miR-20a调控压力超负荷型心肌肥大

miR-20a regulates pressure overload-induced cardiac hypertrophy

背景:心肌肥大是心脏对压力超负荷等生理和病理刺激所做出的适应性反应,早期具有代偿意义,若刺激持续进行可引起心肌病变导致心力衰竭。micro RNAs参与调控了心肌肥大的发生发展,然而mi R-20a在压力超负荷所致心肌肥大中的作用尚未见报道。目的:探究mi R-20a在压力超负荷所致心肌肥大中的作用及其机制。方法:横向主动脉缩窄术诱导心肌肥大小鼠模型,使用血管紧张素Ⅱ诱导心肌肥大H9c2细胞模型。体内实验在小鼠心脏原位注射mi R-20a过表达腺病毒,体外实验将mi R-20a mimic转染至H9c2细胞。通过检测心质量/体质量比值、细胞表面积、心肌纤维化等指标评估心肌肥大,实时荧光定量PCR法检测心房利钠肽、脑利钠肽、β-肌球蛋白重链和mi R-20a的表达水平,Mito Tracker法检测线粒体分裂情况,RNAhybrid软件预测miR-20a的下游靶基因。结果与结论:(1)在心肌肥大细胞模型和动物模型中,mi R-20a的表达水平均显著降低(P<0.05);(2)在动物水平,过表达miR-20a显著抑制了横向主动脉缩窄手术诱导的心肌肥大,包括抑制了肥大标志基因表达水平的升高(P<0.05),抑制了心脏体积的增大,抑制了心质量/体质量比值的升高(P<0.01),抑制了心肌横截面积的增大(P<0.05)以及减轻了心肌纤维化程度(P<0.01);(3)在细胞水平,过表达mi R-20a显著抑制了血管紧张素Ⅱ诱导的心肌细胞肥大,包括抑制了心房利钠肽(P<0.05)、脑钠肽(P<0.01)、β-肌球蛋白重链(P<0.05)等肥大标志基因表达水平的升高,抑制了蛋白质/DNA比值的升高(P<0.01),抑制了细胞表面积的增大(P<0.05);(4)过表达mi R-20a显著抑制了血管紧张素Ⅱ诱导的线粒体分裂(P<0.05);(5)RNAhybrid软件分析结果显示mi R-20a与蛋白质活化酶抑制剂αm RNA的3’非翻译区可以很好地互补配对,且预测的结合位点具有高度保守性;(6)结果表明,在压力超负荷心肌肥大模型中mi R-20a表达水平显著下调,过表达mi R-20a可在细胞水平和动物水平抑制心肌肥大表型,并减轻血管紧张素Ⅱ诱导的心肌细胞线粒体分裂。

BACKGROUND:Cardiac hypertrophy is an adaptive response of the heart to physiological and pathological stimuli such as pressure overload.It is of compensatory significance in the early stage,but if the stimulation continues,it can cause cardiomyopathy leading to heart failure.MicroRNAs are involved in the regulation of cardiac hypertrophy.However,the role of miR-20a in pressure overload-induced cardiac hypertrophy has not been reported.OBJECTIVE:To investigate the role of miR-20a in pressure overload-induced cardiac hypertrophy and the underlying mechanisms.METHODS:Transverse aortic constriction was used to induce cardiac hypertrophy in vivo and angiotensin II was used to induce H9c2 cell models of cardiac hypertrophy in vitro.MiR-20a was overexpressed in vivo by intramyocardial injection of miR-20a overexpressing adenovirus and in vitro by transfecting miR-20a mimic into H9c2 cells.Cardiac hypertrophy was assessed by measuring heart weight/body weight ratio,cell surface area,and myocardial fibrosis.The expression levels of atrial natriuretic peptide,brain natriuretic peptide,β-myosin heavy chain and miR-20a were detected by real-time fluorescence quantitative PCR.Mitochondrial fission was detected by MitoTracker.The downstream target genes of miR-20a were predicted by RNAhybrid software.RESULTS AND CONCLUSION:(1)The expression level of miR-20a was significantly decreased in both hypertrophic cardiomyocytes and hearts(P<0.05).(2)At the animal level,overexpression of miR-20a significantly inhibited transverse aortic constriction-induced cardiac hypertrophy,including decreasing the upregulated expression level of hypertrophic marker genes(P<0.05),reduced the enlarged heart volume,reducing the increased heart weight/body weight ratio(P<0.01),reducing the increased myocardial cross-sectional area(P<0.05),and attenuating fibrosis(P<0.01).(3)At the cellular level,overexpression of miR-20a significantly inhibited angiotensin II-induced cardiomyocyte hypertrophy,including decreasing the upregulated expression levels of atrial natriuretic peptide(P<0.05),brain natriuretic peptide(P<0.01)andβ-myosin heavy chain(P<0.05),reducing the increased protein/DNA ratio(P<0.01),and suppressing the increased cell surface area(P<0.05).(4)Overexpression of miR-20a significantly inhibited angiotensin II-induced mitochondrial fission(P<0.05).(5)The results of RNAhybrid software analysis showed that miR-20a and the mRNA 3’untranslated region of cAMP-dependent protein kinase inhibitor alpha were well complementary and the predicted binding sites were highly conserved.(6)In conclusion,miR-20a is significantly down-regulated in pressure overload-induced cardiac hypertrophy.Overexpression of miR-20a inhibits cardiac hypertrophy at both the cellular level and animal level and attenuates angiotensin II-induced mitochondrial fission.