H_2S对大鼠心肌肥大与miRNA-133a和Ca^(2+)/CaN/NFATc4信号通路的影响

Effects of hydrogen sulfide(H_2S) on cardiac hypertrophy and miRNA-133a-mediated Ca^(2+)/calcineurin/NFATc4 signal pathway in rats

目的:研究硫化氢(H_2S)对心肌细胞肥大的负性调控作用与miRNA-133a介导Ca^(2+)/CaN/NFATc4信号通路的关系。方法:异丙肾上腺素(ISO)诱导体外培养的大鼠心肌细胞肥大模型;Leica图像分析软件测量心肌细胞表面积;qRT-PCR检测脑钠尿肽(BNP)、β-肌球蛋白重链(β-MHC)、H_2S合酶(CSE)、miRNA-133a和钙调神经磷酸酶(CaN)mRNA表达;Western blot检测CaN、活化T细胞核因子c4(NFATc4)蛋白表达;Elisa方法检测心肌细胞H_2S含量;激光共聚焦显微镜检测心肌细胞钙离子浓度;细胞免疫荧光检测NFATc4核转位变化。结果:(1)心肌细胞肥大时,CSE/H_2S水平、miRNA-133a mRNA表达均显著下降。应用NaHS预处理,能上调心肌细胞CSE/H_2S水平,增加H_2S含量和miRNA-133a mRNA表达,并明显抑制心肌细胞肥大。(2)心肌细胞肥大时,细胞内钙离子浓度明显增加,CaN表达和NFATc4胞核蛋白表达增加,NFATc4核转位明显增强;应用NaHS预处理能明显抑制ISO诱导的上述效应。(3)应用antagomir-133a能逆转H_2S抑制心肌细胞肥大的作用,使心肌细胞内钙离子浓度、CaN表达和NFATc4胞核蛋白表达增加,NFATc4核转位增强。结论:H_2S通过负性调控作用抑制心肌细胞肥大,该作用可能与H_2S上调miRNA-133a的表达,抑制其下游的Ca^(2+)/CaN/NFATc4信号通路的激活有关。

Objective： To investigate the effects of hydrogen sulfide（ H2 S） on the negatively regulation of cardiomyocyte hypertrophy and the relationship between the effect of H2 S with miRNA-133 a-mediated Ca2＋/calcineurin/NFATc4 signal pathway. Methods： Cardiomyocyte hypertrophy was induced by isoproterenol（ ISO）. The cell surface area was measured by image analysis system（ Leica）. The expression of brain natriuretic peptide（ BNP）,β-myosin heavy chain（ β-MHC）,cystathionase（ CSE）,miRNA-133 a,calcineurin（ Ca N） were detected by qRT-PCR. The protein expressions of Ca N、nuclear factors of activated T cells（ NFATc4） were detected by Western blot. The concentration of H2 S in the cardiomyocyte was detected by Elisa. The concentration of intracellular calcium was measured by calcium imaging using confocal microscope. The nuclear translocation of NFATc4 was checked by immuno-fluorescence cell staining technique. Results：（1）The level of system of CSE/H2 S and expression of miRNA-133 a were significantly reduced in cardiomyocyte hypertrophy. Pretreatment with Na HS increased the concentration of H2 S and the expression of miRNA-133 a mRNA in cardiomyocytes,and suppressed cardiomyocyte hypertrophy.（2）The concentration of intracellular calcium,the expression of Ca N and nulear protein NFATc4 were significantly increased,and the nuclear translocation of NFATc4 were obviously enhanced in cardiomyocyte hypertrophy. Na HS pretreatment markedly inhibited these effects of ISO induced cardiomyocyte hypertrophy.（3）Application of antagomir-133 a reversed the inhibitory effects of Na HS on cardiomyocyte hypertrophy,and increased the influx of intracellular calcium,and elevated the expression of Ca N and nuclear protein NFATc4,and enhanced the nuclear translocation of NFATc4. Conclusion： H2 S can negatively regulate cardiomyocyte hypertrophy. The effects might be associated with H2 S increasing expression of miRNA-133 a and inhibiting inactivation of Ca2＋/calcineurin/NFATc4 signal pathway.