The Role Of Micro RNA-199 On Effects Of HLXK In Cardiac Fibrosis

BackgroundCardiac fibrosis may refer to an abnormal thickening of the heart valves due to inappropriate proliferation of cardiac fibroblasts but more commonly refers to the proliferation of fibroblasts in the cardiac muscle. Fibrotic cardiac muscle is stiffer and less compliant and is seen in the progression to heart failure. The description below focuses on a specific mechanism of valvular pathology but there are other causes of valve pathology and fibrosis of the cardiac muscle. Postponing or reversing cardiac fibrosis has become one of the most important goals in hypertension and chronic congestive heart failure treatment. Currently, the precise mechanism of cardiac fibrosis still remains to be elucidated. In the meanwhile, specific medical treatment did not gain any productive advancement. Therefore, it is of great significance to further explore the underlying mechanism of cardiac fibrosis and to searching potential target for drug development.Micro RNAs(miRNAs), which are 22-25 nucleotides in length, is the member of non-protein coding RNAs. They silence mRNA by repressing the translation of target RNA by binding to homologous 3’UTR region as an imperfect match. Abnormal expression of mi RNAs was found in cardiac remodeling while its expression pattern and the meaning behind it are still little known.Guanine nucleotide-binding proteins(GP) are in the core position in the pathogenesis of cardiac remodeling. Multiple major signaling in cardiac remodeling must couple GP to further activate downstream signal. During the past decades, our lab have successfully develop a simulated peptide of the α subunit of Gq protein named HLXK, which is proved effective in anti- cardiac hypertrophy on various animal model and cell lines. The present study was designed to determine the curative effect of HLXK on cardiac remodeling. By observing the expression changes of miRNA during the treatment, this study were meant to find out the potential roles that mi RNA plays during the treatment. This study will lay an experimental foundation for renew the pathogenesis of cardiac remodeling and for novel drug target screening.Methods1. Male specific germ free Kunming mice were divided into three groups:(1) control group, animal get intraperitoneal injected with saline daily;(2) animal model group, mice intraperitoneal injected with norepinephrine(1.5mg/kg) daily;(3) losartan treated group, mice intraperitoneal injected with norepinephrine(1.5mg/kg) plus losartan(5mg/kg) daily;(4) HLXK(90μg/kg)treated group, mice intraperitoneal injected with norepinephrine(1.5mg/kg) plus HLXK(90μg/kg) daily;(5) HLXK(30ug/kg)treated group, mice intraperitoneal injected with norepinephrine(1.5mg/kg) plus HLXK(30μg/kg) daily. The mice were sacrificed at day 21 and the heart was collected for weighing, morphological observation(by HE and Picric-Sirius red staining) and mi RNA expression detection(by RT-PCR).2. Primary cardiac fibroblasts were cultured with 1×10-7mol/L Ang II plus HLXK, and detected mi RNA and collagen I /III expressions to find out the effect of HLXK on the expression of miR-199 and on fibrosis formation; observed the miR-199 in cardiac fibroblasts expression changes of collagen I /III m RNA and protein after transfecting mi R-199 inhibitor or mimic.3. Primary rat cardiomyocytes were cultured with 1×10-6mol/L NE plus HLXK, and detected mi RNA and 3H-leucine incorporation to find out the effect of HLXK on the expression of mi R-199 and on myocardial hypertrophy; observed the mi R-199 in myocardial hypertrophy incorporation of 3H-leucine after transfecting mi R-199 inhibitor or mimic.Results1. On the 21 days of modeling of mice, LVMI of model group was up-regulated significantly compared with control group; LVMI of HLXK group(90μg/kg) was down-regulated compared with model group. The Picric-Sirius red staining of Left ventricular myocardial tissue remodeling of all mice showed that HLXK(90μg/kg) myocardial tissue collagen deposition and hydroxyproline content significantly reduced compared with the model group(P< 0.01). In vitro, HLXK(11.3 mg/L) could reduce the collagen I mRNA expression, but little effect on collagen III gene expression. HE staining indicates that the myocardial cells of HLXK group were arranged in substantially neat, myocardial fibrosis was significantly reduced; no significant infiltration of inflammatory cells, suggesting HLXK can relieve NE induced left ventricular remodeling of mice. In the this animal model, mi R-199, miR-499 expression and mi RNA-1-2-5p of model group were significantly increased by 731%, 148%, 242% compared with control group, and HLXK could decrease these mi RNA expression( P< 0.01) when paly a important role in prevention and treatment of cardiac hypertrophy.2. In the cultured cardiac fibroblasts in vitro, miR-199 expression was inincresed significantly which induced by AngⅡ(P< 0.01),and HLXK(11.3 mg/L)could also increase the expression of mi R-199 in cardiac fibroblasts(P< 0.01). The expression of collagen I and Ⅲ m RNA and protein of cardiac fibroblasts cells was increased induced by AngII significantly; miR-199 inhibitor can significantly reduce the synthesis; transfecting mi R-199 mimic alone can significantly increase the m RNA and protein expression of collagen I and Ⅲ(P< 0.01).3. In the cultured neonatal rat cardiomyocytes in vitro, HLXK(11.3 mg/L) could significantly reduce the 3H- leucine incorporation by 30.5% induced by NE. HLXK(11.3 mg/L) could significantly reduce the expression of miR-499, miR-1-2-5p and mi R-199 induced by NE(1×10-6mol/L). mi R-199 inhibitor(30nmol/L) could reduce 3H-leucine incorporation in cardiomyocytes induced by NE(1×10-6mol/L) compared with the control group, the 3H-leucine incorporation of transfected miR-199 mimic(30nmol/L) alone significantly increased in myocardial cells.