A TRPC6-dependent pathway for myofibroblast transdifferentiation and wound healing in vivo

Abstract

After injury or cytokine stimulation, fibroblasts transdifferentiate into myofibroblasts, contractile cells that secrete extracellular matrix for wound healing and tissue remodeling. Here, a genome-wide screen identified TRPC6, a Ca(2+) channel necessary and sufficient for myofibroblast transformation. TRPC6 overexpression fully activated myofibroblast transformation, while fibroblasts lacking Trpc6 were refractory to transforming growth factor β (TGF-β) and angiotensin II-induced transdifferentiation. Trpc6 gene-deleted mice showed impaired dermal and cardiac wound healing after injury. The profibrotic ligands TGF-β and angiotensin II induced TRPC6 expression through p38 mitogen-activated protein kinase (MAPK) serum response factor (SRF) signaling via the TRPC6 promoter. Once induced, TRPC6 activates the Ca(2+)-responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPC6-dependent transdifferentiation and dermal wound healing. These results demonstrate an obligate function for TRPC6 and calcineurin in promoting myofibroblast differentiation, suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, p38 MAPK, and SRF.

Figure 1

TRPC6 overexpression promotes fibroblast to myofibroblast conversion. (A) αSMA-luciferase and Postn-luciferase promoter activity from cultured neonatal rat cardiac fibroblasts transfected with expression vectors encoding TRPC6 or IL6, or treated with recombinant TGFβ(−/−). (B) Immunofluorescent staining of αSMA (red) positive stress fibers and collagen I (green) in 3 different fibroblast cell lines infected with adenovirus (Ad) encoding TRPC6 or stimulated with recombinant TGFβ. Scale bar is 50 µm. (C) Quantification of the experiment shown in B, except that cardiac fibroblasts were used, as was AngII treatment (100 nM). N≥ 700 cells per group. Control (Con.) represents Adβgal infection or no drug. *P<0.05 vs control. (D) Western blot for fibronectin ED-A in AdTRPC6 infected versus uninfected control cardiac fibroblasts (GAPDH serves as a loading control). (E) Photographs and (F) quantification of floating collagen gel matrices seeded with cardiac fibroblasts that have contracted after 36 hrs of TGFβ or AdTRPC6 infection. *P<0.05 vs. control; N=3 independent experiments. Arrows in E show area of gel contraction. (G) Cardiac fibroblast proliferation measured by colormetric MTT (tetrazoleum) assay 48 hrs after AdTRPC6 or Adβgal (con.) infection in serum-free or medium containing 10% fetal bovine serum (FBS). N=3 independent experiments. All data represent the mean ± s.e.m (error bars).

Figure 2

Loss of TRPC6 prevents TGFβ-mediated myofibroblast conversion. (A) Immunofluorescent staining of αSMA (green) positive stress fibers and TOPRO-3 iodide nucleic acid stain (blue) in primary Trpc6^+/+ (Wt) and Trpc6^−/− dermal fibroblasts, with and without recombinant TGFβ stimulation. Scale bar is 50 µm. (B) Photographs and (C) quantification of contraction of floating collagen gel matrices seeded with Trpc6^+/+ or Trpc6^−/− dermal fibroblasts at 36 hours post TGFβ. *P<0.05 vs. control; #P<0.05 vs. Trpc6^+/+ with TGFβ; N=3. The arrow in B shows contraction of the gel. (D) Quantification of in vitro scratch closure rates in Trpc6^+/+ and Trpc6^−/− fibroblast monolayers in culture in serum free and 10% fetal bovine serum conditions (FBS). The scratch area was measured at time 0 and 24 hours later. *P<0.05 vs. Trpc6^+/+; N=3. All data represent the mean ± s.e.m (error bars).

Figure 3

TRPC6 is necessary for dermal and cardiac wound healing. (A) RT-PCR for TRPC6 mRNA from skin wounds of 3 separate mice or uninjured skin of 2 control mice. GAPDH is used as a control. (B) Photographs of 2 full excision 6 mm dorsal coat (skin) punch biopsies taken on 8–10 week old Trpc6^+/+ and Trpc6^−/− littermates at day 0, and the progression shown again at day 5. (C) Quantification of dermal would closure rates. N=8 per group. *P<0.05 vs Trpc6^+/+. (D and E) Wound border zone pictures and quantitation at 3 days post biopsy stained with isolectin (green) to detect epithelial tissue and αSMA (red) to detect myofibroblasts, which was quantified. Scale bar = 0.1 mm. *P<0.05 vs Trpc6^+/+; N=4 per group. (F) Survival curves of Trpc6^+/+ (Wt) and Trpc6^−/− (KO) littermates after sham or permanent coronary ligation surgery (MI). (G) Quantification of scar size at 1-week post coronary ligation in surviving Trpc6^+/+ and Trpc6^−/− mice. *P<0.05 vs Trpc6^+/+; N=5 per group. (H, I) M-mode echocardiographic assessment of left ventricular fractional shortening (FS %) and left ventricular end diastolic dimensions (LVED) in the indicated groups of mice. *P<0.05 vs Wt sham; #P<0.05 vs Wt MI; N=10–14 per group. All data represent the mean ± s.e.m (error bars).

Figure 4

Non-canonical TGFβ-mediated signaling induces TRPC6 gene expression to activate myofibroblasts. (A) Representative RT-PCR for TRPC family members in which PCR product was sampled every 5 cycles starting at cycle 20 from cardiac fibroblasts ± 12 hours of TGFβ treatment. (B) Immunofluorescent staining of TRPC6 (green) and TOPRO-3 iodide nucleic acid stain (blue) in cardiac fibroblasts treated with TGFβ or infected with AdTRPC6. Scale bar is 50 µm. (C) Real time PCR for TRPC6 induction (normalized to 18sRNA) from cardiac fibroblasts that were simultaneously treated for 24 hours with profibrotic molecules (TGFβ or AngII) ± non-canonical (p38 inhibitor, SB731445) or canonical (adenoviral SMAD6/7) inhibitors. *P<0.05 vs media; #P<0.05 vs control with same treatment; N=3 independent experiments run in duplicate. (D) Immunofluorescent staining and (E) quantification of αSMA (green) positive stress fibers and TOPRO-3 iodide nucleic acid stain (blue) in cardiac fibroblasts that were treated for 48 hours with TGFβ. Some groups also received non-canonical (p38 inhibitor, SB731445) or canonical (AdSMAD7 infection) inhibitors as depicted. *P<0.05 vs control; #P<0.05 vs baseline with same treatment; N≥250 cells per group over 2 independent experiments. Scale bar is 50 µm. (F) Immunofluorescent staining of αSMA expression (green) in fibroblasts expressing a scrambled shRNA control or shRNA-anti-ALK5 that were also treated with TGFβ, AngII or infected with AdTRPC6, or AdMKK6. Nuclei are shown in blue. Scale bar is 50 µm. All data represent the mean ± s.e.m (error bars).

Figure 5

SRF mediates TRPC6 gene expression and myofibroblast transdifferentiation. (A) Real time PCR analysis of TRPC6 mRNA in primary cardiac fibroblasts infected with the indicated adenoviruses, with or without p38 inhibitor (SB731445). *P<0.05 vs Adβgal; #P<0.05 vs AdSRF + vehicle. Results are averaged from 3 independent experiments. (B) TRPC6-luciferase promoter activity (-1187 bp) from fibroblasts co-transfected with the indicated plasmids or treated with TGFβ or SB731445. *P<0.05 vs media (untransfected); #P<0.05 versus TGFβ or SRF; †P<0.05 vs SRF alone. (C) ChIP assay from the TRPC6 promoter regions (−1238 to −855 bp) for SRF using a standard or phospho-specific SRF antibody. Cells were previously treated with or without TGFβ or infected with Ad-shSRF or Ad-shGFP as a control. (D) Immunocytochemistry for αSMA (green) in Wt or Trpc6^−/− primary dermal fibroblasts infected with Adβgal or AdSRF. Red staining is SRF. Scale bar is 50 µm. (E) Quantitation of percentage of αSMA converted Wt or Trpc6^−/− fibroblasts treated with the agonist or adenovirus shown. (N=3 experiments; *P<0.05 vs Adβgal Wt; #P<0.05 vs Wt of the same treatment). (F) Western blot for SRF or GAPDH in fibroblasts infected with AdshSRF or Ad-shGFP control. (G) Immunocytochemistry for αSMA (green) or (H) collagen gel contraction assays in control (shGFP) or shSRF adenoviral infected primary dermal fibroblasts treated with TGFβ, AngII, or co-infected with the indicated adenoviruses. Scale bar is 50 µm. (N=3 experiments; *P<0.05 vs control; #P<0.05 vs shGFP of the same co-treatment. NS = not significant). All data represent the mean ± s.e.m (error bars).

Figure 6

TRPC6-mediated Ca²⁺ signaling mediates myofibroblast transdifferentiation. (A) Quantification of store-operated calcium entry (SOCE) for control (no treatment or Adβgal infection, N=24), TGFβ treated (N=25), and AdTRPC6 (N=54) infected cardiac fibroblasts. *P<0.05 vs control; 3 independent experiments were performed. (B) Quantification of SOCE in Trpc6^+/+ and Trpc6^−/− dermal fibroblasts ± 48 hr TGFβ treatment. *P<0.05 vs control Trpc6^+/+; #P<0.05 vs TGFβ in Trpc6^+/+ fibroblasts; N≥20 cells per group derived from 3 independent experiments. (C) Quantification of SOCE from control (N=159), TGFβ treated (N=71), and AdTRPC6 (N=41) infected cardiac fibroblasts ± p38 inhibitor (N=32) or ± AdMKK6 (N=32). *P<0.05 vs media under control conditions; #P<0.05 vs TGFβ with media; consisting of over 3 independent experiments. SOCE is calculated as the difference between the peak Ca²⁺ signal obtained during Ringer’s reperfusion and the steady-state Ca²⁺ level reached after CPA treatment. (D) Immunofluorescent staining and (E) quantification of αSMA (red) positive stress fibers and collagen I (green) in cardiac fibroblasts 48 hrs post adenoviral infection with Adβgal, AdTRPC6, AdΔCnA, AdCain (calcineurin-NFAT inhibitor), or coinfection with AdTRPC6 + AdCain. *P<0.05 vs Adβgal; #P<0.05 vs AdTRPC6; N≥750 cells per group from 2 independent experiments. (F) N-luciferase activity from cardiac fibroblasts infected with the reporter AdNFATx9-Luc, AdTRPC6, AdΔCnA or treated with recombinant TGFβ. *P<0.05 vs control; N=3 independent experiments. (G) Immunofluorescent staining of αSMA (red) positive stress fibers and the localization of NFAT-GFP in cardiac fibroblasts coinfected with AdNFATc1-GFP and AdTRPC6, AdΔCnA, or treated with TGFβ. Arrows indicate fibroblasts that have αSMA stress fibers and NFAT in the nucleus. All data represent the mean ± s.e.m (error bars).

Figure 7

The TRPC6-Calcineurin-NFAT signaling axis is necessary for dermal wound healing. (A) Photographs and (B) quantification of floating collagen gel matrix contraction rates seeded prior with Trpc6^+/+ or Trpc6^−/− dermal fibroblasts at 48 hrs post infection with AdTRPC6, AdΔCnA, or treated with recombinant TGFβ. Arrows indicate the contracted diameter of the collagen matrix. *P<0.05 vs. baseline; #P<0.05 vs. Trpc6^+/+ TGFβ; †P<0.05 vs. TGFβ in Trpc6^−/−; N=3 independent experiments. (C) Photographs of 2 full excision 6 mm dorsal dermal punch biopsies taken on 8–10 week old Trpc6^+/+ or Trpc6^−/− littermates 4 days after the biopsy. Topical application of adenovirus containing GFP, TRPC6 or ΔCnA was applied to the site just after wounding. (D) Time course of wound closure for Trpc6^−/− mice compared with Trpc6^+/+ controls and treated with the indicated adenovirus. N=8 mice per group. *P<0.05 vs Trpc6^+/+ (Wt); #P<0.05 vs Trpc6^−/− AdGFP; †P<0.05 vs Trpc6^−/− alone or Trpc6^−/− AdGFP. (E) Time course of wound closure for Trpc6^+/+, Trpc6^−/−, or Ppp3cb^−/− mice. N=8 per group. *P<0.05 vs Trpc6^+/+ (Wt); #P<0.05 vs Ppp3cb^−/−. (F) Signaling model for myofibroblast transdifferentiation whereby non-canonical TGFβ signaling cascade through p38 signaling to SRF, mediates a transcriptional upregulation of TRPC6 gene expression, enhancing Ca²⁺ entry leading to calcineurin (CnA) activation, sending NFAT to the nucleus to participate in myofibroblast phenotypic conversion. All data represent the mean ± s.e.m (error bars).