TRPV4 Channels Promote Pathological, but Not Physiological, Cardiac Remodeling through the Activation of Calcineurin/NFAT and TRPC6

Abstract

TRPV4 channels, which respond to mechanical activation by permeating Ca²⁺ into the cell, may play a pivotal role in cardiac remodeling during cardiac overload. Our study aimed to investigate TRPV4 involvement in pathological and physiological remodeling through Ca²⁺-dependent signaling. TRPV4 expression was assessed in heart failure (HF) models, induced by isoproterenol infusion or transverse aortic constriction, and in exercise-induced adaptive remodeling models. The impact of genetic TRPV4 inhibition on HF was studied by echocardiography, histology, gene and protein analysis, arrhythmia inducibility, Ca²⁺ dynamics, calcineurin (CN) activity, and NFAT nuclear translocation. TRPV4 expression exclusively increased in HF models, strongly correlating with fibrosis. Isoproterenol-administered transgenic TRPV4-/- mice did not exhibit HF features. Cardiac fibroblasts (CFb) from TRPV4+/+ animals, compared to TRPV4-/-, displayed significant TRPV4 overexpression, elevated Ca²⁺ influx, and enhanced CN/NFATc3 pathway activation. TRPC6 expression paralleled that of TRPV4 in all models, with no increase in TRPV4-/- mice. In cultured CFb, the activation of TRPV4 by GSK1016790A increased TRPC6 expression, which led to enhanced CN/NFATc3 activation through synergistic action of both channels. In conclusion, TRPV4 channels contribute to pathological remodeling by promoting fibrosis and inducing TRPC6 upregulation through the activation of Ca²⁺-dependent CN/NFATc3 signaling. These results pose TRPV4 as a primary mediator of the pathological response.

Keywords: TRP; TRPC6; TRPV4; calcium; exercise; heart failure; mechanoreceptors; mechanotransduction; pathological remodeling; physiological remodeling.

Figure 1

Differential features between physiological (green) and pathological (red) remodeling. From (A–G), results of the four experimental groups generated in mice (N = 6–12/group). (A) Echocardiographic parameters of interventricular septum thickness in diastole (IVSd), left ventricular diameter in diastole (LVDd), and ejection fraction (EF). (B) Heart weight-to-tibial length (HW/TL) ratio. On the right, representative images visually depict the size of the heart. (C) Overall quantification of cardiomyocyte cross-sectional area (CSA) with representative photomicrographs of the four study groups stained with hematoxylin and eosin (H&E). Scale bar represents 50 µm. (D) Percentage of fibrosis measured by collagen deposition with representative images of the four study groups stained with picrosirius red. Scale bar corresponds to 200 µm. (E) Gene expression of fibrotic markers in the four study groups. (F) Gene and protein expression of TRPV4 in the four study groups. (G) Gene expression of TRPV4 in the four experimental groups studied in rats (see the Methods section, N = 12/group). Ex: exercise; Sed: sedentary; HF(iso): HF induced by isoproterenol infusion; HF(TAC): HF induced by transverse aortic constriction. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 2

Time-course changes during the development of pathological remodeling induced by isoproterenol infusion (N = 5–18/group). (A) Echocardiographic parameters of interventricular septum thickness in diastole (IVSd), left ventricular diameter in diastole (LVDd), and ejection fraction (EF). (B) Heart weight-to-tibial length ratio (HW/TL, left) and cardiomyocyte cross-sectional area (CSA, right). (C) Fibrosis quantification by percentage of collagen deposition. (D) Representative microphotographs stained with picrosirius red at each timepoint. Scale bar corresponds to 200 µm. (E) Protein expression of TRPV4 channels over time. (F) Correlation of TRPV4 expression with cardiomyocyte CSA (left) and collagen deposition (right) at all timepoints. Iso 3d, 7d, 14d, and 28d represent mice subjected to isoproterenol infusion for 3, 7, 14 and 28 days, respectively. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 3

Induction of pathological remodeling in TRPV4+/+ and TRPV4−/− mice (N = 6–12/group). (A) Gene and protein expression of TRPV4 channels in the four study groups. (B) Echocardiographic parameters of interventricular septum thickness in diastole (IVSd), left ventricular diameter in diastole (LVDd), and ejection fraction (EF). (C) Heart weight-to-tibial length (HW/TL) ratio. (D) Overall quantification of cardiomyocyte cross-sectional area (CSA), with representative hematoxylin and eosin (H&E) stained images of all experimental groups. Scale bar represents 50 µm. (E) Fibrosis quantification by percentage of collagen deposition with representative microphotographs stained with picrosirius red. Scale bar corresponds to 200 µm. (F) Arrhythmia inducibility under normoxia and ischemia. The number of total ventricular tachyarrhythmias (VTA) are shown for each condition. HF(iso): HF induced by isoproterenol infusion; nd: not detected; * p < 0.05; ** p < 0.01; and **** p < 0.0001.

Figure 4

Functional dynamics of Ca²⁺ in response to specific TRPV4 activation and/or inhibition (N = 4–8 replicates per group from 3–4 independent experiments). (A) Relative expression of TRPV4 in cardiomyocytes (CM) and fibroblasts (FB). (B) Calcium influx recorded in FB from TRPV4+/+ and TRPV4−/− mice (groups HF(iso) and sham) in response to the selective TRPV4 activator GSK1016790A (GSK10, 100 nM) in the absence (left) or the presence (right) of the TRPV4 inhibitor HC067047 (HC, 10 µM). (C) Calcium influx in response to a hypotonic solution (140 mOsm) in the absence (left) or the presence (right) of the TRPV4 inhibitor HC067047 (HC, 10 µM). F/F0 = ratio of fluorescence intensity relative to time 0; HF(iso): HF induced by isoproterenol infusion. In (B,C), all Ca²⁺ measures are expressed as the change with respect to baseline values, which have been normalized to 1. * p < 0.05.

Figure 5

(A) Calcineurin (CN) activity and (B) protein expression in isolated FB from TRPV4+/+ and TRPV4−/− mice of the HF(iso) and sham groups (N = 4–5/group). (C) NFAT cytoplasmatic and nuclear fluorescence quantification expressed as the MGV N/C, the ratio of mean fluorescence intensity (mean gray value) between the nucleus (N) and the cytoplasm (C), n = 202–244. (D) Representative confocal microscopy images of all experimental groups. From top to bottom, each row represents NFAT staining (red), nuclear staining (blue), and merging. The negative control was not incubated with the primary antibody. The positive control was obtained by activation of CN following incubation with a high-calcium medium (4 mM). Scale bar corresponds to 150 µm. (E) Gene expression of col1a1 and acta2 in the four study groups. (F) NFAT nuclear translocation in isolated FB from TRPV4+/+ mice (WT), expressed as the MGV N/C, after specific TRPV4 stimulation with GSK101679A (GSK10 (100 nM), purple), and GSK + pre-incubation with the TRPV4 inhibitor HC067047 (HC (10 µM), gray) in isolated FB. * p < 0.05; ** p < 0.01; and **** p < 0.0001.

Figure 6

(A) TRPC6 protein expression in the HF and Ex models induced in mice (n = 5/group). (B) Gene expression of TRPC6 in the four experimental groups studied in rats (N = 12/group). (C) Time-course protein expression of TRPC6 channels during the development of pathological remodeling (N = 5–11/group). (D) TRPC6 protein expression in TRPV4+/+ and TRPV4−/− mice receiving isoproterenol (HF(iso)) or saline (sham) (N = 6–8/group). (E) Effects of Ca²⁺ overload and calcineurin inhibition with cyclosporine A (CsA) on NFATc3 nuclear translocation (left) and TRPC6 gene expression (right) in FB from TRPV4+/+ and TRPV4−/− mice from the HF(iso) and sham groups. (F) Proximity ligation assay (PLA) for heteromeric channel formation of TRPV4-TRPC1 and TRPV4-TRPC6. PLA was performed with a combination of anti-TRPV4, anti-TRPC1, and anti-TRPC6 antibodies conjugated to PLA PLUS or MINUS probes. The negative control was only incubated with PLA PLUS and MINUS probes. (G) In TRPV4+/+ mice (WT), TRPC6 (right) and TRPV4 (left) expression following exposure to the specific TRPV4 activator GSK10 (100 nM) without and with pre-incubation with CsA (1 µM), a CN inhibitor (N = 5–6/group). (H) NFAT nuclear translocation as a surrogate of activation of the CN/NFAT pathway in cardiac FB from TRPV4+/+ (WT) animals; experimental conditions were as follows: TRPV4 activation by GSK1016790A (GSK10, 100 nM), TRPC6 activation by GSK1702934A (GSK17, 1 µM), simultaneous activation of TRPV4 and TRPC6 (GSK10, 100 nM + GSK17, 1 µM), TRPV4 activation with previous inhibition of TRPC6 (GSK10, 100 nM + BI-749327, 1 µM), TRPC6 activation with previous inhibition of TRPV4 (GSK17, 1 µM + HC067047, 10 µM), and control. All conditions were significantly different (p < 0.0001) compared to controls. Ex: exercise; Sed: sedentary; HF(iso): HF induced by isoproterenol infusion; HF(TAC): HF induced by transaortic constriction; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.