TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation.

Nadine Kirschmer,Erick Miranda-Laferte,Viktor Von Ehrlich-Treuenstätt,Anna-Karina Lamprecht,Sandra Bandleon,Oliver Ritter,Sonja Hartmann,Alice Schaaf,Tanja Langsenlehner,Petra Eder,Alexander G Obukhov

doi:10.1371/journal.pone.0168446

Abstract

The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca2+ signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.

Highlights

Ion channels of the Transient Receptor Potential Channel-canonical type ‘C’ (TRPC) family have been suggested as key players in several cardiac disease conditions [1,2]
For most of the TRPC subunits, low expression levels have been found in the heart which increase after pathological stimulation by transverse aortic constriction (TAC) or myocardial infarction (MI) treatment [2,18,19,20]
Our first aim was to examine whether an upregulation of TRPC4α and TRPC4β as observed after TAC treatment would alter cardiomyocyte Ca2+ signals, signaling and hypertrophy

Summary

Introduction

Ion channels of the Transient Receptor Potential Channel-canonical type ‘C’ (TRPC) family have been suggested as key players in several cardiac disease conditions [1,2] They comprise 7 subunits (TRPC1-7) which are all expressed in the heart depending on the developmental stage, species and occurrence of disease [1,3]. Hypertrophic mouse cardiomyocytes isolated after transverse aortic constriction (TAC) surgery exhibit a sustained Ca2+ influx which can be blocked by the expression of dominant negative (dn)-TRPC3 and TRPC6 mutants This inhibition is coupled with less calcineurin and nuclear factor of activated T-cells (NFAT) activation, an attenuated hypertrophic phenotype as well as an improvement of cardiac function [5]. The lack of TRPC1 function in the heart resulted in reduced calcineurin/NFAT signaling, prevented pathological cardiac hypertrophy but preserved contractility [6]

Methods

Results

Conclusion