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

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.

Keywords: Ca2+ release and Ca2+ entry; TRPC channels; angiotensin II; cardiac fibroblasts (CFs).

Figure 1

Characterization of primary isolated murine cardiac fibroblasts. (A) Isolated primary CFs from adult mice were cultured at high (>300 cells/mm²) and low density as depicted. Phase contrast images showing the typical morphology and confluence of cultured fibroblasts at several time points of the culture are included. (B) Immunocytochemical characterization of primary cultures of CFs using antibodies against α-actinin, α-smooth muscle (SM) actin, P4HB and CD31 (Bi) or additionally DDR2 (Bii) is shown. (C) Expression analysis by qPCR from Trpc channels in four independent (n = 4 hearts) primary cultures of CFs. bp: base pair. * p < 0.05 depicted are according to Dunn’s post-hoc comparisons after Kruskal-Wallis.

Figure 2

Changes in intracellular Ca²⁺ concentration in cardiac fibroblasts depending on the density of the culture. (A,B) AngII-induced Ca²⁺ transients in primary CFs in the presence of 2 mM extracellular Ca²⁺. Cells were analyzed 5 days after isolation and cultivation at low (A) or high density (B). Fluorescence images of Fura-2 loaded cells are included. (C,D) AngII-induced Ca²⁺ release from internal stores and subsequent AngII-induced Ca²⁺ entry were analyzed in fibroblasts maintained at low (C) or high density (D) conditions. Additionally, the expression of α-SM-actin was analyzed on cells cultured on each cell density (upper panels). n = number of independent preparations (hearts).

Figure 3

AngII-induced Ca²⁺ release and Ca²⁺ entry in the absence of TRPC3/C6 or after TGF-β pre-treatment. (A) AngII-induced Ca²⁺ release and Ca²⁺ entry in primary CFs from WT (black) and TRPC3/C6-DKO (red) mice. Ca²⁺ release was measured in the absence of extracellular Ca²⁺ (300 µM EGTA) and Ca²⁺ entry was monitored in the presence of 2 mM extracellular Ca²⁺. Left panels: Original traces and right panels: Mean values from three independent preparations (hearts). (B) Measurements performed as in (A) but in cells pre-incubated (10 min) with the TRPC3/C6/C7 antagonist SAR7334 (1 µM). (C) AngII-induced Ca²⁺ release and Ca²⁺ entry in primary CFs from WT mice cultivated in the presence of 10 ng/mL TGF-β (green) or under control conditions (black). Left panel: α-actin smooth muscle staining after TGF-β treatment, middle panels: Original traces from Ca²⁺ measurements and right panels: Mean values from 3 independent preparations. n = number of independent preparations (hearts). All cells were analyzed 5 days after isolation and were cultured at high density. * p < 0.05 according to the unpaired Student’s t-test.

Figure 4

AngII-induced Ca²⁺ release and Ca²⁺ entry in cardiac fibroblasts in the absence of all seven TRPC proteins. (A) AngII- and (B) thrombin-induced Ca²⁺ transients in primary CFs from WT (black) and TRPC-hepta (Trpc1/2/3/4/5/6/7^–/–) KO (red) mice. Ca²⁺ transients were measured in the presence of 2 mM extracellular Ca²⁺. Left panels: Original traces and right panels: Mean values of three independent preparations (hearts). (C) AngII-induced Ca²⁺ release and Ca²⁺ entry in primary CFs from WT (black) and TRPC-hepta KO (red) mice. Ca²⁺ release was measured in the absence of extracellular Ca²⁺ (300 µM EGTA) and Ca²⁺ entry was monitored in the in the presence of 2 mM extracellular Ca²⁺. Left panels: Original traces and right panels: Mean values of three independent preparations. (D) The effect of the CRAC blocker (10 µM) GSK7975A on the AngII-induced Ca²⁺ release was analyzed in CFs from WT mice like in (C). Left panels: Original traces and right panels: Mean values of three independent preparations. n = number of independent preparations (hearts). All cells were analyzed 5 days after isolation and were cultured at high density. * p < 0.05 and ** p < 0.01 according to the unpaired Student’s t-test.