Effect of efonidipine on TGF-β1-induced cardiac fibrosis through Smad2-dependent pathway in rat cardiac fibroblasts

Abstract

Transforming growth factor beta-1 (TGF-β1) plays a critical role in progression of cardiac fibrosis, which may involve intracellular calcium change. We examined effects of efonidipine, a dual T-type and L-type calcium channel blocker (CCB), on TGF-β1-induced fibrotic changes in neonatal rat cardiac fibroblast. T-type and L-type calcium channel mRNAs were highly expressed in cultured cardiac fibroblasts. TGF-β1 (5 ng/mL) significantly increased Smad2 phosphorylation and [(3)H]-leucine incorporation, which were attenuated by pretreatment with efonidipine (10 µM). Neither R(-)efonidipine (10 µM), selective T-type CCB, nor nifedipine (10 µM), selective L-type CCB, efficaciously inhibited both TGF-β1-induced Smad2 phosphorylation and [(3)H]-leucine incorporation. However, both were markedly attenuated by combination of R(-)efonidipine and nifedipine, EDTA, or calcium-free medium. Pretreatment with Smad2 siRNA significantly attenuated [(3)H]-leucine incorporation induced by TGF-β1. These data suggest that efonidipine elicits inhibitory effects on TGF-β1- and Smad2-dependent protein synthesis through both T-type and L-type calcium channel-blocking actions in cardiac fibroblasts.

Fig. 1

Time-course and dose-dependent effect of TGF-β1 on Smad2 phosphorylation in cardiac fibroblasts. A: TGF-β1 (5 ng/mL) used to stimulate cells. B: Cells stimulated with TGF-β1 for 30 min. Bar graphs represent the mean ± S.E.M. (n = 6), expressed as the fold change in phosphorylation compared with unstimulated cells. *P < 0.05 vs. control cardiac fibroblasts.

Fig. 2

Expression of T-type and L-type calcium channel mRNA in cardiac fibroblasts. A: A subunit of the T-type calcium channel, Cav3.1 (α1G), was highly expressed in cardiac fibroblasts. B: A subunit of the L-type calcium channel, Cav1.2 (CACNA1C), was highly expressed in cardiac fibroblasts. C and D: TGF-β1 (5 ng/mL, 24 h) did not increase T-type or L-type calcium channel in cardiac fibroblasts. Bar graphs represent the mean ± S.E.M. (n = 6), expressed as the fold change in mRNA expression compared to positive controls; mRNA levels are normalized to GAPDH mRNA levels.

Fig. 3

Effect of CCBs on TGF-β1–induced Smad2 phosphorylation in cardiac fibroblasts. Smad2 phosphorylation induced by TGF-β1 was measured in the absence or presence of efonidipine (10 μM) (A); R(−)efonidipine (10 μM), nifedipine (10 μM), calcium-free medium (B); combination of R(−)efonidipine and nifedipine (C); Y27632 (1 μM), wortmannin (100 nM) (D); or PD98059 (30 μM) (E). Bar graphs represent the mean ± S.E.M. (n = 6), expressed as the fold change in phosphorylation compared with unstimulated cells. *P < 0.05, control cardiac fibroblasts vs. TGF-β1 alone; #P < 0.05, TGF-β1 alone vs. TGF-β1 with treatment.

Fig. 4

Effect of TGF-β1 on [³H]-leucine incorporation in the absence or presence of R(−)efonidipine, nifedipine, calcium-free medium, EDTA (A) or the combination of R(−)efonidipine and nifedipine (B). Bar graphs represent the mean ± S.E.M. (n = 6), expressed as the fold change compared with unstimulated cells. *P < 0.05, control cardiac fibroblasts vs. TGF-β1 alone; ^#P < 0.05, TGF-β1 alone vs. TGF-β1 with treatment.

Fig. 5

Effect of Smad2 siRNA on protein expression (A) and TGF-β1–induced [³H]-leucine incorporation (B) in cardiac fibroblasts. Bar graphs represent the mean ± S.E.M. (n = 6), expressed as the fold change compared with unstimulated cells. *P < 0.05, control cardiac fibroblasts vs. TGF-β1 alone; ^#P < 0.05, TGF-β1 alone vs. TGF-β1 with treatment.