Angiotensin-II-Evoked Ca2+ Entry in Murine Cardiac Fibroblasts Does Not Depend on TRPC Channels.

Juan E Camacho Londoño,Christin Richter,Lutz Birnbaumer,Alexander Schürger,Marc Freichel,André Marx,Peter Lipp,Axel E Kraft,Alexander Dietrich

doi:10.3390/cells9020322

Abstract

TRPC proteins form cation conducting channels regulated by different stimuli and are regulators of the cellular calcium homeostasis. TRPC are expressed in cardiac cells including cardiac fibroblasts (CFs) and have been implicated in the development of pathological cardiac remodeling including fibrosis. Using Ca2+ imaging and several compound TRPC knockout mouse lines we analyzed the involvement of TRPC proteins for the angiotensin II (AngII)-induced changes in Ca2+ homeostasis in CFs isolated from adult mice. Using qPCR we detected transcripts of all Trpc genes in CFs; Trpc1, Trpc3 and Trpc4 being the most abundant ones. We show that the AngII-induced Ca2+ entry but also Ca2+ release from intracellular stores are critically dependent on the density of CFs in culture and are inversely correlated with the expression of the myofibroblast marker α-smooth muscle actin. Our Ca2+ measurements depict that the AngII- and thrombin-induced Ca2+ transients, and the AngII-induced Ca2+ entry and Ca2+ release are not affected in CFs isolated from mice lacking all seven TRPC proteins (TRPC-hepta KO) compared to control cells. However, pre-incubation with GSK7975A (10 µM), which sufficiently inhibits CRAC channels in other cells, abolished AngII-induced Ca2+ entry. Consequently, we conclude the dispensability of the TRPC channels for the acute neurohumoral Ca2+ signaling evoked by AngII in isolated CFs and suggest the contribution of members of the Orai channel family as molecular constituents responsible for this pathophysiologically important Ca2+ entry pathway.

Highlights

Remodeling processes comprising the activation of fibroblasts and the consequent changing in the extracellular matrix (ECM) composition are key processes during the development of different pathologies in several organs
To determine the role of TRPC channels for the angiotensin II-induced Ca2+ response in isolated primary murine cardiac fibroblasts (CFs) we first determined the conditions of cell culture and analysis using CFs obtained from adult wild type (WT) mice
After defining that angiotensin II (AngII)-induced Ca2+ entry with a robust Ca2+ release is only observed in fibroblasts kept at high density and TRPC3 and TRPC6 being described as important regulators of fibroblast function [15,47,48,51], we studied the effect of genetic deletion of TRPC3/C6 proteins or its acute inhibition with the described TRPC3/C6/C7 blocker SAR7334 [75] on AngII-induced Ca2+

Summary

Introduction

Remodeling processes comprising the activation of fibroblasts and the consequent changing in the extracellular matrix (ECM) composition are key processes during the development of different pathologies in several organs. Cells 2020, 9, 322 occurs for example during non-alcoholic fatty liver disease (NAFLD) [1,2] or pathological cardiac remodeling presented by the failing heart or after myocardial infarction [3,4]. In the last years an eminent increased effort to characterize such cells has been evident; for example, to define differences between populations of resident fibroblasts, activated fibroblasts and myofibroblasts, the last ones being prominent in ECM deposition, secretion and tissue contraction [9,10,11]. A multiple characterization of fibroblasts models such as primary cultured cells is a necessary step to understand the biology behind fibroblast functioning and required to utilize them in the development of therapeutic strategies, for example for heart disease management [13]

Methods

Results

Conclusion