Background/Aims: Deregulated myocardial fibrosis is associated with a wide spectrum of cardiac conditions, being considered one of the major causes for heart disease. Our study was designed to investigate the role of microRNA-328 (miR-328) in regulating cardiac fibrosis. Methods: We induced cardiac fibrosis following MI by occlusion of the left coronary artery in C57BL/6 mice. Real-time PCR was employed to evaluate the level of miR-328. Masson's Trichrome stain was used to evaluate the development of fibrosis. Luciferase activity assay was performed to confirm the miRNA's binding site in the TGFβRIII gene. Western blot analysis was used to examine TGFβRIII, p-smad2/3 and TGF-β1 at protein level. Results: In this study, we found that miR-328 was significantly upregulated in the border zone of infarcted myocardium of wild type (WT) mice; TGFβRIII was downregulated whereas TGF-β1 was upregulated along with increased cardiac fibrosis. And miR-328 stimulated TGF-β1 signaling and promoted collagen production in cultured fibroblasts. We further found that the pro-fibrotic effect of miR-328 was mediated by targeting TGFβRIII. Additionally, cardiac fibrosis was significantly reduced in infarcted heart when treated with miR-328 antisense. Conclusions: These data suggest that miR-328 is a potent pro-fibrotic miRNA and an important determinant of cardiac fibrosis in diseased heart.
In tissues composed of post-mitotic cells, like heart, new myocytes cannot be regenerated; instead, fibroblasts proliferate to fill the gaps created due to removal of dead cells. In the normal heart, cardiac myocytes are surrounded by a fine network of collagen fibers: two-thirds of the cell population is composed of non-muscle cells, the majority of which are fibroblasts . A growing body of evidence indicates that, along with cardiomyocyte hypertrophy, diffused interstitial fibrosis is a key morphological feature of the structural myocardial remodeling that is a characteristic of all forms of cardiac diseases of different origins (e.g. ischemic, hypertensive, valvular, genetic, and metabolic) [2,3,4,5,6].
Acute myocardial infarction (AMI) due to coronary artery occlusion represents a major cause of morbidity and mortality in humans. Scar formation at the site of the infarct and interstitial fibrosis of adjacent myocardium prevent myocardial repair, contribute to loss of pump function, and predispose individuals to ventricular dysfunction and arrhythmias, which in turn confer an increased risk of adverse cardiovascular events [7,8]. Fibrosis results in the abnormal myocardial stiffness that contributes to diastolic dysfunction, cardiomyocyte loss, arrhythmias, and the progression of heart failure. Developing novel molecular therapeutic strategies for cardiac fibrosis is still a challenge for basic and clinical scientists.
The transforming growth factor-β1 (TGF-β1), a multifunctional cytokine, mediates the signaling pathway known to be essential regulator of matrix deposition and production of collagen in the development of cardiac fibrosis. TGF-β1 stimulates cardiac fibrogenesis through its receptors. There are three prototypical TGF-β1 receptors: TGFβRI, TGFβRII and TGFβRIII (also known as betaglycan) . TGFβRIII is a transmembrane glycoprotein with large extracellular regions that can bind TGF-β1, and small cytoplasmic regions with no clearly identifiable signaling motif. TGFβRIII is considered an ‘accessory' receptor, since it regulates the interaction of TGF-β1 with TGFβRI/TGFβRII . Recent studies showed that TGFβRIII transduces anti-fibrotic signals through binding TGF-β1 [11,12]. However, the molecular mechanisms for the regulation of TGFβRIII in the development of cardiac fibrosis are still poorly understood.
MicroRNAs (miRNAs) are a recently discovered class of endogenous non-coding RNAs that silence expression of protein-coding genes by acting on the 3' untranslated region (3'UTR) of the target genes through a partial base-paring mechanism and have been implicated in a variety of biological and pathological processes including cardiovascular disease [13,14,15]. A subset of miRNAs is enriched in cardiac fibroblasts compared to cardiomyocytes . A number of studies have demonstrated the involvement of miRNAs in regulating myocardial fibrosis in the settings of myocardial ischemia or mechanical overload [7,16,17,18].
In our previous study, we generated a transgenic mouse line with cardiac-specific overexpression of miR-328 and demonstrated that miR-328 plays a critical role in atrial fibrillation and atrial electrical remodeling process . In this study, we demonstrated that miR-328 is a potent pro-fibrotic miRNA and is potentially involved in cardiac fibrosis by targeting TGFβRIII, which provides new insight into the mechanism for regulation of cardiac fibrosis and indicates that inhibition of miR-328 may be a promising therapeutic strategy for intervention of cardiac fibrosis.
Materials and Methods
Mouse model of myocardial infarction
Adult male (8 week) C57BL/6 mice used in this study were kept under controlled conditions (humidity: 55 ± 5%; temperature: 23 ± 1°C and a 12 h light/dark artificial cycle). Mice (25-30 g) were randomly divided into sham and myocardial infarction groups. Five animals were included in each group. Myocardial infarction (MI) was established as previous described in detail . Mice were anesthetized with pentobarbital (40 mg/kg) and subjected to open chest surgery. The left anterior descending coronary artery was occluded and then the chest was closed. Sham animals underwent open-chest procedures without coronary artery occlusion. All surgical procedures were performed under sterile conditions. One week after occlusion, the heart was removed for detecting the level of miR-328 and other molecules as to be specified. All experimental procedures were in accordance with and approved by the Institutional Animal Care and Use Committee of Harbin Medical University, P.R. China. The cholesterol-conjugated miR-328 antisense (antagomiR-328, sequence: 5'-ACG GAA GGG CAG AGA GGG CCAG-3') and mismatch antagomiR-328 (M-antagomiR-328, sequence: 5'-GGC AAG ACG AAA CGA GAC GACA-3') was purchased from RiboBio Co., Ltd. (Guangzhou, China). The antagomiR-328 and M-antagomiR-328 were injected through mouse tail vein per 24 h at a dose of 40 mg/kg for consecutive three days before MI. One week after MI, the hearts were removed from the mice and 1-2 mm area between the infarct region and normal tissue was selected as the peri-infarct region for the following experiments.
Masson Trichrome's stain was used for the detection of collagen fibers in tissues on 4% paraformaldehyde-fixed, paraffin-embedded sections. The collagen fibers were stained in blue and the nuclei in black, and the cytoplasm was stained in red. Fibrous tissue was examined with image analysis software (Image-pro plus 4.0, Meida Cybernetics LP) as previously described [21,22].
Western blot analysis
Protein samples were extracted from the heart tissue as previously described [23,24]. After grinding frozen tissue with RIPA lysis buffer (Beyotime, Jiangsu, China), cells were lysed in standard lysis buffer. After boiling the samples for 5 min, the protein samples were fractionated by SDS-PAGE (10% -15% polyacrylamide gels). Primary antibodies were used to detect TGFβRIII (Cell Signaling, Beverly, MA), p-smad2/3 (Cell Signaling, Beverly, MA), and TGF-β1 (Cell Signaling, Beverly, MA). Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (area × OD) for each group and normalizing to GAPDH (anti-GAPDH antibody from Kangcheng Inc., Shanghai, China) as an internal control.
Total RNA was isolated from cells and cardiac tissue using Trizol reagent (Invitrogen, Carlsbad, CA) for measurements of mRNAs and miRNA, and mirVana qRT-PCR miRNA Detection Kit (Applied Biosystems, Foster City, CA, USA) was used for quantification of miRNAs according to the manufacturer's protocol. Real-time RT-PCR was carried out as described . GAPDH was used as an internal control for mRNAs quantification and U6 was used as an internal control for miRNA. All primers were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co. Ltd (Shanghai, China).
Fibroblast culture and transfection of miRNA
Neonatal mouse myocardial fibroblasts were prepared as previously described . The fibroblasts was incubated with 50 nM SB505124 (Sigma-Aldrich, St. Louis, MO, USA), a selective inhibitor of TGFβRI kinase, for 1 h before transfection of miR-328, AMO-328. miR-328 (5'-CUG GCC CUC UCU GCC CUU CCGU-3'), its antisense oligonucleotides (AMO-328, 5'-ACG GAA GGG CAG AGA GGG CCAG-3'), and a scrambled RNA as negative control for miR-328 (5'-UUC UCC GAA CGU GUC ACG UAA-3') were synthesized by Integrated DNA Technologies, Inc. These constructs were delivered into cardiac fibroblasts by transfection using Lipofectamine-2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's protocols. The miRNA and Lipofectamine-2000 were separately mixed with 500 ml of Opti-MEM I Reduced Serum Medium (Gibco, Grand Island, NY, USA) for 5 min. Then, the two mixtures were combined and incubated at room temperature for 20 min. The Lipofectamine-miRNA mixture was added to the cells and incubated at 37°C for 6 h. Subsequently, 4 ml of fresh medium containing 10% FBS was added to the flask. Then 5 ml fresh medium containing 10% FBS was added to the flasks and the cells were maintained in the culture until following experiments.
Measurement of collagen content
The Sircol Collagen Assay (Biocolor, Belfast, U.K.) is a quantitative dye-binding method for the analysis of total collagen extracted from tissue and cells. Briefly, cells (2 × 105) were cultured for 4 days with various treatments and then were lysed in 0.05 M Tris buffer (pH 7.5). The samples were stirred at 4°C. A transparent solution was obtained, containing salt soluble collagen. After treatments, lysate (100 µl) was stained with 1 µl of Sircol Dye reagent and mixed by rotating for 30 min. The sample was transferred to a microcentrifuge tube and spanned at 10,000 × g for 10 min. The unbound dye solution was removed carefully and 1 ml of the Alkali reagent was added into the tube. When the bound dye had been dissolved by the Alkali (approximately 10 min), the sample (200 µl) was transferred to a 96-well plate for measurement. The dye was solubilized and absorbance was read at 540 nm. Readings were converted into protein units using a linear calibration curve generated from standards (Vitrogen 100, Angiotech Biomaterials, Palo Alto, CA, USA). Total protein was quantified by the bicinchoninic acid (BCA) assay (BCA protein assay kit, Beyotime, Shanghai, China). Quantitative measurement of collagen content was normalized to the total protein. Results are presented as a ratio of collagen content to the total protein.
TGFβRIII 3'UTR containing the conserved miR-328 binding sites was amplified by PCR. The PCR fragment was subcloned into the SacI and HindIII sites downstream of the luciferase gene in PGL3 plasmid (Promega). For luciferase assays, HEK293 cells (1 × 105/well) were transfected with 100 ng target DNA (firefly luciferase vector) and 10 ng PRL-TK (TK-driven Renilla luciferase expression vector) which was used as an internal control to normalize transfection efficiencies. At the same time, miRNA was transfected into HEK293 cells using Lipofectamine-2000 (Invitrogen, Carlsbad, CA) according to manufacturer's instructions, the control group was only transfected with PGL3 plasmid and PRL-TK. Luciferase activity was measured using a luminometer (Lumat LB9507) and luciferase assay kit (Promega, Madison, WI) 24 h after transfection.
Group data are expressed as mean ± SEM. To analyze the differences between two groups, an unpaired Student's t-test was performed once normality had been proven (the Kolmogorov-Smirnov test); a nonparametric test (the Wilcoxon test for unpaired data) was applied when normality could not be determined due to small size sample. To analyze the differences more than two groups, a one-way analysis of variance (ANOVA) followed by a Bonferroni test was performed once normality had been proven (the Kolmogorov-Smirnov test). P < 0.05 was taken to indicate a statistically significant difference.
miR-328 is upregulated in the heart of mouse after myocardial infarction (MI)
To explore the potential role of miR-328 in cardiac fibrosis, we firstly determined if miR-328 expression is altered in the peri-infarct region of mice one week after MI. As shown in Fig. 1A & 1B, compared to sham group, Masson's Trichrome stain showed marked collagen production in MI mice. And, quantitative real-time PCR results showed that miR-328 level was ∼5-fold higher in MI mice than in sham animals (Fig. 1C).
Effects of miR-328 on collagen production in cultured fibroblasts isolated from neonatal WT mice
TGF-β1 signaling pathway plays important roles in modulating cell function and in the progression of fibrosis. We transfected miR-328 into cultured fibroblasts isolated from neonatal mice to evaluate the effects of miR-328 on TGF-β1 signaling pathway, proteins and collagen production. Western blot analyses revealed remarkable upregulation of TGF-β1 and p-Smad2/3 at the protein level in cultured fibroblasts transfected with miR-328 (Fig. 2A & 2B). Moreover, the upregulations were efficiently reversed by AMO-328 and M-miR-328 did not affect the protein levels. Consistently, miR-328 promoted production of collagen in cultured fibroblasts and AMO-328 nearly abolished the effect (Fig. 2C).
TGFβRIII is a direct target of miR-328 in cardiac fibroblasts
The above results suggest that miR-328 can activate TGF-β signaling and promote fibrogenesis in cultured fibroblasts. We wanted to know if the fibrosis-promoting effect of miR-328 is related to the TGF-β1 signaling mechanism.
To test this notion, we first performed computational predictions of target genes for miR-328 using the TargetScan algorithm (http://genes.mit.edu/targetscan/). As anticipated, the 3′UTR of TGFβRIII contains a binding site for miR-328 (Fig. 3A) which is highly conserved among mouse, rat, and human (Fig. 3B). We then experimentally validated the targeting. We constructed a chimeric luciferase reporter gene vector by fusing the 3′UTR of TGFβRIII downstream to the luciferase gene coding sequence. We co-transfected this vector with synthetic miR-328 into HEK293 cells and measured luciferase activities 24 h after transfection. Forced expression of miR-328 resulted in marked reduction of luciferase activities and this effect was abrogated by miR-328 antisense AMO-328 (Fig. 3C). The mismatch miR-328 (M-miR-328) failed to affect luciferase activity, neither did the scrambled miRNA (SC-miRNA) as negative controls (Fig. 3C).
The ability of miR-328 to repress the expression of TGFβRIII was further verified by western blot analysis of TGFβRIII protein in fibroblasts isolated from neonatal mouse hearts. The downregulation of TGFβRIII induced by miR-328 was efficiently prevented by AMO-328. By comparison, the M-miR-328 lost the ability to downregulate TGFβRIII expression (Fig. 3D).
Pro-fibrotic effect of miR-328 is mediated through downregulating TGFβRIII
We subsequently confirmed TGFβRIII as a negative regulator of collagen production. Delivery of the plasmid carrying the TGFβRIII gene into the fibroblasts not only inhibited miR-328 induced expression of TGF-β1, also resulted in decreased TGF-β1 compared with control group. However, negative control (NC) failed to do so (Fig. 4A). As a consequence, when co-transfected, TGFβRIII remarkably attenuated the collagen production induced by miR-328 and when transfected alone, it diminished the collagen content compared with control. Meanwhile, SB505124, a selective inhibitor of TGFβRI kinase, abolished the effects of miR-328 (Fig. 4B). These results indicate that overexpression of miR-328 stimulates TGF-β1 signaling and promotes collagen production through targeting TGFβRIII.
Knockdown of miR-328 attenuates cardiac fibrosis in mice following MI
If miR-328 is indeed crucial to cardiac fibrogenesis, then knockdown of this miRNA should confer a resistance to fibrosis stimulation, being beneficial to improve myocardial interstitial fibrosis. To get onto this point, we assessed whether knockdown of miR-328 alleviates cardiac fibrosis induced by MI. As depicted in Fig. 5A, injection of antagomiR-328 significantly inhibited the expression of miR-328 in the heart of mice. Furthermore, our data showed that administration of antagomiR-328 substantially diminished cardiac fibrosis (Fig. 5B and 5C) and collagen contents (Fig. 5D). As expected, mismatch antagomiR-328 (M-antagomiR-328) had no such effects. In addition, protein level of TGF-β1 was reduced by antagomiR-328 (Fig. 5E). Conversely, TGFβRIII expression was elevated by antagomiR-328 (Fig. 5F).
In this study we evaluated the role of miR-328 in controlling cardiac fibrosis, the underlying signaling mechanisms, and the significance of this cellular function in the pathological fibrogenesis of infarcted myocardium. And for the first time, our results showed that overexpression of miR-328 increased the collagen content in the primary cardiac fibroblasts. On the contrary, normalization of miR-328 level by its antisense reversed this pro-fibrotic effect, which could be a novel approach to protect against cardiac fibrosis. miR-328 repressed the expression of TGFβRIII protein through directly targeting its encoding gene. Knockdown of miR-328 alleviated cardiac fibrosis and rescued the TGFβRIII expression in infarcted myocardium of mice. These findings indicate that (1) miR-328 is a strong pro-fibrotic miRNA in the heart; (2) Repression of the anti-fibrotic signaling molecule TGFβRIII likely underlies the pro-fibrotic action of miR-328; (3) miR-328 is a critical determinant of fibrosis and therefore miR-328 may be considered a new therapeutic target for cardiac disease.
Recent identification of miRNAs as regulators of myocardial fibrosis has forged new frontiers in understanding cardiac conditions associated with fibrogenesis. In this conceptual framework, characterization of individual miRNAs that are specifically associated with myocardial fibrosis might allow us to develop diagnostic tools and innovative therapies for fibrogenic cardiac diseases. van Rooij et al.  reported that miR-29 exerts an anti-fibrotic effect in myocardium. Artificial overexpression of miR-29b in fibroblasts results in reduced collagen expression. Conversely, downregulation of miR-29b by its antisense both in vitro and in vivo induces expression of collagens. Another study from Thum et al. showed that miR-21 is selectively upregulated in cardiac fibroblasts in the later stages of heart failure . In vivo silencing of miR-21 in mice inhibits interstitial fibrosis and attenuates cardiac dysfunction.
In our previous study, we demonstrated that miR-328 controls atrial fibrillation (AF) via targeting CACNA1C and CACNB1 genes encoding L-type Ca2+ channel α1c- and β1-subunits in animal models . Forced expression of miR-328 in canine atrium and in mice recapitulates the phenotypes of AF, diminishes L-type Ca2+ current, and shortens atrial action potential duration. However, knockdown of miR-328 level with its antisense oligonucleotide reverses the effects both in vivo and in vitro. Interestedly, histological examination reveals progressive fibrosis in the heart of miR-328 transgenic mice, which indicates that miR-328 plays an important role in the process of cardiac fibrosis. We also found that miR-133 and miR-590 contribute to nicotine-induced AF by targeting TGF-β1 and TGF-βRII. Upregulation of miR-133 and miR-590 decreases TGF-β1 and TGF-βRII levels and collagen content. These effects are abolished by antagomiRs against miR-133 or miR-590 . In the present study, we presented strong evidence for miR-328 as a pro-fibrotic miRNA critically contributing to the increased cardiac fibrosis in ischemic myocardium through directly repressing TGFβRIII to indirectly upregulate TGF-β1.
TGFβRIII displays different biological functions to mediate TGF-β pathway as an activator  or inhibitor [12,25] dependent on cell and tissue types. Eickelberg et al. reported that TGFβRIII inhibits TGF-β pathway activity in LLC-PK1 renal proximal tubule cells and enhances TGF-β pathway activity in L6 myoblasts . Recent studies provide more evidence that TGFβRIII exhibits antagonistic activities of TGF-β1 signaling by preventing TGFβRI-TGFβRII complex formation. Our previous study showed that TGFβRIII negatively regulates TGF-β pathway by neutralizing TGF-β1 and preventing the formation of TGFβRI/TGFβRII which in turn attenuates collagen production in cardiac fibroblasts . Moreover, a synthetic peptide from TGFβRIII can inhibit TGF-β1-induced collagen I synthesis by blocking the binding of TGF-βl to its type I and type II receptors in fibroblast cells . Hermida et al. further demonstrated that this soluble peptide exerts significant inhibition on TGF-β1 dependent signaling activity, collagen type I synthesis, and myocardial fibrosis, in addition to blocking the binding of TGF-β1 to its type I and II receptors . A study by Vilchis-Landeros et al. demonstrated that this soluble peptide binds TGF-β with high affinity and isoform selectivity to block TGF-β2 and TGF-β1 and therefore inhibits the actions of TGF-β .
Our results show that miR-328 expression is upregulated in the peri-infarct region. Correspondingly, the level of TGFβIII is downregulated whereas that of TGF-β1 is upregulated after MI. More importantly, application of miR-328 antisense increases TGFβRIII level and reduces collagen contents and protein levels of TGF-β1. These findings suggest that miR-328 plays an important role in cardiac fibrogenesis in ischemic heart as a pro-fibrotic miRNA and reveal a novel signaling mechanism of miRNA in controlling the generation of cardiac fibrosis.
There are a couple of limitations in the present study. The experiments were performed with 5 animals in the study. However, adequate sample size can produce more precise results. Myocardial infart size is closely related to the cardiac fibrosis. Measurement of infarct size and heart function will provide more evidence to evaluate role of miR-328 in regulation of cardiac fibrosis induced by MI.
In summary, our results indicate that miR-328 as a pro-fibrotic factor plays a critical role in regulation of cardiac fibrosis following myocardial infarction by targeting TGFβRIII. These findings provide new insight into the mechanism of cardiac fibrosis, and miR-328 may be a new target for therapy of heart disease associated with cardiac fibrosis.
This work was supported in part by the National Basic Research Program of China (973 program, 2013CB531104), the National Nature Science Foundation of China (No. 81530010, 81370245 and 31300943), the Heilongjiang Province Outstanding Youth Foundation (NO. JC201315) and New Century Training Program Foundation for the Talents (1155-NCET-010) of Heilongjiang Province of China.
The authors state no conflict of interest.