Influences Of AT1R-Calcineurin Signaling Pathway In Ion Channel Remodeling Of Ventricular Hypertrophy In Neonatal Rats

Objective: To study the effect of AT1R-Calcineurin signaling pathway on the ion channel remodeling of hypertrophic neonatal rat ventricular myocytes.Methods: Neonatal rat ventricular myocytes were isolated from the ventricles of 1 day-old neonatal Sprague-Dawley rats and cultured for 24 hours before any treatment.Stretch interventions were as follows:(1)Control group,without intervention.(2)Stretch(Str)group,incubation with Str for 24 hours.(3)Losartan(Los)+ Str group,incubation with Los for 2 hours then treated with Str.(4)CyclosporinA(CsA)+Str group,incubation with CsA for 2 hours then treated with Str.Drug interventions were as follows :(1)Control group,without intervention.(2)phenylephrine(PE)group,incubation with PE for 24 hours.(3)Los + PE group,incubation with Los for 2 hours then treated with PE.(4)CsA + PE group,incubation with CsA for 2 hours then treated with PE.RNA interference administration mediated by adenovirus carrying short hairpin RNA to knock down CnA?(CnA?shRNA)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 for 24 hours.(3)Ad-CnA?shRNA1,transfection with the adenovirus containing the CnA?shRNA1 for 48 hours.(4)Ad-CnA?shRNA1+PE group,transfection with the adenovirus containing the CnA?shRNA1 for 24 hours then treated with PE for 24 hours.Immunofluorescence and immunocytochemistry staining were used to identify ventricular myocytes.The protein expression of CaN was determined by western blot.Whole cell patch clamp technique was used to record Ito,IK1 and ICa,L channel current and action potential.Results: Stretch treatment reduced the Ito current density of Neonatal rat ventricular myocytes significantly,acceleratedinactivation,but didn't significantly change activation.Los and CsA intervention significantly inhibited the effect of stretch stimulation.Through PE treatment,Ito and IK1 current density was reduced,ICa,L current density was increased and APD was significantly prolonged.Los,CsA and Ad-CnA?shRNA1 intervention inhibited the effect of PE stimulation.Conclusion:.AT1R-Calcineurin signaling pathway is involved in the regulation of ion channel remodeling and APD changes in isolated neonatal rat ventricular myocyte.