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

Figures

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  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…

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  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.

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  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.

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  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.

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  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.

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  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…