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To explore the role of angiotensin receptor I type (AT1)- calcinerurin signaling pathway on ion channels expression in hypertrophic neonatal rat ventricular myocytes.
Ventricular myocytes were isolated from the ventricles of 1 day-old neonatal Sprague-Dawley rats and cultured for 48 hours before any treatment. Drug interventions were as follows, 1) Control group. 2) Phenylephrine (PE) group, incubation with PE (100μM) for 24 hours. 3) Losartan (Los) + PE group, incubation with Los (10μM) for 2 hours then treated with PE (100μM). 4) CyclosporinA (CsA) + PE group, incubation with CsA 10μM for 2 hours then treated with PE (100μM). The MOI=50 corresponding to the amount of adenovirus was choosen, and adenoviral RNA interference (RNAi) vector administration was as follows, 1) Ad-Null group, transfection with the adenovirus containing the empty expression vector for 48 hours. 2) Ad-Null+PE group, transfection with the adenovirus containing the empty expression vector for 24 hours then treated with PE (100μM) for 24 hours. 3) Ad-CnAβshRNA1, transfection with the adenovirus containing the CnAβshRNA1 for 48 hours. 4) Ad-CnAβshRNA1+PE group, transfection with the adenovirus containing the CnAβshRNA1 for 24 hours then treated with PE (100μM) for 24 hours. Gene expressions of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), myosin heavy chain beta(β-MHC), Nav1.5 and Kv4.2 were detected by real-time reverse transcriptase-polymerase chain reaction(real-time RT-PCR). CnAβ, Nav1.5, Kv4.2 proteins in whole-cell extracts were assayed by western blot analysis.
PE treatment increased the protein-to-DNA ratio and the gene expressions of ANP, BNP and β-MHC. The size of cell surface was also increased after PE treatment. Treatment of neonatal rat ventricular myocytes with PE increased the protein expression of CnAβ, and reduced the protein expression of subunit Nav1.5, but didn't significantly altered its gene expression. The gene and protein expressions of Kv4.2 were reduced by PE treatment. Los and CsA both prevented those effects of PE.Siliencing of CnAβ in neonatal rat Ventricular myocytes using Ad-CnAβshRNA inhibited the ability of PE to increase the gene expression of BNP, and diminished the ability of PE to reduce protein expression of Nav1.5. The ability of PE to reduce the gene and protein expressions of Kv4.2 was also repressed by deletion of CnAβ.
AT1-calcinerurin signaling pathway participates in regulating gene and protein expressions of Nav1.5 and Kv4.2 in hypertrophic ventricular myocytes from neonatal rat.