Chronic inhibition of cyclic guanosine monophosphate-specific phosphodiesterase 5 prevented cardiac fibrosis through inhibition of transforming growth factor β-induced Smad signaling

doi:10.1007/s11684-014-0378-3

Front. Med.

2014, Vol. 8

Issue (4) : 445-455 https://doi.org/10.1007/s11684-014-0378-3

RESEARCH ARTICLE

Chronic inhibition of cyclic guanosine monophosphate-specific phosphodiesterase 5 prevented cardiac fibrosis through inhibition of transforming growth factor β-induced Smad signaling

Wei Gong,Mengwen Yan,Junxiong Chen,Sandip Chaugai,Chen Chen,Daowen Wang(

)

Department of Internal Medicine and Gene Therapy Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

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Abstract

Recent evidences suggested that cyclic guanosine monophosphate-specific phosphodiesterase 5 (PDE5) inhibitor represents an important therapeutic target for cardiovascular diseases. Whether and how it ameliorates cardiac fibrosis, a major cause of diastolic dysfunction and heart failure, is unknown. The purpose of this study was to investigate the effects of PDE5 inhibitor on cardiac fibrosis. We assessed cardiac fibrosis and pathology in mice subjected to transverse aortic constriction (TAC). Oral sildenafil, a PDE5 inhibitor, was administered in the therapy group. In control mice, 4 weeks of TAC induced significant cardiac dysfunction, cardiac fibrosis, and cardiac fibroblast activation (proliferation and transformation to myofibroblasts). Sildenafil treatment markedly prevented TAC-induced cardiac dysfunction, cardiac fibrosis and cardiac fibroblast activation but did not block TAC-induced transforming growth factor-β1 (TGF-β1) production and phosphorylation of Smad2/3. In isolated cardiac fibroblasts, sildenafil blocked TGF-β1-induced cardiac fibroblast transformation, proliferation and collagen synthesis. Furthermore, we found that sildenafil induced phosphorylated cAMP response element binding protein (CREB) and reduced CREB-binding protein 1 (CBP1) recruitment to Smad transcriptional complexes. PDE5 inhibition prevents cardiac fibrosis by reducing CBP1 recruitment to Smad transcriptional complexes through CREB activation in cardiac fibroblasts.

Keywords PDE5 cardiac fibrosis TGF-β CREB

Corresponding Author(s): Daowen Wang

Just Accepted Date: 24 October 2014 Online First Date: 24 November 2014 Issue Date: 18 December 2014

Cite this article:

Daowen Wang,Wei Gong,Mengwen Yan, et al. Chronic inhibition of cyclic guanosine monophosphate-specific phosphodiesterase 5 prevented cardiac fibrosis through inhibition of transforming growth factor β-induced Smad signaling[J]. Front. Med., 2014, 8(4): 445-455.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-014-0378-3
https://academic.hep.com.cn/fmd/EN/Y2014/V8/I4/445

Fig.1 PDE5 inhibition attenuated TAC-induced cardiac hypertrophy and improved the function of failing hearts. (A) Representative images of hearts. Scale bar, 2 mm. (B) Ratio of heart weight to body weight (HW/BW). (C,D) H&E staining of mouse hearts and cardiomyocyte cross-sectional area (mm). Scale bar, 50μm. (E) Expression of BNP and β-MHC analyzed by real-time polymerase chain reaction. n = 6. *P<0.05 vs. Sham. ^#P<0.05 vs. TAC. Data are presented as mean±SEM. Sil: sildenafil.

Tab.1 Anatomic data

Tab.2 Echocardiographic characteristics of Sham, Sil, TAC, and TAC+Sil mice

Fig.2 PDE5 inhibition prevented TAC-induced cardiac fibrosis in mice. (A, B) Representative staining for collagen deposition in LV is presented. Collagen deposition was stained with a saturated solution of picric acid containing 1% Sirius Red as red color and was quantified as percent of cardiac area. Scale bar, 100μm. (C) Expression of collagen I and III analyzed by real-time polymerase chain reaction. n = 6. (D) Immunoblot analysis showed α-SMA was increased in hearts of TAC mice and decreased in PDE5 treatment mice. α-SMA was normalized to GAPDH. n = 4. *P<0.05 vs. Sham. ^#P<0.05 vs. TAC. Results are presented as mean±SEM.

Fig.3 PDE5 inhibition did not block TAC-induced TGF-β1 production or phosphorylation of Smad2/3. (A) Immunoblot analysis showed TGF-β1 was increased in TAC mice, and sildenafil treatment did not reduce TGF-β1 expression. TGF-β1 was normalized to GAPDH. n = 5. (B) Gene expression of TGF-β1 in LV was analyzed by real-time polymerase chain reaction. n = 6. (C) Expression of TGF-β1 in serum measured by ELISA. n = 6. *P<0.05 vs. Sham. ^#P<0.05 vs. TAC. (D) Level of p-Smad2, p-Smad3 and total Smad2/3 measured by Western blot after TAC. n = 4. (E) Densitometric quantification of Smad2 and Smad3 phosphorylation after TAC. *P<0.05 vs. Sham. ^#P<0.05 vs. TAC. Data are presented as mean±SEM.

Fig.4 PDE5 inhibition prevented the TGF-β1-stimulated fibrotic response in cardiac fibroblasts. Cells were pretreated with sildenafil (1μM) for 1 h, and then exposed to TGF-β1 (10ng/ml) for 24 h. (A) Sildenafil alleviated TGF-β1-induced α-SMA expression in cardiac fibroblasts. n = 4. (B) Sildenafil alleviated TGF-β1-induced cardiac fibroblast proliferation. n = 6. (C) Sildenafil pretreatment markedly decreased collagen synthesis (collagen I and III) by TGF-β1 incubation. Results are shown as mean±SEM. n = 5. *P<0.05 vs. control. ^#P<0.05 vs. TGF-β1.

Fig.5 PDE5 inhibition increasd phosphorylation of CREB. Cells were pretreated with sildenafil (0 μM, 0.5 μM, 1 μM) for 24 h. (A) Sildenafil pretreatment markedly increased p-CREB. (B) The level of cGMP and cAMP in cardiac fibroblasts. Results are shown as mean±SEM. n = 6, *P<0.05 vs. control.

Fig.6 PDE5 inhibition disrupted the interaction between Smads and the coactivator CBP1. (A) CBP1 expression was detected by Western blot. (B) Immunoprecipitates of the indicated proteins were separated by SDS-PAGE and probed with antibodies specific for CBP1, p-Smad2, p-Smad3, or p-CREB were used to examine levels of the indicated proteins. Results are shown as mean±SEM. n = 4.

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