TRPV4 channels mediate cardiac fibroblast differentiation by integrating mechanical and soluble signals

Abstract

The phenotypic switch underlying the differentiation of cardiac fibroblasts into hypersecretory myofibroblasts is critical for cardiac remodeling following myocardial infarction. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extracellular matrix (ECM) during the wound healing process. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. TGF-β has been shown to promote differentiation and this, combined with the robust mechanical environment in the heart, lead us to hypothesize that the mechanotransduction and TGF-β signaling pathways play active roles in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF-β1-induced differentiation of cardiac fibroblasts into myofibroblasts. We found that the TRPV4-specific antagonist AB159908 and siRNA knockdown of TRPV4 significantly inhibited TGFβ1-induced differentiation as measured by incorporation of α-SMA into stress fibers. Further, we found that TGF-β1-induced myofibroblast differentiation was dependent on ECM stiffness, a response that was attenuated by TRPV4 blockade. Finally, TGF-β1 treated fibroblasts exhibited enhanced TRPV4 expression and TRPV4-mediated calcium influx compared to untreated controls. Taken together these results suggest for the first time that the mechanosensitive ion channel, TRPV4, regulates cardiac fibroblast differentiation to myofibroblasts by integrating signals from TGF-β1 and mechanical factors.

Figure 1. TRPV4 channels are required for TGF-β1-induced differentiation of cardiac fibroblasts

A) RT-PCR and Western blot analysis showing the expression of TRPV4 mRNA and protein in rat left ventricular fibroblasts. B) Calcium transients showing that a specific activator of TRPV4, GSK1016790A (10, 100 and 300 nM), induced calcium influx in rat cardiac fibroblasts. Arrow indicates addition of the stimulator. C) Quantitative analysis of relative changes (Δ F/F0) in calcium influx in CFs loaded with Fluo-4 and stimulated with TRPV4 specific activator, GSK1016790A. D) Immunofluorescence images of fibroblast showing the differentiation as evidenced by the incorporation of α-SMA (green) in to stress fibers. Nuclei were stained with DAPI (blue). CFs were serum starved and stimulated with TGF-β1 for 24 h in the presence or absence of TRPV4 antagonist AB159908 (AB1). E) Quantitative analysis of the fibroblast differentiation. Cells (n=300) were counted from independent images and the percentage of α-SMA positive cells was presented as % myofibroblasts. The results shown are mean ± SEM from 3 independent experiments (* p < 0.05).

Figure 2. siRNA knockdown of TRPV4 channels inhibits TGF-β1-induced differentiation of cardiac fibroblasts

A) Western blot analysis showing the TRPV4 protein expression in control siRNA and TRPV4 siRNA treated CFs. Tubulin served as a loading control. B) Quantitative analysis showing the specific knockdown of the expression of TRPV4 in rat CFs. C) Immunofluorescence images showing the significant inhibition of TGF-β1-induced CF differentiation in TRPV4 knockdowned cells as evidenced by the incorporation of α-SMA (green) in to stress fibers. Nuclei were stained with DAPI (blue). CFs were transfected with control or TRPV4 specific siRNAs and, serum starved and stimulated with TGF-β1 for 24 h. E) Quantitative analysis of the fibroblast differentiation in control and TRPV4 knockdown cells. Cells (n=300) were counted from independent images and the percentage of α-SMA positive cells was presented as % myofibroblasts. The results shown are mean ± SEM from 3 independent experiments (* p < 0.05). NS=Non-significant).

Figure 3. TRPV4 channels mediate TGF-β1-ECM stiffness-induced differentiation of cardiac fibroblasts

A) Photomicrographs of cardiac fibroblasts cultured on transglutaminase-linked gelatin hydrogels of varying stiffness (98, 370 and 2280 Pa). B) The histogram shows the average projected cell areas of cells (n=200) calculated using Metamorph and Image J software. C) Immunofluorescence images of fibroblast differentiation (the incorporation of α-SMA in to stress fibers (green) on gelatin hydrogels of varying stiffness (98–2280 Pa). Note: CFs were differentiated only on high stiffness gels. D) Immunofluorescence images showing TRPV4-dependent differentiation of CFs on high stiffness gels. CFs were serum starved and stimulated with TGF-β1 for 24 ( C, D ) in the presence or absence of TRPV4 antagonist AB159908 (AB1, D). Cells (n=300) were counted from independent images and the percentage of α-SMA positive cells was presented as % myofibroblasts. The results shown are mean ± SEM from 3 independent experiments (* p < 0.05).

Figure 4. TRPM7 channels are not required for TGF-β1-induced ECM stiffness dependant differentiation of cardiac fibroblasts

A) RT-PCR analysis showing the expression of TRPM7 mRNA B) Calcium transients showing that an activator of TRPM7 2-APB (5 mM), induced calcium influx in rat cardiac fibroblasts which is inhibited by TRPM7 blocker, Carvacrol (500 µM). Arrow indicates addition of the stimulator. C) Quantitative analysis of relative changes in calcium influx (Δ F/F0) in CFs loaded with Fluo-4 and stimulated with TRPM7 activator, 2-APB. D) Immunofluorescence images of fibroblast showing the differentiation as evidenced by the incorporation of α-SMA (green) in to stress fibers. CFs cultured on high stiffness gelatin hydrogel (2280 Pa) were serum starved and stimulated with TGF-β1 for 24 h in the presence or absence of TRPM7 antagonist Carvacrol (500 µM) E) Quantitative analysis of the fibroblast differentiation. Cells (n=300) were counted from independent images and the percentage of α-SMA positive cells was presented as % myofibroblasts. The results shown are mean ± SEM from 3 independent experiments (* p < 0.05). NS=Non-significant).

Figure 5. TGF-β1 induces increased TRPV4 expression and activity during cardiac fibroblast differentiation

A) Calcium transients showing that an increase in GSK1016790A (100 nM), induced calcium influx in TGF-β1 treated rat cardiac fibroblasts compared to controls. Arrow indicates addition of the stimulator. B) Quantitative analysis of relative changes (Δ F/F0) in calcium influx in CFs (n=300) pre-treated with TGF-β1 and stimulated with TRPV4 specific activator, GSK1016790A in the presence or absence of TRPV4 antagonist, AB159908 (AB1). RT-PCR (C) and Western blot (D) analysis of TRPV4 mRNA and protein levels in control and TGF-β1 treated CFs. The results shown are mean ± SEM from 3 independent experiments (*p < 0.05).