AT_1R-CaN信号通路在乳鼠肥大心室肌细胞Nav1.5蛋白表达调控中的作用

目的:探讨血管紧张素Ⅱ1型受体(AT_1R)调神经磷酸酶(CaN)信号通路在乳鼠肥大心室肌细胞Nav1.5 mRNA和蛋白表达调控中的作用。方法:分离1日龄SD乳大鼠心室获心室肌细胞,分为对照(control)组、苯肾上腺素(PE)组、氯沙坦(Los)+PE组和环孢素A(CsA)+PE组;重组腺病毒shRNA干扰载体介导CaN A亚基β亚型(CnAβ)基因沉默分为腺病毒空载体(Ad-Null)组、Ad-Null+PE组、重组腺病毒CnAβshRNA1(AdCnAβshRNA1)组和Ad-CnAβshRNA1+PE组。实时荧光定量逆转录PCR检测脑钠尿肽(BNP)、β-肌球蛋白重链(β-MHC)和Nav1.5的mRNA表达。Western blot法检测全细胞提取蛋白CnAβ和Nav1.5的表达。结果:PE干预24 h明显增加心室肌细胞蛋白/DNA比值、细胞BNP和β-MHC的mRNA表达以及细胞面积;上调CnAβ蛋白表达,下调Nav1.5蛋白表达。CsA和Los干预明显抑制PE干预的上述效应。PE下调Nav1.5的mRNA表达,但Los和CsA不能抑制此种效应。Ad-CnAβshRNA1沉默乳鼠心室肌细胞CnAβ基因抑制了PE对BNP mRNA的上调作用,抑制了PE对Nav1.5蛋白表达的下调作用。结论:AT_1R-CaN信号通路参与调控培养的乳鼠肥大心室肌细胞Nav1.5蛋白表达的调控。

Role of AT1R-CaN signaling pathway in regulation of Nav1.5 protein expression in hypertrophic ventricular myocytes from neonatal rats

AIM: To investigate the effect of angiotensin II type 1 receptor (AT₁R)-calcineurin (CaN) signaling pathway on the expression of sodium current channel Nav1.5 at mRNA and protein levels in the hypertrophic ventricular myocytes from neonatal rats.METHODS: The ventricular myocytes were isolated from the ventricles of 1-day-old neonatal Sprague-Dawley rats and were divided into 4 groups according to different drug intervention as control group, phenylephrine (PE) group, losartan (Los)+PE group and cyclosporin A (CsA)+PE group. The method of RNA interference mediated by adenovirus carrying short hairpin RNA (shRNA) was used to knock down the gene which encodes the beta subtype of CaN A subunit (CnAβ) and the cells were divided into 4 groups as Ad-Null group, Ad-Null+PE group, Ad-CnAβshRNA1 group and Ad-CnAβshRNA1+PE group. The mRNA expression of brain natriuretic peptide (BNP), β-myosin heavy chain (β-MHC) and Nav1.5 was detected by RT-qPCR. The protein levels of CnAβ and Nav1.5 in the whole-cell extracts were determined by Western blot analysis.RESULTS: Treatment of the neonatal rat ventricular myocytes with PE for 24 h increased the protein-to-DNA ratio and the mRNA expression of BNP and β-MHC. The size of the cell surface was also increased after PE treatment. Treatment of the cells with PE increased the protein expression of CnAβ, and reduced the protein expression of Nav1.5. Both Los and CsA prevented those effects of PE. The mRNA expression of Nav1.5 was reduced by PE, and no significant difference of Nav1.5 mRNA expression among PE group, Los+PE group and CsA+PE group was observed. Silencing of CnAβ in the neonatal rat ventricular myocytes using Ad-CnAβshRNA1 inhibited the ability of PE to increase the mRNA expression of BNP, and diminished the ability of PE to reduce the protein expression of Nav1.5.CONCLUSION: AT₁R-CaN signaling pathway participates in regulating protein expression of Nav1.5 in the hypertrophic ventricular myocytes from neonatal rats.