Atrial fibrillation induces myocardial fibrosis through angiotensin II type 1 receptor-specific Arkadia-mediated downregulation of Smad7

Abstract

Rationale: Tachycardia-induced atrial fibrosis is a hallmark of structural remodeling of atrial fibrillation (AF). The molecular mechanisms underlying the AF-induced atrial fibrosis remain unclear.

Objective: To determine the role of angiotensin II (Ang II)/Ang II type 1 (AT(1)) receptor-coupled transforming growth factor (TGF)-β(1)/Smad signaling pathway in the AF-induced atrial fibrosis.

Methods and results: Rapid atrial pacing (1000 ppm) was applied to the left atrium of rabbit heart to induce atrial fibrillation and fibrosis. Quantitative PCR and Western blot analysis revealed that rapid atrial pacing caused a marked increase in the expression of Ang II, TGF-β(1), phosphorylated Smad2/3 (P-Smad2/3), Arkadia, and hydroxyproline synthesis. However, the expression of Smad7, a key endogenous antagonist of the TGF-β(1)/Smad-mediated fibrosis, was significantly decreased. These changes were dose-dependently reversed by AT(1) receptor antagonist losartan, implicating the involvement of AF-induced release of Ang II and activation of AT(1) receptor-specific pathway. In the adult rabbit cardiac fibroblasts, Ang II increased the expression of TGF-β(1), P-Smad2/3, Smad4, Arkadia, and collagen I synthesis and significantly reduced Smad7 expression. These effects of Ang II were reversed by losartan but not by the AT(2) antagonist (PD123319). In addition, extracellular signal-regulated kinase inhibitor and anti-TGF-β(1) antibody also blocked the Ang II-induced downregulation of Smad7. Silencing of Smad7 gene by small interfering RNA abolished the antagonism of losartan on the fibrogenic effects of Ang II on cardiac fibroblasts, whereas overexpression of Smad7 blocked Ang II-induced increase in collagen I synthesis.

Conclusions: Ang II/AT(1) receptor-specific activation of Arkadia-mediated poly-ubiquitination and degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-β(1)/Smad signaling and serves as a key mechanism for AF-induced atrial fibrosis.

Figure 1. Rapid atrial pacing-induced atrial fibrillation and changes in collagen contents in the left atrium of rabbit heart in the absence and presence of losartan

A. Representative surface ECG recordings before (a) and after (b) the rapid atrial pacing (RAP). a. ECG recordings in leads avR, avL and avF show that the rabbit was in normal sinus rhythm before RAP. The mean sinus heart rate was 173±18 bpm (n=8). b. ECG recordings in leads avR, avL and avF show that after 4 weeks of RAP the rabbit displayed an irregular atrial rhythm with irregular ventricular response: disappearance of P-wave, and absolute irregularity of the RR interval. The mean heart rate was 293±16 bpm (n=8). B. Masson trichrome-staining of rabbit atrial samples subjected to control (N), sham (N), RAP (P), and RAP in the presence of three different doses (D₁, D₂, and D₃) of AT₁ receptor antagonist losartan. Myocardium was stained red and collagens were stained blue. Bar = 50 ⎧m. C. Mean data of collagen content in the left atrium of the 6 groups (n = 8 per group). RAP caused significant deposition of collagens in the left atria, which can be attenuated by losartan in a dose-dependent manner. * P<0.05 compared with Group N, # P<0.05, ## P<0.05 compared with Group P.

Figure 2. Expression of TGF-β₁ and Smads in the left atrium of rabbit heart

A. Quantitative analysis of transcriptional expression of TGF-β₁ and Smad7 by real-time RT-PCR. a. The TGF-β₁ mRNA levels relative to GAPDH expression ratio (TGF-β₁/GAPDH in arbitrary unit) was normalized to that of Group N and Group S and the relative expression levels (in fold expression) were calculated (* P<0.05 vs Group N, # P<0.05 vs Group P, n=5 for each group). b. The relative Smads mRNA expression ratio (Smad7/GAPDH in arbitrary unit) in left atria was normalized to that of Group N and Group S and the relative expression levels (in fold expression) were calculated (* P<0.05, ** P<0.01, *** P<0.001 vs Group S, # P<0.05 vs Group P, n=5 for each group). B. a. Representative Western blot gel depicts the protein expression of TGF-β₁, Smad7, P-Smad2/3, and Arkadia. b-e. Mean value of the protein expression level of TGF-β₁ (b), Smad 7 (c), P-Smad2/3 (d) and Arkadia (e) in control (Group N), sham (Group S), RAP (Group; P) and RAP in the presence of three different doses (Group D₁, D₂, D₃) of AT₁ receptor agonist losartan. RAP caused a significant increase in expression of TGF-β₁, P-Smad2/3, and Arkadia but decreased the expression of Smad7. Losartan reversed the effect of RAP on the expression of these proteins in a dose-dependent manner (* P<0.05, ** P<0.01, *** P<0.001 vs Group S, # P<0.05 vs Group P, n=5 for each group).

Figure 3. AngII induced TGF-®₁ and Smads expression in cultured cardiac fibroblasts

A. Representative Western blot gel depicts the protein expression of TGF-β₁, P-Smad2/3, Smad4, Arkadia, and collagen I. B-F. Mean value of the protein expression level of TGF-β₁ (B), P-Smad2/3 (C), Smad4 (D), Arkadia (E) and collagen I (F) under control, AngII alone, AngII+losartan, and AngII+PD123319 conditions (mean±S.E., n=4, * P<0.05, ** P<0.01, *** P<0.001 vs control; # P<0.05, ### P<0.001 vs AngII).

Figure 4. AT1 receptor antagonist induced Smad7 expression in cardiac fibroblasts

A. a. The fibroblasts were treated without (-) or with (+) specific proteasome inhibitor lactacystin (10_-4 mmol/L) for 3 h before cells treated with AngII, losartan and PD123319. b. Mean value (mean±SEM) of Smad7 protein expression obtained from densitometric analysis and expressed as ratio of Smad7 /β-actin under the corresponding conditions as indicated in a In the absence of lactacystin, AngII decreased Smad7 expression, which could be reversed by losartan, but not by PD123319. In contrast, in the presence of lactacystin, AngII caused no changes in Smad7 (n = 6, * p<0.001 vs control; # p<0.001 vs AngII). B. a. Representative Western blot gel depicts the effects of anti-TGF-β₁ antibody and ERK1/2 inhibitor on AngII-induced Smad7 expression. Cardiac fibroblasts were incubated with AngII (10_-6 mmol/L), AngII+losartan (10_-5 mmol/L), AngII+PD123319 (10_-5 mmol/L), AngII+anti-TGF-β₁ antibody (10 μg/ml), or AngII+ PD98059 (10_-4 mmol/L) for 48 h. Losartan, PD123319, anti-TGF-β₁ antibody, and PD98059 were added 1 hour before the addition of AngII. b. The mean values (mean±S.E.) from 5–6 separate experiments (*P<0.001 vs control, # P<0.001 vs AngII). C. Immunohistochemistry staining of Smad7. Bar = 50 um. Cells treated with the same conditions as Western bolt described in B.

Figure 5. Effect of knockdown of Smad2 or Smad3 on Smad7 and collagen I expression in cardiac fibroblasts

A. Cardiac fibroblasts were transfected with control siRNA, Smad2 siRNA, or Smad3 siRNA. a. Representative Western blot gel depicts the protein expression of Smad2 in cells without transfection (non-transf.), or with transfection of control siRNA or Smad2 siRNA. Smad2 siRNA successfully knocked down the expression of Smad2. b. Representative Western blot gel depicts the protein expression of Smad3 in cells without transfection (non-transf.), or with transfection of control siRNA or Smad3 siRNA. Smad3 expression was successfully knocked down by Smad3 siRNA. B. Representative Western blot gels depicts the protein expression of collagen I and Smad7 in cardiac fibroblsts transfected with control siRNA, Smad2 siRNA or Smad3 siRNA after the cells were treated with AngII (10_-6 mmol/L), losartan (10_-5 mmol/L), or AngII+losartan. C. The mean values (mean±S.E., n=10) of relative expression levels (normalized to β-actin) of collagen I and Smad7 under corresponding conditions (* P<0.05, *** P<0.001 vs non-transfection; ### P<0.001 vs Control siRNA).

Figure 6. Effects of Smad7 knockdown or overexpression on collagen I expression in cardiac fibroblasts

A. Cardiac fibroblasts were transfected with control siRNA and Smad7 siRNA. a. Representative Western blot gel depicts the protein expression of Smad7 in cells without transfection (non-transf.), or with transfection of control siRNA or Smad7 siRNA. Smad7 siRNA successfully knocked down the expression of Smad7. b. Representative Western blot gels depicts the protein expression of collagen I and Smad7 in cardiac fibroblsts transfected with control siRNA or Smad7 siRNA when exposed to AngII (10_-6 mmol/L), losartan (10_-5 mmol/L), or AngII+losartan. c. The mean values of expression ratio of collagen I or Smad7 over β-actin under corresponding conditions (mean±S.E., n=7, *** P<0.001 vs non-transf; ### P<0.001 vs control siRNA). B. a. Representative Western blot gel depicts the protein expression of Smad7 in cardiac fibroblasts transfected with empty pcDNA3 vector (V) or pcDNA3-FLAG-Smad7 (OE). Overexpression of Smad7 caused a significant increase in Smad7 protein expression. Representative Western blot gels (b) and the mean values (mean±SEM) of collagen I protein expression (c) obtained from densitometric analysis and expressed as ratio over β-actin in cardiac fibroblasts treated with either AngII (10_-6 mmol/L), losartan (10_-5 mmol/L), or AngII+losartan. Overexpression (OE) of Smad7 caused a significant decrease in collagen I expression under basal conditions (in the absence of AngII) compared to the control (C, without transfection) cells and the cells transfected with empty vectors (V) (n=7, * P<0.05 vs no transfection or empty vector).