Rapid atrial pacing induces myocardial fibrosis by down-regulating Smad7 via microRNA-21 in rabbit

Abstract

Tachycardia-induced atrial fibrosis is a hallmark of the structural remodeling of atrial fibrillation (AF). The mechanisms underlying tachycardia-induced atrial fibrosis remain unclear. In our previous study, we found that Smad7-downregulation promoted the development of atrial fibrosis in AF. Fibroblasts are enriched in microRNA-21 (miR-21), which contributes to the development of fibrosis and heart failure in the cardiovascular system. Our study was designed to test the hypothesis that miR-21 reinforces the TGF-β1/Smad signaling pathway in AF-induced atrial fibrosis by down-regulating Smad7. Rapid atrial pacing (RAP, 1000 ppm) was applied to the left atrium of the rabbit heart to induce atrial fibrillation and fibrosis. qRT-PCR and northern blot analysis revealed that RAP caused a marked increase in the expression of miR-21. Transfection with a miR-21 inhibitor significantly increased the expression of Smad7, while the expression of collagen I/III significantly decreased. These changes were implicated in the AF-induced release of miR-21 and down-regulation of Smad7. Adult rat cardiac fibroblasts treated with TGF-β1 showed increased miR-21 expression and decreased Smad7 expression. Pretreatment with a TGF-β1 inhibitor reduced the TGF-β1-induced up-regulation of miR-21. Pretreatment with pre-miR-21 and a miR-21 inhibitor significantly decreased and increased Smad7 expression, respectively. This result was negatively correlated with the expression of collagen I/III in fibroblasts. Moreover, the results of a luciferase activity assay suggest that Smad7 is a validated miR-21 target in CFs. Our results provide compelling evidence that the miR-21 specific degradation of Smad7 may decrease the inhibitory feedback regulation of TGF-β1/Smad signaling and serves as a new insight of the mechanism of atrial fibrosis in atrial fibrillation.

Keywords: Atrial fibrillation; Atrial fibrosis; MicroRNA-21; Smad7; Transforming growth factor-β1.

Fig. 1

MiRNA expression profiling in atrial samples from a rabbit model of AF. a Quantitative real-time RT-PCR (qRT-PCR) verification of the miRNA expression profile in pacing rabbits. b Representative Northern blot depicts the expression of miR-21 mRNA. c Mean miR-21 expression levels in the control group (CR), sham group (SH), and rapid atrial pacing group (RAP) groups. RAP caused a significant increase in miR-21 expression. d Quantitative analysis of TGF-β₁ and Smad7 expression by real-time RT-PCR. The relative TGF-β₁/Smad7 mRNA levels (TGF-β₁/GAPDH or Smad7/GAPDH in arbitrary units) were normalized to the expression levels of group CR and group SH and the relative expression levels (in fold expression) were calculated. e Representative Western blot gel depicting the levels of TGF-β₁ and Smad7 protein. f Mean TGF-β₁ and Smad7 protein levels in the control group (CR), sham group (SH), and RAP group (RAP) groups. RAP caused a significant increase in expression of TGF-β₁, but decreased the expression of Smad7 (*p < 0.05 vs. CR, unpaired t test; n ≥ 9 independent samples for each group)

Fig. 2

The effect of a miR-21 inhibitor in RAP-induced myocardial fibrosis in vivo. a miR-21 mRNA levels were examined by qRT-PCR. The miR-21 inhibitor decreased miR-21 expression in the heart of experimental rabbits (rabbits were treated with the miR-21 inhibitor and RAP for 4 weeks). b Representative Northern blot showing miR-21. c Mean miR-21 expression levels in the CR group and the RAP+miR-21 inhibitor group. d Mean incidence of AF. In contrast to the RAP group (89 % incidence of AF), treatment with the miR-21 inhibitor decreased the incidence of AF to 50 % (*p < 0.05). e Masson trichrome staining of rabbit atrial samples from the CR, RAP, and RAP+miR-21 inhibitor groups. The myocardium was stained red and collagens were stained blue. Bar 50 μm. f Mean collagen content in the left atria of the 3 groups (n ≥ 9 per group). RAP caused significant collagen deposition in the left atria that was attenuated by the miR-21 inhibitor (*p < 0.05 vs. CR, ^# p < 0.05 vs. RAP, unpaired t test; n ≥ 9 independent samples for each group)

Fig. 3

Effect of the miR-21 inhibitor on RAP-induced Smad7 and collagen I/III expression in vivo. a Quantitative analysis of Smad7 expression by qRT-PCR. The relative Smad7 mRNA expression ratio (Smad7/GAPDH in arbitrary units) in the left atria was normalized to the expression in group CR, and the relative expression levels (in fold expression) were calculated. b Representative Western blot depicts the expression of Smad7 protein. c Mean Smad7 protein levels in the CR, RAP and RAP + miR-21 inhibitor groups. The level of Smad7 was significantly lower in the left atria of the RAP group. After treatment with the miR-21 inhibitor, the level of Smad7 increased significantly compared with the CR group and the RAP group. d Representative immunohistochemical staining images for Smad7 (magnification: ×400). e Graphical representation of the abundance of Smad7 protein in the sections. f Quantitative analysis of the expression of collagen I and III transcripts by qRT-PCR. The relative collagen I and III mRNA levels (collagen I and III/GAPDH in arbitrary units) in the left atria were normalized to the levels in group CR and the relative expression levels (in fold expression) were calculated. g Representative Western blot depicts the expression of collagen I and III protein. h Mean collagen I and III protein levels in the CR, RAP and RAP + miR-21 inhibitor groups. The collagen I and III level was significantly higher in the left atria of the RAP group. After treatment with the miR-21 inhibitor, the level of collagen I and III decreased significantly compared with the CR and RAP group (*p < 0.05 vs. CR; ^# p < 0.05 vs. RAP, n ≥ 9 independent protein samples for each group)

Fig. 4

Effect of miR-21 on TGF-β₁-induced collagen I/III expression. a qRT-PCR demonstrating that TGF-β1 (10 ng/ml) and TGF-β1 inhibitor(10 μg/ml) induces and decrease miR-21 expression at 48 h. b Representative Northern blot showing miR-21. c Values of miR-21 expression are the mean ± SE. d Northern blot verifying the upregulation of miR-21 in rats fibroblast after transfection with pre-miR-21. e qRT-PCR demonstrating that miR-21 levels increased in rat fibroblasts after transfection with pre-miR-21. f Northern blot verifying the down-regulation of miR-21 in rat fibroblasts after transfection with the miR-21 inhibitor. g qRT-PCR demonstrating that miR-21 levels deceased in rat fibroblasts after transfection with the miR-21 inhibitor. h, i Quantitative analysis of collagen I and III expression by qRT-PCR. The relative collagen I and III mRNA level (collagen I and III/GAPDH in arbitrary units) in the left atria was normalized to the expression in group CR and the relative expression levels (in fold expression) were calculated. j Representative Western blot depicting he expression of collagen I and III protein. k Mean collagen I and III levels in the control (CR), miR-control (M), pre-miR-21 (PM), TGF-β₁ (T), TGF-β₁ + pre-miR-21 (TP) and TGF-β₁ + miR-21 inhibitor (TI) groups (*p < 0.05 vs. CR, ^# p < 0.05 vs. T, unpaired t test; n = 10 independent samples for each group)

Fig. 5

Effect of miR-21 on the TGF-β₁-mediated down-regulation of Smad7. a Alignment of hsa-miR-21 and mmu-miR-21 with human Smad7 3′-UTR and mouse Smad7 3′-UTR based on TargetScan and Pictar software (http://www.targetscan.org/ and http://pictar.mdcberlin.de/). Several nucleotides in the 50-region of miR-21 (human and mouse) contain a perfect match with the 3′-UTR sequences of the human and mouse Smad7 genes. b The results of luciferase reporter assays. c Representative immunocytochemical images of Smad7 and propidium iodide (PI) staining. Cardiac fibroblasts were treated with TGF-β₁ in the presence or absence of miR-21 for 48 h. d Quantitative analysis of Smad7 expression by qRT-PCR. The relative Smad7 mRNA expression ratio (Smad7/GAPDH in arbitrary units) was normalized to that of group CR and the relative expression levels (in fold expression) were calculated. e Representative Western blot depicting the expression of Smad7 protein. f Mean Smad7 protein levels in the control (CR), miR-control (M), pre-miR-21 (PM), TGF-β₁ (T), TGF-β₁ + pre-miR-21 (TP) and TGF-β₁ + miR-21 inhibitor (TI) groups (*p < 0.05 vs. CR, ^# p < 0.05 vs. T, unpaired t test; n = 10 independent samples for each group)