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Smad3 Inactivation and MiR-29b Upregulation Mediate the Effect of Carvedilol on Attenuating the Acute Myocardium Infarction-Induced Myocardial Fibrosis in Rat

Carvedilol, a nonselective β-adrenoreceptor antagonist, protects against myocardial injury induced by acute myocardium infarction (AMI). The mechanisms underlying the anti-fibrotic effects of carvedilol are unknown. Recent studies have revealed the critical role of microRNAs (miRNAs) in a variety of cardiovascular diseases. This study investigated whether miR-29b is involved in the cardioprotective effect of carvedilol against AMI-induced myocardial fibrosis. Male SD rats were randomized into several groups: the sham surgery control, left anterior descending (LAD) surgery-AMI model, AMI plus low-dose carvedilol treatment (1 mg/kg per day, CAR-L), AMI plus medium-dose carvedilol treatment (5 mg/kg per day, CAR-M) and AMI plus high-dose carvedilol treatment (10 mg/kg per day, CAR-H). Cardiac remodeling and impaired heart function were observed 4 weeks after LAD surgery treatment; the observed cardiac remodeling, decreased ejection fraction, and fractional shortening were rescued in the CAR-M and CAR-H groups. The upregulated expression of Col1a1, Col3a1, and α-SMA mRNA was significantly reduced in the CAR-M and CAR-H groups. Moreover, the downregulated miR-29b was elevated in the CAR-M and CAR-H groups. The in vitro study showed that Col1a1, Col3a1, and α-SMA were downregulated and miR-29b was upregulated by carvedilol in a dose-dependent manner in rat cardiac fibroblasts. Inhibition of ROS-induced Smad3 activation by carvedilol resulted in downregulation of Col1a1, Col3a1, and α-SMA and upregulation of miR-29b derived from the miR-29b-2 precursor. Enforced expression of miR-29b significantly suppressed Col1a1, Col3a1, and α-SMA expression. Taken together, we found that smad3 inactivation and miR-29b upregulation contributed to the cardioprotective activity of carvedilol against AMI-induced myocardial fibrosis.


Introduction
Acute myocardial infarction (AMI) is one of the most serious cardiovascular conditions. The acute loss of myocardium post-AMI triggers a cascade of intracellular signaling processes, contributing to left ventricular (LV) remodeling, scar expansion, and heart failure. Beta-adrenergic receptor antagonists (βblockers) effectively ameliorate post-AMI LV remodeling. Carvedilol, a third-generation, non-selective β-adrenoreceptor antagonist, possesses antioxidant, anti-apoptotic, anti-inflammatory, and anti-fibrotic properties [1]. Numerous studies have shown that carvedilol treatment improves the left ventricular ejection fraction (LVEF) and attenuates left ventricular remodeling in patients with chronic heart failure (CHF) [2]. The protective effects of carvedilol against AMIinduced myocardial injury have been attributed to the reduction of fibrosis [3,4]. However, the precise mechanisms underlying the anti-fibrotic effect of carvedilol is unknown.

Ethics Statement
Male sprague-dawley (SD) rats weighing 180-240 g, license number SCXK (YUE) 2004-0011 (Department of Experimental Animal Research Center, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou, China) were used. All animals were housed on a 12-h light/dark cycle under pathogen-free conditions and kept on standard mouse chow with free access to tap water. This study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th Edition, National Research Council, 2011). The present program was also approved by the research ethics committee of Guangdong General Hospital, the approval number was No. GDREC2010093A.

A rat model of AMI and carvedilol treatment
AMI was induced by ligating the left anterior descending coronary artery as described [18]. Electrocardiography was used to demonstrate ST elevation and thereby confirm the success of surgery. Forty surviving rats that underwent ligation were randomly divided into five groups (N=8): (1) The Sham control group (Sham), (2) Saline treated AMI model group (AMI), (3) Low dose of carvedilol treated AMI group (CAR-L, 1 mg/kg/day), (4) Medium dose of carvedilol treated AMI group (CAR-M, 5 mg/kg/day), (5) High dose of carvedilol treated AMI group (CAR-H, 10 mg/kg/day). The second day after LAD surgery, four-week-oral dosing was conducted in rats in all groups except the sham group. All rats were fed ad libitum (the dose of standard rat fodder for one rat over 30 g/day).

Echocardiographic study
Left ventricular (LV) function variables were assessed by transthoracic echocardiography. After the induction of light general anesthesia, the rats underwent transthoracic twodimensional (2D) guided M-mode echocardiography with a 8.5-MHz transducer (Acuson, Mountain View, CA). From the cardiac short axis (papillary level), the LV anterior wall enddiastolic thickness (LVAWd), the systolic LV anterior wall thickness(LVAWs), the LV internal dimension at end-diastole (LVIDd), the LV internal dimension at end-systole(LVIDs), the LV posterior wall end-diastolic thickness (LVPWd), the LV posterior wall end-systolic thickness(LVPWs), the ejection fraction (EF) and fractional shortening (FS) were measured. Echocardiographic measurements were averaged from at least three separate cardiac cycles.

Histological analysis
Rats were killed by an intraperitoneal injection of 2 mL of pentobarbital. The heart was excised, and the LV myocardium fixed overnight in 10% formalin. Samples were embedded in paraffin and cut into 4 µm thick sections. They were mounted on normal glass slides and stained with Masson trichrome for histological examination. For the collagen volume fraction (CVF) analysis in the border zone of the infarcted region, eight separate views (magnification=original×400) were selected and assessment of CVF used the following formula: CVF=collagen area/ total area.

Rat cardiac fibroblasts (CFs) isolation, culture and treatment
CFs were isolated from 1-3 days old SD rats by using a modification of previous report [19]. CFs were separated from cardiomyocytes by gravity separation and grown to confluency on 10-cm cell culture dishes in growth media (DMEM/LG 10% FBS 1% penicillin 1% streptomycin) at 37 °C in humid air with 5% CO2. CFs from the third passage were used for experiments. The passage 3-4 neonatal SD rat CFs were used for mechanism study in vitro. Fifty nM Smad3 siRNA, 5 µM Smad3 inhibitor SIS-3, 80 µM Smad3 inhibitor naringenin (Nar) were used to treat rat CFs.

Imaging of DCF Fluorescence
Intracellular oxidants in mouse cardiac fibroblasts were measured by using the probe dichlorofluoroscin diacetate (DCFH-DA) and confocal microscopy as described [20]. In brief, cells were incubated with DCFH-DA (10 µM) for 15 min, and subsequently treated with Hoechst33342 (1 µg/mL) for 15 min before being imaged. Cells were washed twice with culture medium, and images were acquired from five or more randomly chosen fields using Leica SP5 Spectral scanning laser confocal microscope (Leica Microsystems, Wetzlar, Germany). The levels of DCF fluorescence were analyzed with Leica Application Suite 2.02 software.

Quantitative real-time PCR
Total RNA was extracted using TRIzol reagent (Gibco-BRL, Grand Island, NY, USA) according to manufacturer's instruction. Methods for coding gene and miR-29b precursor expression detection were as follows: First-strand cDNAs were synthesized using a mixture of oligo (dT) 15 and random primers with Superscript reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed with the following thermal cycling profile: 95°C for 5 min, followed by 40 cycles of amplification (94°C for 25 s, 60°C for 25 s, and 72°C for 25 s). The absorption values of the SYBR Green I fluorescence in each tube were detected at the end of each Smad3 Signal and MiR-29b Modulated by Carvedilol PLOS ONE | www.plosone.org cycle. The housekeeping gene GAPDH was used as an internal control. PCR primers for coding genes and miR-29b precursors used in this study, as well as the size of fragments amplified, are shown in Table S1. Methods for mature miR-29b level detection were as follows: the total RNA was used to detect mature miRNA level using Bulge-Loop miRNA qRT-PCR kit (Guangzhou Ribobio, China). In brief, the real-time miRNA assay has two steps: miRNA RT reaction and real-time PCR detection. miRNA RT primer binds to the 3′end of miRNA molecules and are transcribed with reverse transcriptase. RT product is quantified using real-time PCR with the following thermal cycling profile: 95 °C for 20 sec, followed by 40 cycles of amplification (95 °C for 10 s, 60 °C for 20 s, 70 °C for 10 s). To normalize RNA content, the U6 snRNA was the internal control. The above two kinds of real-time PCR assays were performed using the vii A7 Quantitative PCR System (Applied Biosystems, Carlsbad, CA, USA). The relative expression values of coding genes and mature miRNAs of interest were calculated using the 2 -∆∆Ct method [21].

Statistical analysis
The data represent the mean ± standard deviation (SD). In each experiment, all determinations were performed at least in triplicate. Differences between experimental groups were analyzed using Student's t-test. A value of p< 0.05 indicated significance.

ECM-related genes and miR-29b expression in AMIinduced fibrotic myocardium treated with carvedilol
Consistent with the echocardiography data, Masson's trichrome staining showed that the collagen volume fraction (CVF) in the AMI border zone was dramatically lower in the CAR-M and CAR-H AMI groups than in the AMI and CAR-L group (p < 0.01 and p < 0.001, p < 0.05 and p < 0.01, respectively) ( Figure 1). Quantitative real-time PCR showed that Col1a1, Col3a1, and α-SMA mRNA were significantly decreased in the AMI border zone in the CAR-M and CAR-H groups (p < 0.05 and p < 0.01, respectively) ( Figure 2A). Western-blot results showed that Col1a1, Col3a1, and α-SMA protein expression was also significantly lower in the AMI border zone in the CAR-M and CAR-H groups ( Figure 2B, S1).
The quantity of mature miR-29b in the AMI border zone was significantly higher in the CAR-M and CAR-H groups (p < 0.05 and p < 0.01, respectively) than in the untreated AMI group ( Figure 2C). In the sham surgery control group, the expression level of the mir-29b-2 precursor was much higher than that of the mir-29b-1 precursor (p < 0.001) ( Figure 2D). Only the expression of the miR-29b-2 precursor was significantly higher in all 3 carvedilol-treated AMI groups (p < 0.05 and p < 0.01, respectively) ( Figure 2D).

ECM-related genes and miR-29b expression in carvedilol-treated rat cardiac fibroblasts in vitro
A dose-course study of the effect of carvedilol on Col1a1, Col3a1, and α-SMA expression was demonstrated by quantitative real-time PCR and Western-blotting assay, respectively ( Figure 3A, Figure 3B, S2). These data demonstrated that 4µM carvedilol inhibited Col1a1, Col3a1, and α-SMA expression at both the mRNA and protein level. Compared with the control group, mature miR-29b was significantly up-regulated in the 2 µM and 4 µM carvediloltreated rat cardiac fibroblasts (p < 0.05 and p < 0.05, respectively) ( Figure 3C). Quantitative real-time PCR showed that the expression of the mir-29b-2 precursor was much higher than that of the mir-29b-1 precursor (p < 0.01) ( Figure  3D). Expression of the miR-29b-2 precursor, but not the miR-29b-1 precursor, increased significantly in a dosedependent manner in carvedilol-treated rat cardiac fibroblasts (p < 0.05) ( Figure 3D).

miR-29b modulated expression of ECM-related gene in cardiac fibroblasts
To study the expression of ECM-related genes after miR-29b mimic treatment, we transfected rat cardiac fibroblasts with the miR-29b mimic or scrambled oligonucleotide. After a 24-h incubation, real-time PCR analysis indicated that the miR-29b mimic inhibited Col1a1, Col3a1, and α-SMA expression at the mRNA level (p < 0.05, p < 0.01, and p < 0.001, respectively) ( Figure 5A). Consistent with these results, miR-29b also inhibited Col1a1, Col3a1, and α-SMA protein expression compared to that observed using the scrambled oligonucleotide (p < 0.05 and p < 0.001, respectively). (Figure 5B, S3.)

Discussion
In this study, we demonstrated the anti-fibrotic effect of carvedilol in vivo and in vitro by inhibiting fibrosis-related genes Smad3 Signal and MiR-29b Modulated by Carvedilol PLOS ONE | www.plosone.org expression. The fibrosis regulator, miR-29b, was validated to be modulated by carvedilol, contributing to the anti-fibrotic effect of carvedilol. Mechanism study revealed that carvedilol inhibited ROS-induced Smad3 activation, and Smad3 signal negatively modulated miR-29b expression, miR-29b could efficiently inhibit fibrosis-related genes expression in cardiac fibroblasts.
LV remodeling after AMI contributes to heart failure. βblockers are first-line therapy for AMI and heart failure. Initiation of β-blockers within the first 24 h of the ischemic episode has been recommended in clinical practice guidelines [22]. Carvedilol is a β-blocker that also has α-blocking effects, reducing the mortality rate of patients with moderate to severe chronic cardiac failure by 35% to 65% and lowering rehospitalization rates [23,24]. The underlying mechanism whereby carvedilol attenuates AMI-induced cardiac remodeling seems to be multifactorial. Carvedilol has antioxidant activity [25], attenuates inflammatory mediators, and activates NF-kB [26]. Carvedilol treatment has been shown to delay oxidativestress-induced apoptosis by improving Ca 2+ handling [27] and increasing the expression of anti-apoptosis proteins [28].
Consistent with previous reports [32], the echocardiography results of the present study show that carvedilol treatment can rescue AMI-induced cardiac changes in LVAWd, LVAWs, LVIDd, and LVIDs. In addition, medium-and high-dose carvedilol treatment significantly increased cardiac EF% and FS% compared to those in the untreated AMI group (Table 1). Masson's staining demonstrated that the collagen volume  Quantitative real-time PCR and Western-blot assay revealed that Col1a1, Col3a1, and α-SMA expression were significantly lower in the border zone of the infarcted region in the mediumand high-dose carvedilol treatment groups compared with those in the untreated AMI group (Figure 2 A, 2B,S1). This result is similar to that of a previous report that found carvedilol inhibited Col1a1 and Col3a1 mRNA expression in fibrotic kidney [33]. To investigate the anti-fibrotic effect of carvedilol, SD rat cardiac fibroblasts were cultured and treated with carvedilol in vitro. Quantitative real-time PCR and Western-blot results revealed that carvedilol inhibited Col1a1, Col3a1, and α-SMA expression in a dose-dependent manner ( Figure 3A, 3B,S2).
Activation of the renin-angiotensin system (RAS) has been implicated in the pathogenesis of acute myocardial infarction (AMI) [34]. Angiotensin II (Ang II) played a pivotal role in cardiac fibrosis through increasing the intracellular generation of ROS and stimulating the collagen production in cardiac fibroblasts [35], and ROS could activate TGF-β/smad3 pathway leading to fibrosis [36]. In the present study, we confirmed that Ang II increased ROS generation and Smad3 activation in cardiac fibroblasts ( Figure 4A, 4B), however, NAC and cardvedilol could consistently abrogate the effect of Ang II on ROS generation and Smad3 activation ( Figure 4A, 4B). Therefore, carvedilol, possessing ROS scavenging and ROS suppressive effects, could inhibit ROS-activated Smad3 signaling involved in cardiac fibrosis.
We further demonstrated that Smad3 siRNA and two Smad3 inhibitors, SIS-3 and Nar, inhibited Col1a1, Col3a1, and α-SMA mRNA expression in rat cardiac fibroblasts ( Figure 4C, 4F). These results revealed that inactivation of the Smad3 pathway mediates the anti-fibrotic effect of carvedilol in AMI-induced cardiac fibrosis. miRNAs have been shown to play roles in cardiac remodeling after AMI [37]. miR-29b was shown to regulate myocardial fibrosis [14,16], but whether carvedilol could modulate miR-29b expression was unknown. In this study, we found that miR-29b was upregulated in the border zone of infarcted myocardium in the rat AMI model treated with medium-and high-dose carvedilol (Fig. 2C). miR-29b was also upregulated by carvedilol in rat cardiac fibroblasts in vitro (Fig.  3C). Mature rat miR-29b can be derived from miR-29b-1 and miR-29b-2 precursors, which are transcribed from two different loci in the rat genome (www.mirbase.com). We also confirmed that miR-29b-2 precursor expression was much higher than miR-29b-1 in myocardium (Fig. 2D) and cardiac fibroblasts  3D); miR-29b-2, but not miR-29b-1, could be modulated by carvedilol in vivo (Fig. 2D) and in vitro (Fig. 3D). Using Smad3 siRNA and 2 Smad3 inhibitors, SIS-3 and Nar, we demonstrated that Smad3 signaling negatively regulates miR-29b expression derived from only from the miR-29b-2 precursor ( Figure 4E, 4H). This result was consistent with previous reports that Smad3 signaling promotes renal fibrosis by inhibiting miR-29 [15,38].
The anti-fibrotic effect of carvedilol contributed to the amelioration of AMI-induced cardiac remodeling and impaired cardiac function in rats. We showed that carvedilol specifically inhibits expression of the ECM-related proteins Col1a1, Col3a1, and α-SMA and promotes miR-29b expression in infarcted myocardium and cardiac fibroblasts in vitro. Taken together, these data demonstrate that inactivation of ROSinduced Smad3 signaling and miR-29b upregulation mediate the anti-fibrotic effect of carvedilol in MI-induced cardiac fibrosis ( Figure 6). Table S1. Primers used in real-time qRT-PCR. (DOC) Figure S1. Col1a1, Col3a1, and α-SMA protein expression in the border zone of the infarcted region by Western-blot assay. A. Significant inhibition of Col1a1 protein by carvedilol treatment. ※ p < 0.001 vs. sham surgery control group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. AMI group, N = 6-8. B. Significant inhibition of Col3a1 protein by carvedilol treatment. ※ p < 0.01 vs. sham surgery control group, * p < 0.01, ** p < 0.001 vs. AMI group, N = 6-8. C. Significant inhibition of α-SMA protein by medium-and high-dose carvedilol treatment. ※ p < 0.001 vs. sham surgery control group, # p < 0.01 vs. AMI group, N = 6-8.