Mirna-221 Regulation Mechanisms Of Myocardial Remodeling

Heart failure (HF) involves changes in myocyte deformation, cardiac structure, myocardial composition, and multiple molecular alterations that impact heart function and reserve capacity. Collectively, these changes have been considered as cardiac remodeling. Understanding the components of this process with the goal of stopping or reversing its progression becomes a major challenge, and may provide promising to prevent the development of heart failure. Recently, miRNAs have been established as important regulators of cardiac remodeling. Dozens of miRNAs have been reported to modulate or control the development of cardiac remodeling. Nevertheless, previous reports and microarray data have implied that several other miRNAs are dysregulated in cardiac hypertrophy, and their roles in cardiac hypertrophy have yet to be elucidated.MiRNA-221has been identified in heart and is upregulated in a mouse model of transverse aortic constriction (TAC). However, these results were based on microarray data and have not yet been experimentally and clinically validated. Evidences in cancer field revealed that miRNA-221could be induced by stimulators of cardiac remodeling and targeted by suppressor of cardiac remodeling,implying that miRNA-221is involved in cardiac remodeling. In this study, we aimed to explore whether miRNA-221regulates cardiac remodeling, and if any, what the mechanism that may be invovled in this process.To determine the potential involvement of miRNA-221in cardiac remodeling, we performed qRT-PCR analysis to examine the expression levels of miRNA-221in the left ventricular tissue of TAC mouse models and hypertrophic cardiomyopathy (HCM patients). Increased expression levels of miRNA-221were observed in hypertrophic and failured mouse at2-week and9-week after TAC (1.5-fold and1.6-fold, respectively; P<0.05). Moreover, the expression level was up-regulated2-fold in patients with HCM, indicating that miRNA-221is involved in cardiac remodeling.To investigate the role of miRNA-221in cardiac remodeling in vitro, we overexpressed miRNA-221in primary neonatal rat cardiomyocytes by transfection with miRNA-221mimics. The overexpression of miRNA-221induced cardiomyocyte hypertrophy, which is characterized by enlargement of cardiomyocyte size (53%upregulation, p<0.01) and reexpression of fetal genes (atrial natriuretic factor (ANP) and b-type natriuretic protein (BNP), upregulated3.1-fold and3.2-fold, respectively, p<0.01). While inhibition of endogenous miRNA-221resulted in significantly decreased fetal gene reexpression, with ANP and ACTA1were downregulated by30%and50%, respectively (p<0.01). Furthermore, Western blotting analysis and Luciferase reporter assays verified that p27is a target for miRNA-221in cardiomyocytes. These in vitro results suggested that miRNA-221might regulate cardiac remodeling through modulating of p27levels.To investigate the role of miRNA-221in cardiac remodeling in vivo, we generated two lines of transgenic mice with cardiac-restricted overexpression of miRNA-221, which carry46(TG-221-L) and127(TG-221-H) copies of the transgene, respectively.Cardiac remodeling and left ventricular dysfunction were detected in all miRNA-221transgenic mice, including female and male transgenic mice. The degree of cardiac remodeling and left ventricular dysfunction is positively correlated with the number of integrated transgenic copies.At the age of3months, histomorphologic assay showed that compared to the non-transgenic littermates, the ratio of heart/body weight were significantly increased in the miRNA-221transgenic mice (12%for transgenic mice with low copy number and44%for transgenic mice with high copy number, respectively, female mice, p<0.01;7%for transgenic mice with low copy number and64%for transgenic mice with high copy number, respectively, male mice, p<0.01), indicating that miRNA-221was sufficient to induce the cardiac remodeling.In transgenic female mice with high-copy-number miRNA-221, the left ventricular thickness was dramatically decreased in the end of systolic stage (20%and19%decrease in LVAW and LVPW, respectively, P=0.06, P<0.05), while not in diastolic stage. The left ventricular chamber dimension was significantly increased (26%increase in LVEDD, P<0.01, and55%increase in LVESD, P<0.01, respectively), as measured by M-mode echocardiography. Fractional shortening significantly decreased by38%(P<0.05), indicating the left ventricular dysfunction occurred in transgenic female mice.In transgenic male mice with high-copy-number miRNA-221, while the alteration of LVAW, LVPW were not observed, LVEDD, LVESD and Fractional shortening decreased more dramatically, with a reduction of34%(p<0.001),55%(p<0.001),64%(p<0.01), respectively, compared to the non-transgenic littermates, indicating that cardiac remodeling and left ventricular dysfunction developed in male mice to heart failure.Upregulation of fetal genes were observed, indicating the progressive of cardiac remodeling. Calcium-related proteins were decreased significantly, indicating that the left ventricular dysfunction exists. Meanwhile, p27was also decreased in transgenic mice, negative correlated with the miRNA-221transgenic copy numbers, indicating that miRNA-221might regulate cardiac remodeling and left ventricular dysfunction through modulating p27expression level in vivo. Furthermore, autophagic markers were inhibited in transgenic mice, indicating the involvement of autophagic regulation in miRNA-221transgenic mice.In conclusion, our results indicate that miRNA-221regulates cardiac remodeling. The overexpression of miRNA-221promotes cardiac remodeling in vivo and in vitro, partially by inhibiting p27expression and autophagic signaling pathway.