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

This study complied with the Declaration of Helsinki and the ethics committee of Guangdong Cardiovascular Institute, Guangdong General Hospital approved the study protocol. Informed consent from all participants was obtained before the initial coronary angiography. All of the experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were in compliance with the guidelines specified by the Chinese Heart Association policy on research animal use and the Public Health Service policy on the use of laboratory animals.

Male New Zealand rabbits (2.0–2.5 kg) were randomly divided into 4 groups (n = 10 for each group): normal control (CR), sham control (SH), rapid atrial pacing (RAP) and RAP + miR-21 inhibitor(the miR-21 inhibitor was administered to this group through a lentiviral vector, Genechem, Shanghai, China). All of the rabbits except for one in the RAP group survived to the end of the study. The data generated from the dead rabbit was not included in the results presented in this paper.

All the rabbits were intravenously anesthetized with 30 mg kg⁻¹ pentobarbital sodium before being intubated and placed on mechanical ventilation with a volume-cycled ventilator (Model HX-200, TAIMENG, Chengdu, China). The heart was exposed at the center of the breastbone and a custom-designed set of electrodes, comprising a pair of electrodes with a distal hook for pacing and a pair of electrodes was sutured to the epicardial surface of the left atrium. The electrodes contain an interelectrode distance of 15 mm aligned proximally that serves for recording. The distal ends of these electrodes leads were tunneled subcutaneously and exposed at the animal’s back, where the electrodes were then connected to a pacemaker (output of 6 V with 1.0 ms pulse duration, Guangzhou Academy of Sciences, China) in the jacket. The pacemaker was programmed to provide RAP at 1000 ppm, and this pacing rate was maintained continuously for 4 weeks with a brief breaking period for the measurement of electrophysiological and mechanical parameters (Supplementary Table 1) [10, 26]. The rabbits in Group CR were not subjected to surgery while those in Group SH (sham control) experienced identical surgical procedure, but no RAP. When surgery was completed, the rabbits were given antibiotics and allowed to recover for 5 days. Postoperative care included the administration of antibiotics and analgesics.

MiR-control lentivirus vectors (109 TU/ml, 4 μl/day) and miR-21 inhibitor lentivirus vectors (109 TU/ml, 4 μl/day) were injected 2 times/per week for 4 weeks. The transcript for the miR-21 inhibitor was PCR purified and inserted into the lentiviral vector under the control of the CMV promoter. The GFP reporter gene was inserted at the 3′ end of the miR-21 inhibitor gene. Lentiviral vectors pseudotyped with the VSVg coat were produced. One day prior to pacing, the miRNA lentiviral vectors were injected into the jugular vein. At the end of the experiment, all of the rabbits were euthanized, and the left atria were examined histopathologically and collected for mRNA and protein analysis.

Masson trichrome staining of the paraffin section prepared from the Bouin-fixed samples was performed as previously described [27]. Adobe Photoshop 5.5 and Scion Images for Windows Beta 4.0.2 software were used for quantitate atrial collagen content [25].

The experimental procedure to isolate cardiac fibroblasts was approved by the Animal Care Committee of Guangzhou, China and was performed according to the method of Meszaros et al. Briefly, the ventricles of 2–3 hearts from adult male Sprague–Dawley rats (250–300 g) were minced, pooled, and placed in a collagenase/protease (Sigma, St Louis, MO, USA) digestion solution. The cells dissociated in the first treatment were discarded. After three digestions lasting 6 min each, the cells were pooled. The debris and myocytes were removed by unit gravity sedimentation. The fibroblasts were suspended in Dulbecco’s Modified Eagle Medium (DMEM) containing 1 % penicillin/streptomycin and 10 % fetal bovine serum. After a 60-min period of attachment to uncoated culture plates, those cells that were weakly attached or unattached were rinsed free and discarded. The attached cells were cultured in an incubator with 5 % CO₂ at 37 °C. The culture medium was changed every other day. These cultures contained N95 % cardiac fibroblasts (CFs) as indicated by positive vimentin expression and negative myoactin expression. Those cells in passages 2 and 3 were used in the following studies.

Cells were plated on 60 mm² plates at a density of 1 × 10⁶ cells per plate. When the cells reached 70–80 % confluency, they were transfected with pre-miR-21 or control pre-miR (Ambion, Austin, TX, USA) using the TransIT TKO transfection reagent (Mirus Bio, Madison, WI, USA). To investigate the role of miR-21 in TGF-β₁-induced collagen expression, we performed miR-21 transfection experiments in CFs. For these experiments, the cells were seeded at a density of 2 × 10⁴ cells/cm² in serum-free DMEM/F12. In this study, the cells were divided into the following groups: cells without transfection (blank control group, Group CR), cells transfected with the miR-control lentiviral vector (30 μM) (miR-control group, Group M), cells transfected with the pre-miR-21 (miR-21 over-expression) (30 μM) lentiviral vector (pre-miR-21 group, Group PM), cells treated with 10 ng/ml TGF-β₁ (TGF-β₁ group, Group T), cells treated with 10 ng/ml TGF-β₁ plus the pre-miR-21 vector (30 μM) (TGF-β₁ + pre-miR-21 group, Group TP), and cells treated with 10 ng/ml TGF-β₁ plus the miR-21 inhibitor (30 μM) (TGF-β₁ + miR-21 inhibitor group, Group TI).

Total RNA was extracted with TRIzol reagent (Gibco-BRL Life Technologies, New York, USA). cDNA was synthesized with the SYBR ExScript RT-PCR kit (TOYOBO, Japan) according to the protocol provided by the manufacturer. PCR primers for TGF-β₁ (forward: 5′-ACA TTG ACTTCC GCA AGG AC-3′; reverse: 5′-TAG TAC ACG ATG GGC AGTGG-3′), Smad7 (forward: 5′-GTG GCA TAC TGG GAG GAGAA-3′; reverse: 5′-GAT GGA GAA ACC AGG GAA CA-3′), collagen I (forward: 5′-TGG CAA GAA CGG AGA TGAC-3′; reverse: 5′-TCC AAA CCA CTG AAA CCT CTG-3′), and collagen III (forward: 5′-TTC CTT TGT GGG CTG TGT CT-3′; reverse: 5′-TTG GCT TCT CTC ACT TTC CAG-3′) were designed with Primer Express software (Applied Biosystems). GAPDH (forward: 5′-GCA CCG TCA AGG CTG AGA AC-3; reverse: 5′-ATG GTG GTG AAG ACG CCA GT-3′) was used as a reference to normalize the input amount of RNA for all samples. Real-time PCR was performed using an ABI7300 Real-Time PCR system (Applied Biosystems) with the SYBR green fluorophore. All of the reactions were performed in at least duplicate for every sample. Threshold cycle (Ct) data were collected using the Sequence Detection Software version 1.2.3 (Applied Biosystems). The fold change in mRNA level relative to GAPDH was calculated using the 2^∆∆Ct method [28].

TaqMan MicroRNA Reverse transcription reactions and TaqMan MicroRNA quantitative polymerase chain reactions (qPCR) were performed to detect miR-21 and an endogenous control, RNA U6 small nuclear (RNU6B) expression using the MicroRNA TaqMan Reverse Transcription Kit and the TaqMan MicroRNA Assays (Applied BioSystems, Carlsbad, CA, USA) according to manufacturer’s instructions. The miRNA detection conditions were: 95 °C for 10 min and 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The miRNA expression levels were calculated as the cycle threshold (-delta CT) of miR-21 and normalized with an endogenous control. MiR-21 (forward: 5′-GCA CCG TCA AGG CTG AGA AC-3; reverse: 5′- CAG CCC ATC GAC TGG TG-3′) and RUN6 (forward: 5′-CTC GCT TCG GCA GCA CA-3; reverse: 5′- AAC GCT TCA CGA ATT TGC GT-3′) were used as a reference to normalize the input amount of RNA for all samples. Real-time PCR was performed using an ABI7300 Real-Time PCR system (Applied Biosystems) with the SYBR green fluorophore. All of the reactions were performed in at least duplicate for every sample. Threshold cycle (Ct) data were collected using the Sequence Detection Software version 1.2.3 (Applied Biosystems). The fold change in mRNA level relative to GAPDH was calculated using the 2^∆∆Ct method [29].

RNA was extracted by standard trizol method and suspended in DNAse- and RNAse-free water. Samples were run on 15 % 7 M urea-acrylamide gels. Electrophoresis was performed at 20 mA for about 3 h at room temperature. The oligonucleotides to be used as probes were purchased from Sigma (Milan, Italy). After electrophoresis on urea-acrylamide gels, the RNA was transferred to hybond nylon filters at 10 V overnight at 4 °C. After the ultraviolet cross-linking, the filters were prehybridized in 6 X SSPE, 5 X DENHARDT and 0.5 % SDS solution. Ten picomoles probes were radiolabelled using P³² γATP by standard reaction at 37 °C. Microspin G25 columns (Amersham) were used to separate radiolabelled probe from unincorporated γATP. The prehybridization and hybridization were performed in the same buffer, at 37 °C overnight. The filters were washed once in prewarmed washing solution containing 6 X SSPE and exposed in cassettes containing phosphor screen overnight. The signals were read by Amersham Typhoon 9200 phosphoimager and densitometry was performed with IMAGEQUANT software (Amersham). The filters were stripped in a solution containing 0.2 × SSPE and 0.2 % SDS for 10 min at 95 °C and subsequently rehybridized with another probe [30].

To examine whether Smad7 is a valid target of miR-21, a single copy of the putative miR-21-recognition element from the 3′-UTR of the Smad7 gene was cloned into the GV306 plasmid vector downstream of the dual luciferase reporter gene (Genechem, Shanghai, China). CFs were co-transfected with the GV306 vector containing the Smad7 3′-UTR and the miR-21 overexpression plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Co-transfection with a non-targeting negative control RNA was performed as a control. At 48 h post transfection, the cells were lysed and assayed for luciferase activity with a dual-luciferase reporter assay kit (Promega, Madison, WI, USA) on a luminometer (Lumat LB9507).

After fixed in 10 % neutral formalin for 24 h, the middle ring of left atrium was embedded in paraffin and cut into 4 μm thick sections. The section was incubated with primary antibodies (goat polyclonal anti-Smad7 antibody from Santa Cruz Biotechnology at a 1:150 dilution) at 4 °C overnight. Incubation with the secondary antibody (biotinylated rabbit anti-goat antibody also from Boster Biotect CO). was performed at 37 °C for 30 min. Staining was generated with the streptavidin biotin-peroxidase complex immunohistochemical staining kit from Boster Biotect CO. Negative controls were performed by leaving out the primary antibody. Sections were viewed with 400× magnification and the intensity of the collagen staining was analyzed using the IBAS2.5 Image Analytical System (Institute of Biomedical Engineering, Beijing, China) as described before (Sheng, 1995). Five random fields per section were analyzed to produce a single number for the section [31].

The experimental procedure was performed as described previously [9], the cells were incubated with a primary antibody (anti-vimentin, myoactin, or Smad7) (1:100) in 5 % BSA overnight at 4 °C. The cells were then washed and incubated with a rhodamine-conjugated secondary antibody (1:500). The intensity of the Smad7 staining was analyzed using the IBAS2.5 Image Analytical System (Institute of Biomedical Engineering, Beijing, China) as previously described [10].

Western blot was performed as described previously [12]. The primary antibodies (Santa Cruz Biotechnology) were diluted as follows: TGF-β₁, 1:200; Smad7, 1:250; collagen I, 1:200; collagen III, 1:200; and β-actin, 1:500. Horseradish peroxidase–labeled secondary antibodies (Cell Signaling Technology) were diluted 1:1000 with 0.2 % TBS-T and 1 % skim milk. The protein bands on the Western blots were visualized using ECL Plus (Amersham, Arlington Heights, IL, USA). The relative band densities were normalized against β-actin.

The data are expressed as mean ± SE. ANOVA and the Student’s t test were used to determine statistical significance. A two-tailed probability value of 0.05 was considered statistically significant.

Earlier reports have shown that miR-21 is extensively involved in myocardial fibrosis, and miR-21 is considered to be associated with TGF-β₁-induced myocardial fibrosis [22, 32]. However, there are few reports about the role of miR-21 in AF. We have reported that TGF-β₁ is up-regulated and promotes myocardial fibrosis in AF. In this study, we explored the relationship between miR-21 and TGF-β₁ in cardiac fibroblasts.

Several animal models of chronic sustained AF have been developed to assess the atrial electric remodeling and arrhythmias of AF. In AF, atrial effective refractory period (AERP) was decreased, and the animals became more vulnerable to AF [4, 34]. It has been reported that rapid pacing induces AF with much higher incidence at the left atrial site than at the right atrium or any other sites, partly because of the inhomogeneous dispersion of AERP. When atrial electrical remodeling returns back to normal, the underlying reasons for persistent AF and structural remodeling are still present. Thus, atrial electrical remodeling alone is sufficient to induce AF, while structural remodeling is responsible for maintaining AF [35, 36]. In this study, we have successfully established the electrical stimulation rapid pacing left atrium-induced AF model. We demonstrated that RAP alone induced profound changes in gene expression of collagens. In addition to the increase in the hydroxyproline content and left atrial weight, a significant deposition of cardiac collagen I and III was observed in the atria subjected to rapid pacing.

Tachycardia-induced atrial fibrosis is a hallmark of AF-induced structural remodeling. This condition is characterized by the accumulation of extracellular matrix (ECM) and myocardial fibrosis [10, 37]. In this study, we demonstrated that RAP alone induced profound changes in the expression of collagen I/III in the heart, strongly suggesting that tachycardia during AF might cause atrial remodeling resulting from atrial fibrosis. In addition to the increase in hydroxyproline content and left atrial weight, a significant deposition of cardiac collagen I and III was observed in the atria subjected to rapid pacing. These results also support our previous observations that atrial fibrosis consisting of collagen types I and III is a typical feature of AF.

Increasing evidence indicates that targeting TGF-β₁/Smad-specific miRNAs related to fibrosis may be a better approach for combating heart disease [38–40], with miR-21 being the best characterized miRNA associated with TGF-β₁-mediated fibrosis [41]. It reported that miR-21 is expressed much higher in cardiac fibroblasts and accompanied by the increased expression of collagen in pathological states of heart failure [42]. In a rat model of myocardial infarction, the down-regulation of miR-21 can reduce the degree of atrial fibrosis and the incidence of AF [43]. Adam recently confirmed miR-21 levels were significantly higher in patients with atrial fibrillation, and the up-regulation of miR-21 target genes can increase fibrin deposition [19]. We have observed previously that RAP may cause atrial fibrosis through the AngII/AT1 receptor–specific activation of the TGF-β₁/Smad pathway and that TGF-β₁ overexpression induces myocardial fibrosis in AF [10]. However, whether miR-21 over-expression enhanced TGF-β₁-induced myocardial fibrosis in AF remained elusive. In vivo, we found that RAP induced TGF-β₁ and miR-21 up-regulation with the increased expression of collagen. Transfection with the miR-21 inhibitor, we found it reduce the incidence of AF and ameliorated RAP-induced atrial fibrosis. In vitro, we found that miR-21 over-expression increased collagen I/III expression and that miR-21 over-expression after treatment with TGF-β₁ further increased collagen I/III expression. In addition, blocking miR-21 expression reverses the TGF-β_1-induced up-regulation of collagen I/III. Thus, our study further suggested that miR-21 may be involved in the regulation of RAP-induced myocardial fibrosis, and we speculate that miR-21 over-expression significantly enhances TGF-β₁-induced collagen expression.

The down-regulation of Smad7 may be key in the increased activation of the TGF-β₁/Smad signaling pathway that is responsible for increased atrial fibrosis during AF [10, 44]. Smad7 not only exhibits a protective role in Ang II-induced cardiac fibrosis and inflammation, but also possesses therapeutic potential for hypertensive cardiac disease [11, 16]. In our previous study, we reported that the overexpression of Smad7 blocked AngII-induced increases in collagen I synthesis and that the overexpression of TGF-β₁ decreased Smad7 expression [10]. In this study, we also found that RAP decreased Smad7 expression, which is negatively correlated with TGF-β₁ and collagen I/III expression. This finding further confirmed that the down-regulation of Smad7 might cause myocardial fibrosis in AF. miR-21 is the best characterized miRNA associated with TGF-β₁-mediated fibrosis [41]. For example, miR-21 and Smad7 are critical regulators of TGF-β₁ signaling during the induction of carcinoma-associated fibroblast formation [33, 45, 46]. miR-21 overexpression enhances the TGF-β₁-induced epithelial-to-mesenchymal transition by targeting Smad7 and aggravates renal damage in diabetic nephropathy. However, the regulation mechanism linking miR-21 and Smad7 in RAP-induced myocardial fibrosis was unclear. To further demonstrate our observations in this study and gain more information about how Smad7 is down-regulated during RAP, we examined growth-arrested adult rat fibroblast cells. Moreover, bioinformatics and the luciferase reporter assays showed that Smad7 is a validated target of miR-21. We found that miR-21 over-expression decreases Smad7 expression, which was similar to the change that occurred with TGF-β₁ treatment. Furthermore, miR-21 over-expression significantly enhanced TGF-β₁-induced Smad7 down-regulation. Interesting, inhibiting miR-21 expression weakened the TGF-β₁-mediated down-regulation of Smad7 expression. This study further confirmed our previous research that the down-regulation of Smad7 may be responsible for RAP-induced myocardial fibrosis. More interestingly, the molecular mechanism underlying this phenomenon is as follows: the expression of TGF-β₁ leads to miR-21 up-regulation, and miR-21 over-expression directly down-regulates Smad7 expression, which leads to amplification of TGF-β₁ signaling and ultimately results in RAP-induced fibrosis. Thus, we speculate that miR-21 over-expression significantly enhances TGF-β₁-induced collagen expression and that Smad7 can directly inhibit collagen expression in RAP-induced myocardial fibrosis.

In summary, our data demonstrate that TGF-β₁-induced miR-21 over-expression enhances TGF-β₁-induced collagens expression by directly down-regulating Smad7. These results may provide new insights into the mechanisms underlying AF-induced atrial fibrosis and myocardial remodeling and provide valuable information for novel therapeutic targets for AF.

Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 14 kb)