genes and HCM phenotype by screening all the three members of MuRF subfamily in a large HCM cohort and matched healthy controls.METHODS AND RESULTSIn this study we screened all the three members of the MuRF family, MuRF1, MuRF2and MuRF3, in594unrelated HCM patients and307healthy controls by targeted resequencing. Identified rare variants were confirmed by capillary Sanger sequencing. The prevalence of rare variants in both MuRF1and MuRF2in HCM patients was higher than that in control subjects (MuRFl13/594[2.2%] versus1/307[0.3%], P=0.04; MuRF222/594[3.7%] versus2/307[0.7%];P=0.007). Patients with rare variants in MuRF1or MuRF2were younger (P=0.04) and had greater maximum left ventricular wall thickness (P=0.006) than those without such variants. In contrast, rare variants in MuRF1were detected with equal frequency in patients and control individuals. PolyPhen2and SIFT analysis showed21rare variants of MuRFl and MuRF2were potentially pathogenic, including18variants among22(3.7%) HCM patients and3variants among3(1%) control individuals (P=0.02). Mutations in genes encoding sarcomere proteins were present in19(55.9%) of the34HCM patients with rare variants in MuRF1and MuRF2. These data strongly supported that rare variants in MuRF1and MuRF2are associated with higher penetrance and more severe clinical manifestations of HCM.CONCLUSIONSIn conclusion, rare variants in the genes encoding MuRFl and MuRF2act as modifiers that increase the risk of development of HCM and lead to more severe disease phenotypes. Our study is consistent with the hypothesis that impaired ubiquitin-proteasome system contributes to the pathogenesis of HCM.