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

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

Introduction

Heart failure (HF) is one of the most frequent causes of death worldwide [ 1]. One of the main causes of heart failure is cardiac fibrosis characterized by a pathological accumulation of extracellular matrix throughout the myocardium [ 2].

Collagen production and fibrosis in the myocardium is produced primarily by cardiac fibroblasts. In heart failure, stress induced cardiac fibroblasts proliferation and transformation into myofibroblasts [ 3]. Myofibroblasts express pro-contractile protein α-smooth muscle actin (α-SMA) and are responsible for the excessive accumulation of fibrillar collagen under pathological conditions [ 3, 4].

Transforming growth factor-β1 (TGF-β1) is a major cause of cardiac fibroblasts transformation into myofibroblasts [ 5, 6]. Under pathological conditions, TGF-β1 binds to membrane-bound heteromeric type I (TGFβRI) and type II (TGFβRII) receptors that transduce intracellular signals via phosphorylation and nuclear translocation of receptor activated Smad2 and Smad3 proteins, which recruit CBP1 to become transcriptional complexes and modulate many fibrosis-related genes [ 3, 7, 8]. Therefore, inhibition of TGF-β1 signals is a potential target for suppressing cardiac fibroblasts transformation into myofibroblasts and cardiac fibrotic remodeling.

Phosphodiesterase gene family (PDE family) is widely distributed in the body, and has 11 types [ 9]. The main function of the PDE family is hydrolysis of the second messenger cyclic 3′,5′-adenosine monophosphate (cAMP) and/or cyclic 3′,5′-guanosine monophosphate (cGMP), thus affecting its downstream protein kinase activity (PKA or PKG), and then regulate various biological effects. Phosphodiesterase inhibitors have clinical applications for the treatment of cardiovascular diseases such as pulmonary hypertension, erectile dysfunction, and asthma [ 9, 10]. Phosphodiesterase 5 (PDE5) selectively hydrolyzes cGMP, and selective inhibition of PDE5 can increase cGMP bioavailability. Sildenafil is a specific inhibitor of PDE5 [ 11]. Recently, some studies demonstrated that sildenafil markedly attenuated the left ventricular (LV) hypertrophy and dysfunction [ 11, 12], and reduced myocardial infarct-induced LV remodeling [ 13]. In addition, PDE5 expression was increased in hypertrophied human right ventricles and left ventricles from humans with heart failure [ 14, 15]. However, there is little information on the impact of PDE5 inhibition on the progression of cardiac fibrosis. And, we hypothesized that TGF-β1 signals play a major role in the effects of PDE5 inhibitor on cardiac fibrosis.

The present study was designed to investigate the effect of PDE5 inhibitor on TGF-β1 signals in mice subjected to transverse aortic constriction (TAC) and in cultured cardiac fibroblasts (CFs). We also studied the detailed signaling mechanism by which PDE5 inhibitor regulates TGF-β1 signals.

Materials and methods

Materials

DMEM and FBS were obtained from Gibco BRL (Life Technologies, Inc., Grand Island, NY). TGF-β1 and sildenafil were supplied by Sigma-Aldrich Chemical Co. (St. Louis, MO). Antibodies against PDE5, CBP1, p-Smad2, p-Smad3, Smad2/3, p-CREB, CREB, and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-α-SMA antibody was purchased from Boster (Boster Bio-Engineering Limited Company, Wuhan, China). Horse radish peroxidase-conjugated secondary antibodies were from Pierce Biotechnology (Thermo Fisher Scientific, Rockford, IL). Trans StartTM SYBR Green qPCR Supermix was from TransGen Biotech (TransGen Biotech, Beijing, China). All other chemicals and reagents were purchased from Sigma-Aldrich Company unless otherwise specified.

Animals

Male C57BL/6 mice (22–25 g, 8 weeks) were obtained from the Experimental Animal Center of Beijing (Beijing, China). Mice were housed in temperature-controlled cages under 12h/12h light/dark cycles at the animal care facility of Tongji Medical College and given free access to water and normal mice chow throughout the study period. All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. The protocol was approved by the Institutional Animal Research Committee of Tongji Medical College.

Mice were randomly divided into 4 groups: sham (n = 10), sildenafil (n = 10), TAC (n= 15) and TAC+ sildenafil (n = 15) treatment group. Oral sildenafil was provided in soft diet. In sildenafil group and TAC+ sildenafil group, treatment with sildenafil at a dose of 100 mg/(kg·d) was started when mice were subjected to TAC and continued for 4 weeks. Finally, mice were sacrificed by overdose of anesthesia.

Induction of pressure overload by transverse aortic constriction

Pressure overload-induced cardiac fibrosis was induced by transverse aortic constriction (TAC) in mice according to the method described previously [ 11, 16]. Briefly, mice were anesthetized by 2% isoflurane mixed with 0.5–1.0 L/min 100% O₂. The mouse was placed in a supine position atop a heating pad in order to maintain body temperature. Endotracheal intubation was performed using PE 90 tubing. Then, the endotracheal tube was connected to rodent ventilator cycling at 125–150 breaths/min and a tidal volume of 0.1–0.3 ml. During the procedure, anesthesia was maintained at 1.5%–2% isoflurane with 0.5–1.0 L/min 100% O₂. The mouse was carefully monitored from the point of view of body temperature, respiratory rate and circulation, airway problems for the adequacy of anesthesia. Partial thoracotomy to the second rib was performed under a surgical microscope and the sternum retracted using a chest retractor. The transverse aorta was isolated and constricted by a 6–0 silk suture ligature tied against a 271/2 gauge blunt needle. The needle was removed to form a constriction of 0.4 mm in diameter. Sham-operated mice underwent a similar surgical procedure without constriction of aorta.

Analysis of cardiac function by echocardiography

Three weeks after TAC, echocardiography (VisualSonics Vevo 2100 System with a 40 MHz high resolution transducer) was used to detect the alterations of cardiac functions and structure. Mice were anaesthetized by 1.5% isoflurane. Measurements included left ventricular ejection fraction (EF), fractional shortening of left ventricular diameter (FS) and left ventricular posterior wall (LVPW) under long axis M-mode. All data were analyzed by the Vevo 2100 Imaging System software version 1.0.0.

Histological analysis of cardiac hypertrophy and fibrosis

H&E was used to assess the degree of hypertrophy, and Sirius Red stain was used to assess the degree of fibrosis. The mice hearts were dissected, fixed in formalin for 16 h, then embedded in paraffin and sectioned into 4 μm slices. Sections were stained with H&E and Sirius Red, as described previously [ 17].

Isolation and culture of neonatal rat cardiac fibroblasts

Primary cardiac fibroblasts from newborn (1- to 2-day-old) Sprague-Dawley rats were isolated as described [ 18]. Briefly, neonatal hearts were rapidly minced and placed in a collagenase/pancreatin digestion solution for 10 min. After four to five digestion periods, the cell suspension was collected and centrifuged. The cells were resuspended in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (GibcoBRL, Life Technologies, Inc., Grand Island, NY). A single pre-plating step was used to allow cardiac fibroblasts to attach to culture plates. Nonadherent or weakly adherent cells were removed, fresh medium was added. Cardiac fibroblasts were maintained at 37°C in constant humidified incubator containing 95% air and 5% CO₂ atmosphere.

Analysis of protein level by Western blot

Cell lysates and lysates from heart tissue were prepared and Western blot was performed as described previously [ 19]. Briefly, cells and frozen animal heart tissue were homogenized in ice-cold lysis buffer containing protease inhibitors. Cell lysates and lysates from heart tissue were centrifuged at 12 000 g at 4 °C for 20 min, and supernatant was collected. The protein concentration was measured using the BCA protein assay reagent kit (Boster Bio-Engineering Limited Company, Wuhan, China). Lysates (50 μg protein/lane) were separated on 10% SDS-PAGE gels electrophoresis and transferred to PVDF membranes. Next, the membranes were blocked with nonfat milk, incubated overnight with primary antibodies and incubated with appropriate secondary antibody. Finally, the bands were visualized with the enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL) according to the manufacturer’s recommendations. Antibodies against TGF-β1, p-Smad2, p-Smad3, Smad2/3, GAPDH (Santa Cruz), p-CREB and CREB (Cell Singling Technology) were used.

Analysis of RNA level by real-time quantitative PCR

Total RNAs were extracted from rat neonatal cardiac myocytes in cell culture or from the left ventricles of mice using TRIzol (Invitrogen, Carlsbad, CA) according to the manufacture’s protocol. Reverse transcription of total mRNA were done starting from equal amounts of RNA (1μg) using Easy Script First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) according to manufacturer’s instructions. The BNP, β-MHC, TGF-β1, collagen I and II levels were quantified by real-time quantitative PCRs using Trans StartTM SYBR Green qPCR Supermix (TransGen Biotech, Beijing, China), and with GAPDH as an internal normalized reference. The qRT-PCR results were analyzed and expressed as relative mRNA levels of the CT (cycle threshold) value, which were then converted to fold change.

ELISA analysis of TGF-β1

Blood samples were collected into tubes. After coagulation, serum was collected by centrifugation and stored at -80 °C. The levels of TGF-β1 in mice serum was assayed by corresponding ELISA kits (Boster Bio-Engineering Limited Company, Wuhan, China) following the manufacturer’s instruction.

Immunoprecipitation

Cells were pretreated with 1μM sildenafil for 1 h before treatment with 10 ng/ml TGF-β1 for 24 h. Cells were washed twice with ice-cold PBS, scraped in 1ml of lysis buffer, and then homogenized. Samples were incubated with primary antibodies for the target protein (or IgG control antibody) for 24h and then precipitated by incubating with protein A+ G-agarose 3h. Pellets were washed once in lysis buffer followed by three washes in wash buffer. And immunoprecipitated proteins were analyzed by immunoblot analysis.

Statistical analysis

All values are expressed as mean±standard error. Differences between data groups were compared by unpaired data or one-way analysis of variance (ANOVA) and Bonferroni post-test. P<0.05 was considered as statistically significant.

Results

PDE5 inhibition prevented pressure overload-induced cardiac dysfunction

To investigate the effects of PDE5 inhibition on cardiac fibrosis in heart failure, we first established cardiac fibrosis model by transverse aortic constriction (TAC) in mice for 4 weeks. TAC induced cardiac hypertrophy was assessed by increase of the ratio of heart weight to body weight (HW/BW) and heart size. Sildenafil treatment prevented cardiac hypertrophy induced by TAC (Fig. 1A and 1B, Table 1). H&E staining also showed that TAC induced a marked increase in myocyte hypertrophy, and this was significantly reduced in the hearts of sildenafil-treated mice (Fig. 1C and 1D). And we also detected the hypertrophic markers, and results showed that BNP and β-MHC were induced in the failing hearts, and sildenafil treatment markedly suppressed the hypertrophic markers in the hearts (Fig. 1E).

Cardiac function was measured by echocardiography. As shown in Table 2, PDE5 inhibition significantly attenuated TAC-induced cardiac hypertrophy and dysfunction.

All the data suggest that PDE5 inhibition prevents TAC-induced cardiac hypertrophy and heart dysfunction.

PDE5 inhibition blunted TAC-induced fibrosis

To determine whether PDE5 inhibition prevents TAC-induced cardiac fibrosis, tissue was stained with Picrosirius Red to detect collagen distribution in the left ventricle. As expected, TAC significantly increased collagen deposition in TAC mouse hearts. Sildenafil reduced TAC-induced collagen deposition (Fig. 2A and 2B). We then assessed collagen I and III expression in the hearts and results showed that PDE5 inhibition substantially lowered collagen I and III accumulation induced by TAC (Fig. 2C). α-SMA plays an important role in cardiac fibrosis and we found PDE5 inhibitor inhibited the upregulation of α-SMA induced by TAC in mice (Fig. 2D). These findings indicate that PDE5 inhibition prevents cardiac fibrosis induced by TAC in mice.

PDE5 inhibition did not block TAC induced TGF-β1 production or phosphorylation of Smad2/3

TGF-β pathway plays a crucial role in cardiac fibrosis. To investigate whether the action of PDE5 participates in TAC-induced TGF-β production, we detected TGF-β1 protein and mRNA levels in hearts and serum concentration of TGF-β1. The results showed that PDE5 inhibitor, sildenafil, did not block TAC-induced TGF-β1 production (Fig. 3A to 3C). We also measured Smad2 and Smad3 phosphorylation levels by Western blot. TAC increased Smad2 and Smad3 phosphorylation and sildenafil did not change the level of TAC-induced p-Smad2 and p-Smad3 (Fig. 3D and 3E). Therefore, PDE5 inhibition did not inhibit TAC-induced TGF-β1 production and Smad2/3 phosphorylation.

PDE5 inhibition prevented the TGF-β1-stimulated fibrotic response in cardiac fibroblasts

To further examine the action of PDE5 inhibitor on cardiac fibrosis, we pretreated cardiac fibroblasts with TGF-β1 and found that TGF-β1 treatment increased transformation (α-SMA expression), cardiac fibroblast proliferation and collagen synthesis (collagen I and III) (Fig. 4). Sildenafil pretreatment markedly decreased the expression of α-SMA, and cardiac fibroblast proliferation and collagen synthesis by TGF-β1 incubation (Fig. 4).

PDE5 inhibition increased phosphorylation of CREB and reduced CBP1 recruitment to Smad transcriptional complexes

We treated the cells with sildenafil and measured CREB phosphorylation with Western blot. As shown in Fig. 5A, sildenafil dose-dependently increased the level of phospho-CREB in cardiac fibroblasts. CREB is a cAMP-responsive element. So we detected cAMP level, and found that sildenafil had no effect on cAMP (Fig. 5B).

To investigate whether PDE5 inhibition is able to interfere with the recruitment of CBP1/Smad transcriptional complexes, we used immunoprecipitations of nuclear proteins to assess the interaction between these endogenous transcriptional proteins in cardiac fibroblasts after treatment with TGF-β1 (Fig. 6). We detected CBP1 expression in cardiac fibroblasts and found that CBP1 was not affected by either sildenafil or TGF-β1 (Fig. 6A). Treatment with sildenafil and TGF-β1 resulted in a reduction of p-Smad2 and p-Smad3 in CBP1 immunoprecipitates and an increase of the complex of p-CREB and CBP1 compared with TGF-β1 alone (Fig. 6B). Taken together, these results imply that PDE5 inhibition represses TGF-β signaling by activating CREB, which recruits CBP1, effectively competing with Smad transcriptional complexes for binding to CBP1.

Discussion

Although PDE5 inhibition has been demonstrated to exert profound beneficial effects in the failing heart, the mechanisms underlying therapeutic effect of PDE5 inhibition remain unclear. In the present study, we have demonstrated that PDE5 inhibition prevents TAC-induced cardiac fibrosis in mice. We have also provided evidence that PDE5 inhibition blocks the TGF-β1-stimulated cardiac fibroblast proliferation, transformation, and collagen synthesis in vitro and in vivo. Finally, we found that PDE5 inhibition attenuated cardiac fibrosis dependent on CREB activation, which reduces CBP1 recruitment to Smad transcriptional complexes.

Cardiac fibrosis is a major cause of diastolic dysfunction and death in heart failure patients [ 3, 20, 21]. Previous studies have demonstrated that TGF-β pathway plays a crucial role in cardiac fibrosis. Three different TGF-β isoforms have been described, TGF-β1, TGF-β2, and TGF-β3. TGF-β1 is the most important isoform for the cardiovascular system [ 22]. Under pathological conditions, TGF-β1 is released and binds to type II (TGFβRII) and type I (TGFβRI) receptors, leading to Smad2 and Smad3 phosphorylation [ 23]. Phosphorylated Smad2/3 binds Smad4 and translocates into the nucleus, where it recruits CBP1 to form transcriptional complexes and promotes the transcription of several genes important for fibrosis, such as procollagens, fibronectin, CTGF and PAI-1 [ 4, 24– 26]. It has been reported that Smad3 is required for TGF-β-induced gene expression in adult fibroblasts [ 27], indicating that Smad3 plays an important role in fibrosis. And another study reported that overexpression of some Smad proteins activate transcription of some of these genes, like PAI-1, even in the absence of TGF-β [ 28]. Therefore, anti-TGF-β strategies occupy an important position in the treatment of cardiac fibrosis. Recently, cGMP has been identified as a negative regulator of fibroblast proliferation and differentiation [ 3, 7, 29]. The cGMP/PKG signaling pathway attenuates TGF-β1-induced cardiac fibrosis by blocking TGF-β1-induced nuclear translocation of phospho-Smad3 through PKG-induced phosphorylation of Ser309 and Thr388 sites in the MH2 domain of the Smad3 protein [ 7, 29].

PDE5 is the predominant enzyme responsible for cGMP hydrolysis and inhibition of its activity by sildenafil which is a specific inhibitor of PDE5, ultimately increases intracellular cGMP concentration and activates cGMP-dependent kinase (PKG). Previous studies have demonstrated that PDE5 expression was upregulated in human hypertrophied and failing hearts, and its inhibitor sildenafil stimulated PKG activity [ 14, 30], attenuating cardiac hypertrophy and contractile dysfunction [ 11, 13]. Sildenafil has also been confirmed to attenuate TAC-induced cardiac fibrosis [ 31]. However, the role of PDE5 in the pathophysiological process of cardiac fibrosis is still not fully understood. Previous research [ 11] has suggested that PDE5 plays cardioprotective roles by ERK, JNK and Akt signaling pathways. These could also mediate the anti-fibrotic effects of PDE5 inhibition. But PDE5 inhibition does not attenuate hypertrophy induced by overexpression of calcineurin in vitro and Akt in vivo [ 11, 32], suggesting other mechanisms are involved in these pathways. We hypothesized that the effects of sildenafil on cardiac fibrosis may depend on TGF-β signals. However, whether there is a link between PDE5 inhibition and TGF-β signals has not been reported. In this work, we utilized classical animal model (TAC) and cardiac fibroblasts to explore the link. Here, we found that TAC significantly increased the levels of active TGF-β1 protein, phosphorylation of Smad2/3, transformation of cardiac fibroblasts, and the levels of collagen I and III mRNA. We also detected that PDE5 inhibition prevented the increase of cardiac fibroblasts transformation, and collagen I and III mRNA without altering the levels of active TGF-β1 protein and phosphorylation of Smad2/3. Therefore, our findings indicate that PDE5 inhibition may affect downstream of TGF-β1 and prevent cardiac fibrosis.

Using cultured cardiac fibroblasts, we further identified the antifibrotic signaling of PDE5 inhibition and its interaction with the fibrotic signaling of TGF-β1. In the in vitro study, we observed that PDE5 inhibition prevented TGF-β1-induced proliferation, transformation, and collagen synthesis in cardiac fibroblasts. We also found that PDE5 inhibitor, sildenafil, caused phosphorylation of CREB in cardiac fibroblasts.

cAMP responsive element binding protein (CREB) is a member of a large family (CREB/ATF) of structurally related transcription factors that bind to promoter cAMP responsive element (CRE) sites [ 33]. The group of proteins shows many structural and functional variations, and they are expressed in a wide range of cell types and tissue. The crucial event in the activation of CREB is the phosphorylation of Ser133 in the P-box (flanking a cluster of phosphorylation sites that regulate the activity of CREB), or kinase-inducible domain (KID) [ 34, 35]. And the phosphorylated CREB can interact with CREB binding protein (CBP). The CREB-CBP complex can recruit basal transcription factors and initiate transcription. Based on the theory, recent studies showed that phosphorylated CREB reduces Smad-mediated transcription by recruiting CBP1/p300, making them unavailable for recruitment to Smad transcriptional complexes [ 33]. cAMP-elevating agents suppress collagen gene expression by inducing phosphorylation of the transcription factor CREB [ 36]. Consistent with this evidence, we found that PDE5 inhibition caused phosphorylation of CREB in cardiac fibroblasts. Previous studies have also found that PDE5 inhibitors induce p-CREB in B16 melanoma cells [ 37]. And we also showed that sildenafil markedly increased the level of cGMP, but had no effect on cAMP level. Moreover, we detected that PDE5 inhibition can interfere with the recruitment of CBP1 to Smad transcriptional complexes in cardiac fibroblasts by immunoprecipitation. CREB phosphorylation and suppression of TGF-β/Smad pathway in cardiac fibroblasts may be an important mechanism of the protective effect of PDE5 inhibition against cardiac fibrosis.

In summary, our results demonstrate that PDE5 plays a crucial role in cardiac fibrosis, and chronic PDE5 inhibition markedly prevented the profibrotic effects of TGF-β in cardiac fibroblasts and failing hearts by reducing Smad-mediated recruitment of transcriptional coactivators through the activation of CREB. Thus, alleviation of TGF-β pathway may be an important mechanism underlying the therapeutic effect of PDE5 inhibition on cardiac fibrosis and heart failure.

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