Study On The Mechanism Of Qiliqiangxin On Cardiomyocyte Hypertrophy In Neonatal Rats

Background/Aims:Qiliqiangxin (QL), a traditional Chinese medicine, has long been used to treat chronic heart failure. Previous studies demonstrated that QL could prevent cardiac remodeling and hypertrophy in response to hypertensive or ischemic stress. However, little is known about whether QL could modulate cardiac hypertrophy in vitro, and (if so) whether it is through modulation of specific hypertrophy-related microRNA.Methods:1.To establish the model that QL attenuates cardiac hypertrophy in vitro: (1).The primary neonatal rat ventricular cardiomyocytes were isolated, cultured, and treated with phenylephrine (PE,50 μmol/L,48 h) to induce hypertrophy in vitro, in the presence or absence of pretreatment with QL (0.5 μg/ml,48 h).(2)The cell surface area was determined by immunofluorescent staining for a-actinin. The mRNA levels of hypertrophic markers including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and β-myosin heavy chain (MYH7) were assayed by qRT-PCRs. The protein synthesis of cardiomyocytes was determined by the protein/DNA ratio.2.The expression of miR-199a-5p in different pathophysical hypertrophy models:The miR-199a-5p expression level was quantified in heart samples from patients with dilated cardiomyopathy (DCM), PE-treated cardiomyocytes and acute myocardial infarction (AMI) mouse model, in the presence of absence of qiliqiangxin using real time PCR.3.The role of miR-199a-5p in anti-hypertrophic effects of qiliqiangxin: Primary cardiomyocytes was first treated with QL for 48h, and then transfected the cells with miR-199a-5p mimics or miRNA negative control (NC mimics) for 8 h before PE induction.4. Statistical analysis All data were expressed as mean± SEM. The main statistical methods are one-way ANOVA and t test, using GraphPad statistical software for statistical analysis A value of p<0.05 was considered statistically significant.Results:(1)QL effectively reversed Phenylephrine-induced CMs hypertrophy. PE-induced cardiomyocyte enlargement and the increase in hypertrophic markers (ANP, BNP, and. MYH7) and protein/DNA ratio, were markedly reversed by treatment with QL (Fig.2A-C), indicating that QL was protective against PE-induced cardiac hypertrophy. (2)miR-199a-5p is increased during cardiac hypertrophy, while reduced by treatment with QL. Our data showed that miR-199a-5p was considerably induced in human DCM heart samples in contrast with normal ones. Moreover, miR-199a-5p was markedly increased in the hearts suffered from AMI adverse remodeling and PE-treated cardiomyocytes, while the induced expression of miR-199a-5p was efficiently reversed by treatment with QL. (3) miR-199a-5p suppression is essential for the protective effect of QL against cardiomyocyte hypertrophy. Our data showed that miR-199a-5p mimics could efficiently abolish the protective effect of QL against cardiomyocyte enlargement as compared with QL-treated hypertrophic cardiomyocytes transfected with NC mimics. These data clearly indicate that the inhibition of miR-199a-5p is necessary for the protective effect of QL on hypertrophic cardiomyocytes.Conclusion:QL prevents PE-induced cardiac hypertrophy. miR-199a-5p is increased in cardiac hypertrophy, while reduced by treatment with QL. miR-199a-5p suppression is essential for the anti-hypertrophic effect of QL on cardiomyocytes.