TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis

Figures

• 

  Figure 1. Inhibition of TRPC3 channel activity suppresses cardiac fibrosis but not hypertrophy in pressure overloaded mouse hearts. (a) Representative images of wheat germ agglutinin (WGA) staining for cross-sectional areas (CSA) measurement 6 weeks after TAC (left). Green; WGA, blue; DAPI. Quantitative results are shown in right panel. TRPC3(+/+)-sham (n = 6), TRPC3(−/−)-sham (n = 6), TRPC3(+/+)-TAC (n = 13), TRPC3(−/−)-TAC (n = 12). (b) Representative images of fibrosis 6 weeks after TAC. (c) Expression levels of hypertrophy-related and fibrosis-related mRNAs in mouse hearts 1 week after TAC. Sham groups (n = 3), TAC groups (n = 5). (d,e) Correlation between fibrosis and hypertrophy 6 weeks after TAC. TRPC3(+/+)-TAC (n = 13), TRPC3(−/−)-TAC (n = 12), Vehicle-TAC or pyrazole-3 (Pyr3)-TAC (n = 15). Error bars, s.e.m. *P < 0.05, **P < 0.01.

• 

  Figure 2. Involvement of GEF-H1 in TRPC3-mediated cardiac fibrosis in pressure overloaded mouse hearts. (a) Activation of RhoA in TRPC3(+/+) and TRPC3(−/−) mouse hearts 1 week after TAC (n = 3). (b) Representative image of SYPRO Ruby staining of gels loaded with RhoAG17A-pulled down proteins. Fibroblasts were stimulated with TGF-β2 (2 ng/mL) for 6 h. (c) Activation and expression of GEF-H1 in mouse hearts 1 week after TAC (n = 3). (d) Expression of LARG in mouse hearts 1 week after TAC (n = 3). Error bars, s.e.m. *P < 0.05, **P < 0.01.

• 

  Figure 3. Requirement of TRPC3 in mechanical stress-induced activation of GEF-H1 and RhoA, and induction of fibrosis-related mRNA expressions in NRCMs. (a) Effect of pyrazole-3 (Pyr3) on RhoA activation induced by mechanical stretch (MS) in NRCMs. NRCMs were treated with Pyr3 (1 μM) 20 min before MS (20% static-MS for 10 min). Lower panel is the quantitative result of RhoA pulldown assay (n = 3). (b) Effect of TRPC3 knockdown on MS-induced increase in fibrosis-related genes (CTGF and TGF-β2). NRCMs were subjected to 20% static-MS for 6 h. (c) Effects of TRPC3 knockdown on GEF-H1 activation. NRCMs were subjected to 20% static-MS for 10 min (n = 3). (d,e) Effects of GEF-H1 siRNAs (#42, 43 and 44) on GEF-H1 protein (d) and CTGF mRNA (e) expressions induced by MS (20% static MS for 6 h) in NRCMs (n = 4). Error bars, s.e.m., *P < 0.05, **P < 0.01.

• 

  Figure 4. Inhibition of TRPC3 attenuates TGF-β-induced myofibroblast transdifferentiation of neonatal rat cardiac fibroblasts. (a) Representative α-SMA-stained images of rat cardiac fibroblasts. Cells were treated with Pyr3 (1 μM) for 20 min prior to treatment with TGF-β2 (2 ng/mL) for 48 h (n = 3). (b,c) Effect of Pyr3 (1 μM) on TGF-β2 (2 ng/mL for 6 h)-induced CTGF mRNA expression (b) and [3H] proline incorporation (c) in fibroblasts (n = 3). (d) Effects of TRPC3 knockdown on [3H] proline incorporation in fibroblasts with or without Ang II (100 nM), endothelin-1 (ET-1; 100 nM) or TGF-β2 (n = 3). (e,f) Effects of TRPC3 knockdown on GEF-H1 (e) and RhoA (f) activation induced by TGF-β2 for 6 h. (n = 3) (g) Effects of GEF-H1 knockdown on the TGF-β2-induced increase in α-SMA expression (n = 3). Error bars, s.e.m. *P < 0.05, **P < 0.01.

• 

  Figure 5. Involvement of microtubule stability in TRPC3/Nox2-mediated GEF-H1 activation. (a) Representative images of microtubule organization in rat cardiac fibroblasts with or without TGF-β2 (2 ng/mL) for 6 h, in the presence or absence of Pyr3 (1 μM). Right panels were magnified views indicated with white boxes. Bar graph represents the semi-quantitative results of microtubule densities (n = 6). (b,c) Effects of knockdown (b) or pharmacological inhibition (c) of Nox2 on GEF-H1 activation induced by MS. NRCMs were subjected to 20% static-MS for 10 min (n = 3). (d) Involvement of tubulin stability on MS-induced GEF-H1 activation in NRCMs. NRCMs were treated with taxol (1 μM) 30 min prior to 20% static-MS for 10 min (n = 3). (e) Effect of Nox2 knockdown on MS-induced GEF-H1 activation in NRCMs. NRCMs were treated with okadaic acid (0.1 μM) 30 min prior to 20% static-MS for 10 min (n = 3). Error bars, s.e.m. *P < 0.05, **P < 0.01.

• 

  Figure 6. Inhibition of TRPC3 attenuates fibrotic response in human iPSC-derived cardiomyocytes or human cardiac fibroblasts. (a–c) Effect of Pyr3 (1 μM) on induction of fibrosis-related mRNAs (CTGF and TGF-β1 or -β2) by MS in human iPSC-derived cardiomyocytes (n = 3). Cells were subjected to 20% static-MS for 3 h. GAPDH and 18 S ribosomal RNA were used as internal controls. (d–g) Effects of knockdown (d) or pharmacological inhibition (e) of TRPC3 on CTGF mRNA expression (d,e) and α-SMA protein expression (f,g) induced by TGF-β2 stimulation in human cardiac fibroblasts (n = 3). Error bars, s.e.m. *P < 0.05, **P < 0.01. (h) Schema for the mechanism of TRPC3-mediated cardiac fibrosis induced by pressure overload. TRPC3 mediates MS-induced expression of profibrotic factors (CTGF and TGF-β) in cardiomyocytes and TGF-β-induced fibrotic responses of cardiac fibroblasts, partially through Nox2-dependent GEF-H1 activation.