| Cardiac hypertrophy is one of the common features of cardiovascular diseases, which are the major risk factors for human health and the leading causes of deaths worldwide. Cardiac hypertrophy is the compensatory response of hearts to various biomechanical and neurohormonal stimuli, and characterized by the increased cardiac mass and ventricle wall thickness. The extent of cardiac hypertrophy can be used as a predictor of progressive cardiac diseases and is inversely correlated with the prognosis. Pathological cardiac hypertrophy will eventually progress to heart failure, which is the major cause of mortality for patients with it. Despite several signaling pathways involved in the cardiac hypertrophy have been identified, the underlying mechanisms are still largely unknown. To search for therapeutic targets by finding new genes regulating cardiac hypertrophy is of significance for the prevention and treatment of cardiac diseases.The development of pathological cardiac hypertrophy is accompanied with the imbalance of cardiac homeostasis. Recent studies have demonstrated that autophagy plays important roles in the maintenance of cardiac homeostasis. Autophagy refers to the process in which intracellular proteins or organelles are firstly surrounded by double-membraned vesicles to form autophagosomes and then fuse with lysosomes to form autolysosomes for the degradation of the components. Autophagy helps to meet the requirements of cells for the energy metabolism and organelle renewal, making it a significant process for the cellular homeostasis. Disruption of autophagy has been shown to be related to cardiac hypertrophy, but there exists controversy concerning the effects of autophagy on cardiac hypertrophy due to the complexity of autophagy. Different conclusions are drawn by using genetic mouse models of different autophagy related genes.micro RNAs(mi RNAs) are a class of single-stranded noncoding RNAs with a length of about 22 nucleotides. mi RNAs can bind to their target genes’ m RNA 3’ untranslational regions(3’UTR) by complementary base pairing, and regulate their expression in a post-transcriptional manner through inhibition of m RNA translation or promotion of m RNA degradation. mi RNAs broadly participate in many cell biological processes. In recent years, researchers have found a serious of mi RNAs that play important roles in cardiac hypertrophy by regulating hypertrophy-related signaling pathways. These studies provide valuable clues that mi RNAs may act as novel clinical diagnostic criteria and therapeutic targets.mi R-199 a is an important regulatory gene of cardiomyocyte size. Previous studies have found that mi R-199 a is significantly upregulated in human heart failure samples and mouse models of pathological cardiac hypertrophy, and that mi R-199 a could regulate cardiomyocyte size and apoptosis in vitro. However, the in-vivo function of mi R-199 a in cardiac homeostasis and its underlying mechanisms are still lacking. Therefore, this study aims to investigate the in-vivo function and the mechanism of mi R-199 a in pathological cardiac hypertrophy.We first validated the upregulation of mi R-199 a in the mouse models of cardiac hypertrophy and human heart failure samples. Then, we constructed cardiomyocytespecific mi R-199a-overexpressing transgenic mice and found that transgenic mice developed pathological cardiac hypertrophy and heart failure. mi R-199 a activated autophagy-inhibitor m TOR and inhibited cardiomyocyte autophagy both in vitro and in vivo, which were partially mediated by mi R-199 a target gene GSK3β. Activation of autophagy by rapamycin treatment in vivo or Atg5 overexpression in vitro recued mi R-199a-induced cardiac hypertrophy, suggesting that mi R-199 a induced cardiac hypertrophy possibly by inhibiting autophagy.As shown above, cardiomyocyte-specific mi R-199 a overexpressing transgenic mice developed pathological cardiac hypertrophy. Previous study has found that cardiomyocyte-specific mi R-199 b overexpressing transgenic mice also exhibit more exaggerated cardiac hypertrophy than wild-type mice when subjected to transverse aortic constriction(TAC), indicating that mi R-199 family plays an important regulatory role in cardiac hypertrophy. However, the role of endogenous mi R-199 in vivo has not been elucidated yet. By using a “mi RNA sponge†strategy, we constructed a cardiomyocyte-specific mi R-199-sponge transgenic(mi R-199-SP) mice to inhibit mi R-199 family in vivo. Both mi R-199 a and mi R-199 b were reduced in mi R-199-SP mice. The expression levels of their verified targets genes, such as GSK3β, HIF1α, and DYRK1 A, were increased in mi R-199-SP mice. 3-month-old mi R-199-SP mice showed increases in heart mass and cardiomyocyte size, but the extent of the hypertrophy was not exaggerated with age. Echocardiography results indicated that the cardiac function of mi R-199-SP mice was normal. The hypertrophic marker ANF was significantly inhibited, while BNP was unchanged in mi R-199-SP mice compared with sex-matched littermate controls. Also, no fibrosis was observed in the hearts of mi R-199-SP mice. Further study demonstrated that PGC1α was the target gene of the mi R-199 family, and PGC1α and its downstream metabolic genes were also increased in mi R-199-SP mice. These data suggested that mi R-199-SP mice developed physiological cardiac hypertrophy, indicating a significant role of endogenous mi R-199 in cardiac homeostasis maintenance.Finally, to test the potential of mi R-199 a as the therapeutic target of cardiac hypertrophy, we designed and performed a therapeutic study in a mouse model of TAC. Pharmacological inhibition of mi R-199 a by mi R-199-SP expressing adenovirus injection partially rescued the increased heart mass and cardiomyocyte size induced by TAC. The upregulation of hypertrophic marker and the impairment of cardiac systolic function were also rescued in the treatment group. These data indicated that inhibition of mi R-199 could effectively treat cardiac hypertrophy and failure induced by TAC.Our study provides the first genetic evidence to show the in vivo function of mi R-199 family in cardiac hypertrophy and homeostasis maintenance. We revealed a novel mechanism that mi R-199 a inhibits autophagy via targeting GSK3β in the process of cardiac hypertrophy using the cardiomyocyte-specific mi R-199 a overexpressing transgenic mice. We also demonstrated that inhibition of endogenous mi R-199 family results in physiological cardiac hypertrophy using the cardiomyocyte-specific mi R-199 sponge transgenic mice. The treatment experiment by using mi R-199-SP adenovirus indicates that mi R-199 a might be a new therapeutic target of cardiac hypertrophy. This study provides new insight into the mechanism of cardiac hypertrophy, and provides a new theoretical basis and experimental animal model for the treatment of cardiac hypertrophy. |
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