MicroRNA-208b Alleviates Post-Infarction Myocardial Fibrosis in a Rat Model by Inhibiting GATA4.

BACKGROUND Myocardial infarction affects the health of many people. Post-infarction myocardial fibrosis has attracted much attention, but details of the mechanism remain elusive. In this study, the role of microRNA-208b (miR-208b) in modulating post-infarction myocardial fibrosis and the related mechanism were investigated. MATERIAL AND METHODS A rat model of myocardial infarction induced by ligating the left anterior descending artery was used to analyze the expression and roles of miR-208b by overexpression with the lentivirus vector of pre-miR-208b. Myocardial function was assessed and the expression of fibrosis-related factors type I collagen (COL1) and ACTA2 (alias αSMA) was detected. Myocardial fibroblasts isolated from newborn rats were transfected with luciferase reporter vectors containing wild-type or mutant Gata4 3' UTR to verify the relationship between Gata4 and miR-208b. We then transfected the specific small interference RNA of Gata4 to detect changes in COL1 and ACTA2. RESULTS miR-208b was down-regulated in hearts of model rats (P<0.01). Overexpressing miR-208b improved myocardial functions, such as reducing the infarction area (P<0.05) and promoting LVEF and LVFS (P<0.01), and inhibited COL1 and ACTA2 (P<0.01). Luciferase reporter assay proved Gata4 to be the direct target of miR-208b, with the target sequence in the 3'UTR. Inhibiting GATA4 resulted in the down-regulation of COL1 and ACTA2, suggesting that the role of miR-208b was achieved via regulating GATA4. CONCLUSIONS This study demonstrates the protective function of miR-208b via GATA4 in post-infarction myocardial fibrosis, providing a potential therapeutic target for treating myocardial fibrosis.

Background

Myocardial infarction damages heart muscle by causing blood flow stoppage, which may lead to heart failure or cardiac arrest, and causing discomforts similar to heartburn. As a manifestation of coronary artery disease, myocardial infarction impacts the health of many people, with about 90% of the cases attributable to modifiable risk factors such as smoking and hypertension [1]. Environmental pollution, including noise and air pollution, also contribute to myocardial infarction [2]. In terms of the pathogenic mechanism, the most evident feature of myocardial infarction is the accumulation of collagen, which in the heart is primarily synthesized by cardiac fibroblasts, leading to post-infarction myocardial fibrosis. Collagen is expressed at low levels in healthy hearts, but is increased at infarction sites and the peripheral zone with the progression of myocardial infarction [3]. Myocardial fibrosis can be clearly observed at the infarction sites at day 7 after myocardial infarction in rats, contributing to heart dysfunction [4]. Promising detection and evaluation techniques, such as ultrasonic backscatter, have been reported [5]. Improved therapy for cardiac diseases, especially the stem cell transplantation strategy [6], has attracted attention, and related molecular mechanisms, like the role of C3G protein, have been revealed [7]. However, there is still a need for more detailed information on myocardial infarction and fibrosis.

microRNAs are short RNAs encoded by the genome, inhibiting translation or promoting mRNA degradation via binding to the 3′ untranslated region (UTR) of mRNAs [8]. A single microRNA can recognize various target mRNAs, and a single mRNA can be bound by several microRNAs simultaneously, constituting an elaborate regulatory system. Recent studies have suggested the association and use of microRNAs in myocardial infarction and fibrosis. Overexpression of microRNA-21 (miR-21) decreases myocardial infarction area [9], and miR-1 and mir-206 are up-regulated in myocardial infarction [10], with miR-1 also being a potential biomarker for acute myocardial infarction [11]. miR-29 is a repressor of myocardial fibrosis after infarction [12], as well as miR-24, which modulates myocardial fibroblast functions after infarction via the furin-TGF-β pathway [13]. It thus appears that exploitation and utilization of these powerful microRNAs are of great significance in treatment of myocardial infarction and myocardial fibrosis.

miR-208 is primarily expressed in the heart and its elevated concentration in plasma is correlated with myocardial injury [14], which can be a biomarker of acute myocardial infarction [15,16]. However, its relationship with post-infarction myocardial fibrosis and the mechanism remain unclear. This study aimed to reveal the role of miR-208b, a member of the miR-208 family, in modulating post-infarction myocardial fibrosis and the possible regulatory mechanism. The expression of miR-208b was examined in rat hearts in an induced myocardial infarction rat model. miR-208b was overexpressed by the lentivirus vector of pre-miR-208b, and the myocardial function after the model construction was monitored. Fibrosis-related factors, including type 1 collagen (COL1) and actin alpha 2, smooth muscle, and aorta (ACTA2, alias αSMA), were detected to analyze the influence of miR-208b on myocardial fibrosis. Myocardial fibroblasts isolated from newborn rats were used to verify the direct regulatory relationship of miR-208b and GATA binding protein 4 (GATA4). All these experiments were performed to provide valid information regarding the regulatory role of miR-208b in post-infarction myocardial fibrosis.

Material and Methods

Animals

The specific pathogen-free grade Sprague-Dawley (SD) rats used in this study were purchased from Vital River Laboratories (Beijing, China), and raised at 24°C and 50% humidity. Sixty adult rats (weight 250±20 g) were used for the construction of a myocardial infarction model – 15 for the sham-operated group and 45 for the operated group. Ten newborn rats (age 2 days) were prepared for the culture of myocardial fibroblasts. The animal experiments were approved by a local ethics committee and performed according to the rules of our institute.

Myocardial infarction rat model

The rats for model induction were anesthetized by diethyl ether supplemented with intraperitoneal injection of ketamine (75 mg/kg). Atropine (20 μg/kg) was injected to reduce the airway secretion. After being affixed to the operating table, the rats were connected to an electrocardiograph and a ventilator with respiratory rate of 80 times per min, inspiration and expiration ratio of 1: 2, and tidal volume of 18 mL/kg. The pleura were opened to expose the heart, and the left anterior descending (LAD) was ligated with Prolene and a needle (0.5 mm in diameter). Then the chest and skin were closed and sterilized with iodophor. The ventilator was removed after the rats revived. Electrocardiograph indicating an elevation of J point at 10 min after the operation indicated successful model induction. Rats in the sham-operated groups underwent all of the same procedures detailed above except for the ligation step, making a ringer instead of the ligation. To prevent infections, all rats were injected with penicillin (Thermo Scientific, Carlsbad, CA) during the 3 days after the operation.

Myocardial fibroblast culture

The newborn rats were anesthetized and sacrificed for heart sampling. The heart samples were cut into pieces of about 1 mm3 and digested by trypsin (Gibco, Carlsbad, CA) at 37°C for 15 min. Then cells were collected and cultured in Dulbecco’s modified Eagles medium (DMEM)/F12 (Gibco) supplemented with fetal bovine serum (Gibco) at 37°C with 5% CO2 for adherence. After 90 min, the cell suspension was removed and the adherent cells were collected and cultured. The purity of myocardial fibroblasts was examined by anti-vimentin, anti-Von Willebrand factor, and anti-desmin (ABcam, Cambridge, UK), and the fibroblasts were used for further analysis when the purity reached 95%.

Lentivirus transfection

Lentivirus to overexpress pre-miR-208b and blank lentivirus were designed and constructed by GenePharma (Shanghai, China). For animal transfection, the lentivirus was injected through the coronary artery of the operated group after the induction of the rat model. The rats were raised for 4 weeks for further sampling and detection. For cell transfection, the cells were pre-cultured in 24-well plates (1×105/well) for 24 h and then treated with 6 μg/mL Polybrene (Sigma-Aldrich, Shanghai, China) and lentivirus suspension, and cultured for 24 h. Then the medium was replaced by fresh medium, and BD FACSCanto (BD Biosciences, San Jose, CA) was used to detect the transfection efficiency after 48 h.

Myocardial function assessment

At 14 d after the operation, the rats were anesthetized, and left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were examined by ultrasonic cardiogram (10S probe, image depth of 2–4 cm, frequency of 11.4 MHz). Then 3 rats from each group were sacrificed for sampling. The remaining heart samples were cut and fixed in 4% paraformaldehyde for 8 h, embedded in paraffin, cut into 6-μm slices, and stained with the Masson staining method. The slides were then deparaffinized, stained in iron hematoxylin for 7 min, immersed in 1% hydrochloric acid for 30 s, stained in ponceau for 5 min, immersed in phosphomolybdic acid buffer and aniline blue for 5 min each, and immersed in phosphomolybdic acid buffer for 1 min. After treatment in 1% acetic acid for 1 min and dehydration, the slides were integrated with neutral balsam and observed with an optical microscope (Leica Microsystems, Wetzlar, Germany). Three visual fields were randomly chosen for each sample, and the infarction area was calculated with Qwin software (Leica Microsystems).

Luciferase reporter assay

The regulatory relationship between Gata4 and miR-208 was predicted by use of the online database TargetScanHuman 7.0. The binding site in the Gata4 3′ untranslated region (UTR) was mutated by use of the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA). Then the mutant-type or wild-type of Gata4 3′UTR was ligated into pGL3-basic vector (Promega, Madison, WI) and transfected to cultured myocardial fibroblasts (overexpressed miR-208b or not) in 12-well plates (3×104/well). phRL-TK vector (Promega) was co-transfected as the internal control. Luciferase activity was measured by GloMax (Promega).

siRNA transfection

The specific knockdown of Gata4 was achieved by transfecting its siRNA or siRNA control designed by RiboBio (Guangzhou, China) to the cultured myocardial fibroblasts using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The cells were pre-cultured in 24-well plates (1×105/well) for 24 h before transfection, and then in the serum-free medium during transfection. Transfected cells were collected for further analysis at 36 h after transfection.

Real-time quantitative PCR (qPCR)

At 1, 2, and 4 weeks after model construction, myocardial infarction (I) zone and infarction border (B) zone of rat heart samples were collected. The siRNA-transfected cells were also collected. TRIzol (Invitrogen) was used for total RNA extraction, and RNAiso for Small RNA (TaKaRa, Dalian, China) was used for miRNA extraction. PrimeScript 1st Strand cDNA Synthesis (TaKaRa) was used in reverse transcription, and the specific reverse primer for miR-208b-3p was 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG ACC TTT TG-3′. qPCR was conducted on a LightCycler 480 (Roche, Basel, Switzerland) with specific primers for miR-208b-3p (Fw: 5′-ACA CTC CAG CTG GGA TAA GAC GAA CA-3′ and Rv: 5′-TGG TGT CGT GGA GTC G-3′), Col1a1 (Fw: 5′-CAA GAT GGT GGC CGT TAC TAC-3′ and Rv: 5′-GCT GCG GAT GTT CTC AAT CT-3′), Acta2 (Fw: 5′-AGG GAG TGA TGG TTG GAA TG-3′ and Rv: 5′-GGT GAT GAT GCC GTG TTC TA-3′) and Gata4 (Fw: 5′-GGA GCT GGC CAG GAC TGT CG-3′ and 5′-TGC GCA GGC CTT CGG ATC AC-3′). U6 and Gapdh were used as the endogenous controls. Data were analyzed with the 2−ΔΔCt method.

Western blot

Tissue samples of I zone at 2 weeks after model induction and transfected cells were collected for protein extraction. The protein samples were lysed with RIPA Lysis Buffer (Beyotime, Shanghai, China) and quantified using the BCA Protein Assay kit (Beyotime). Protein samples of 20 μg were loaded in each lane and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein bands on the gel were then transferred to a polyvinylidene fluoride membrane, which was then blocked in 5% skim milk for 2 h at room temperature. The blot was incubated in specific primary antibodies (anti-COL1, anti-αSMA, and anti-GATA4, ABcam) overnight at 4°C and then in horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Positive signals were developed by ECL Plus Western Blotting Substrate (Thermo Scientific) and analyzed with ImageJ software version 1.49 (National Institutes of Health, Bethesda, MD) using GAPDH as the endogenous control.

Statistical analysis

All the experiments were performed at least 3 times and data are shown as the mean ± standard deviation. The t test was performed using SPSS 20 (IBM, New York, USA) and differences were considered significant at P<0.05.

Results

miR-208b is down-regulated in hearts of myocardial infarction rats

The changes in miR-208b-3p were detected at 1, 2, and 4 weeks postoperatively (Figure 1). Compared to the sham-operated group, miR-208b-3p expression in the operated group was significantly down-regulated (P<0.001 or P<0.01) in both I zone and B zone. The reduced expression was the most significant at 2 weeks postoperatively and had slightly increased when detected at 4 weeks after the operation. The reduction was more severe in I zone than in B zone, indicating that miR-208b was reduced in myocardial infarction tissues, which suggests its potential regulatory role in myocardial infarction.

Figure 1

miR-208b-3p is down-regulated in myocardial infarction tissues of the rat model. Detection of miR-208b-3p expression by qPCR was performed in heart samples of the sham-operated group and I zone and B zone of the operated group at 1, 2, and 4 weeks after the operation. Significant expression reduction was observed in I zone and B zone compared to the sham-operated group. ** P<0.01. *** P<0.001. U6 was used as the endogenous control. Sham, the sham-operated group. I zone, myocardial infarction zone. B zone, infarction border zone.

miR-208b overexpression mitigates myocardial infarction damages

To analyze the role of miR-208b in myocardial infarction, miR-208b was overexpressed by transfecting its precursor, pre-miR-208b, and was verified at 2 weeks after the operation and transfection (Figure 2A). qPCR results indicated miR-208-3p levels were significantly increased compared to the transfection control group (P<0.001), suggesting the successful overexpression of miR-208b and the suitability of the transfected samples for further tests.

Figure 2

Overexpression of miR-208b in rat hearts mitigates myocardial infarction damage. Detection was conducted at 2 weeks after the operation. miR-208b was overexpressed by transfecting the lentiviral vector overexpressing pre-miR-208b, and blank lentiviral vector was used in the control group. * P<0.05; ** P<0.01; *** P<0.001. Sham, the sham-operated group. Lv-control, the transfection control group with blank lentivirus. Lv-pre-miR-208b, the transfection group with pre-miR-208b lentivirus. (A) Increase in miR-208b-3p levels indicates the successful overexpression of miR-208b. (B) Percentage of infarction area was reduced by miR-208b overexpression. (C) Myocardial infarction operation reduced LVEF, which was alleviated by overexpression of miR-208b. LVEF, left ventricular ejection fraction. (D) Myocardial infarction operation reduced LVFS, which was alleviated by overexpression of miR-208b. LVFS, left ventricular fractional shortening.

The status of heart samples was measured in 3 aspects: infarction area, LVEF, and LVFS. Infarction area was assessed using the Masson staining method and results showed that the infarction area of the transfection control group was approximately 50%, but after the overexpression of miR-208b-3p the infarction area was significantly reduced to approximately 30% (P<0.05, Figure 2B). LVEF and LVFE are used to reflect the conditions of left ventricular and the prognosis of myocardial infarction. LVEF in the sham-operated groups was approximately 60%, which was significantly reduced to approximately 20% after the operation (P<0.001, Figure 2C), and increased to over 30% with the overexpression of miR-208b (P<0.01). Similarly, LVFS was significantly reduced from approximately 30% in the sham-operated group to approximately 15% percent in the operated group (P<0.01, Figure 2D), and was obviously recovered by miR-208b (P<0.01). These results indicate that overexpression of miR-208b can alleviate the effects of myocardial infarction, suggesting its vital role in modulating myocardial infarction progression.

miR-208b inhibits Col1a1 and Acta2

Because miR-208b improved the heart functions and reduced infarction area after the induction of myocardial infarction, it was assumed to affect the synthesis of fibrosis-related factors, such as COL1 and ACTA2, which were detected in this study. Expression of Col1a1, a gene encoding the pro-alpha1 chains of COL1, and Acta2 in I zone, were detected by qPCR (Figure 3A), and COL1 and ACTA2 were detected by Western blot (Figure 3B). Their mRNA levels were significantly up-regulated after myocardial infarction induction (P<0.001), and both were inhibited when miR-208b was overexpressed (P<0.01). The protein expression level of these 2 factors showed consistently changing patterns, indicating that miR-208b might inhibit the expression of Col1a1 and Acta2, and further suppress the synthesis of protein COL1 and ACTA2, thus mitigating post-infarction myocardial fibrosis.

Figure 3

Overexpression of miR-208b inhibits the synthesis of myocardial fibrosis-related factors COL1 and ACTA2. Sham, the sham-operated group. Lv-control, the transfection control group with blank lentivirus. Lv-pre-miR-208b, the transfection group with pre-miR-208b lentivirus. Col1a1, gene encoding the pro-alpha1 chains of COL1. COL1, type 1 collagen. ACTA2, actin, alpha 2, smooth muscle, aorta, alias αSMA. (A) qPCR showing that Col1a1 and Acta2 are up-regulated in myocardial infarction and inhibited by miR-208b. ** P<0.01; *** P<0.001. (B) Western blot showing that expression of COL1 and ACTA2 were up-regulated in myocardial infarction and inhibited by miR-208b. GAPDH was the endogenous control.

miR-208b functions via targeting Gata4

The functional mechanism of miR-208b in myocardial fibroblasts was further analyzed from the molecular level. Gata4 was predicted to be a target of miR-208b-3p by use of the online database TargetScan, which was verified in cultured myocardial fibroblasts by dual-luciferase reporter assay with the wild-type (wt) and the mutant-type (mut) of Gata4 3′UTR (Figure 4A). miR-208-3p was supposed to bind to position 1363 to 1369 of wt-Gata4 3′UTR, but not mut-Gata4 3′UTR. As predicted, the luciferase activity of wt-Gata4 3′UTR vector could be inhibited by miR-208b overexpression (P<0.01, Figure 4B). However, when the target sequence was mutated (mut-Gata4 3′UTR), overexpression of miR-208b had little effect on the luciferase activity (no significant difference was found). These results show that Gata4 is a direct target of miR-208b, with the sequence GUCUUA in its 3′UTR being the interaction sites. Overexpression of miR-208b led to the down-regulation of GATA4 protein expression (P<0.01, Figure 4C), suggesting that miR-208 is capable of directly binding to Gata4 to suppress its expression.

Figure 4

Gata4 was a direct target of miR-208b and inhibited COL1 and ACTA2. Lv-control, the transfection control group with blank lentivirus. Lv-pre-miR-208b, the transfection group with pre-miR-208b lentivirus. si-GATA4, small interference RNA to inhibit Gata4. si-control, the control group of GATA4 inhibition. UTR, untranslated region. wt-Gata4 3′UTR, wild-type Gata4 3′UTR. mut-Gata4 3′UTR, mutant Gata4 3′UTR. (A) miR-208b-3p is predicted to bind to the GUCUUA sequence in wt-Gata4 3′UTR, and mutations in this sequence are supposed to cause failure in miR-208b and Gata4 interaction. The 4 mutated sites are underlined. (B) Luciferase activity detection indicated only wild-type-Gata4 3′UTR can be bound by miR-208b. No significant difference was found between the 2 groups using mut-Gata4 3′ UTR. GAPDH was the endogenous control. (C) si-GATA4 led to the down-regulation of Gata4 mRNA levels. (D) GATA4 and these factors was analyzed to verify whether miR-208b acted via GATA4, before which Gata4 was inhibited by the small-interference RNA (si-GATA4), and the effects were examined. Data showed that si-GATA4 could effectively down-regulate Gata4 mRNA levels compared to the si-control group (P<0.001). (E) Western blot showing COL1 and ACTA2 were inhibited when GATA4 was suppressed. GAPDH was the endogenous control. COL1, type 1 collagen. ACTA2, actin, alpha2, smooth muscle, aorta, alias αSMA.

Based on these demonstrated roles of miR-208b in inhibiting factors related to myocardial fibrosis, the association between GATA4 and these factors was analyzed to verify whether miR-208b acted via GATA4, before which Gata4 was inhibited by the small-interference RNA (si-GATA4), and the effects were examined (Figure 4D). Data showed that si-GATA4 could effectively down-regulate Gata4 mRNA levels compared to the si-control group (P<0.001). Then the protein levels of COL1 and ACTA2 were detected within these cell samples (Figure 4E), both of them being inhibited when GATA4 was suppressed. Taken together, these findings show that GATA4 was directly inhibited by miR-208b, and was able to suppress the expression of the fibrosis-related factors COL1 and ACTA2, which might be the regulatory mechanism of miR-208b in post-infarction myocardial fibrosis.

Discussion

In this study, miR-208b was found to be down-regulated in hearts of the myocardial infarction rat model and its roles in modulating myocardial fibrosis post infarction were revealed; besides the alleviating effects in myocardial infarction, miR-208b can also inhibit the expression of fibrosis-related factors, including COL1 and ACTA2. Further mechanism analysis revealed that miR-208b directly targets Gata4, through which it suppresses the progression of post-infarction myocardial fibrosis.

Two fibrosis-related factors, COL1 and ACTA2, were used as the indicators of myocardial fibrosis in this study. COL1 consists of a triple helix composed of two alpha 1 chains (encoded by Col1a1) and one alpha 2 chain (encoded by Col1a2). It is generally used as a marker of fibrosis in various diseases, including myocardial fibrosis [17,18]. Low levels of COL1 are found in normal rat hearts, but after induction of myocardial infarction, COL1 is increased [19]. In the present study, Col1a1 was detected from the mRNA level and COL1 from the protein level using an antibody recognizing the helical COL1, and results showed that both the gene Col1a1 and protein COL1 were up-regulated in I zone after induction of myocardial infarction, indicating aggravated myocardial fibrosis. Overexpression of miR-208b inhibited Col1a1 transcription and COL1 synthesis. Similarly, ACTA2, which is highly correlated with fibrosis, as proven in different diseases [20], was also promoted at both mRNA and protein levels after myocardial infarction, and was repressed by miR-208b overexpression. Taken together, these results show that the fibrosis-related factors COL1 and ACTA2 were both up-regulated by the induction of myocardial infarction, suggesting anabatic myocardial fibrosis, which was nevertheless mitigated by miR-208b overexpression.

The regulation of COL1 and ACTA2 by miR-208b was shown to be achieved via GATA4. GATA4 is an important transcription regulator, playing extensive roles in regulating genes involved in heart developmental processes and diseases [21,22], and serving as an indicator of myocyte hypertrophy and fibrosis [23,24]. Human GATA4 binds to the DNase I hypersensitive sites of COL1A2 gene and down-regulates COL1A2 expression [25]. Consistently, results of this study showed GATA4 was inhibited by miR-208b overexpression, which was also correlated with down-regulated COL1 and ACTA2 in si-GATA4 myocardial fibroblasts. Together with the protective roles of miR-208b overexpression in myocardial fibrosis, these results suggest the promoting effects of GATA4 in post-infarction myocardial fibrosis. However, GATA4 may play distinct roles as a fibrosis suppressor in other cell types or tissues, such as the hepatic mesenchymal cells of fetal mice, in which Gata4 knockout leads to advanced liver fibrosis [26]. A possible explanation for the distinct functions of GATA4 in different cells might be the disparity in regulatory pathways involved. Influences of GATA4 on myocardial fibrosis in the present study were exerted via COL1 and ACTA2, but its roles in other cells might be achieved via some other factors.

miR-208b was detected to be down-regulated in I zone and B zone of the heart after myocardial infarction in this study, while its up-regulation in plasm after myocardial infarction has been demonstrated in previous studies. For example, the increasing level of miR-208b can serve as a biomarker of myocardial injury [14] and left ventricular remodeling after acute myocardial infarction [16,27]. The decreased level in myocardial fibroblasts and the increased level in plasm might be caused by the stimulated exportation and release of miR-208b from cells. The existence of microRNAs in plasm has been found in various animal species besides humans, and is derived from circulating blood cells and other cells affected by diseases [28,29]. Cells selectively release some premature and mature microRNAs to the blood [30] via exosomes, microvesicles, or protein complexes [31]. From this point of view, the down-regulated miR-208b level found in this study did not contradict its up-regulated level in the plasm; therefore, it could be conjectured that the release of miR-208b to the plasm might be promoted during myocardial infarction. However, it was uncertain whether the altered intracellular miR-208b level during myocardial infarction resulted from its transcription changes or the cellular release activities, and this needs to be investigated by further comprehensive studies.

The protective roles of miR-208b are undeniable. In addition to its functions in cardiovascular diseases, as shown in previous studies [32], it was further demonstrated by the present study to have anti-fibrosis functions during myocardial infarction that reduce damage to the heart. Now that numerous microRNAs have been identified to be the possible therapeutic targets of diseases [33,34], the potential use of miR-208b is an alternative in alleviating post-infarction myocardial fibrosis, provided that a comprehensive understanding of the regulatory network is achieved.

Conclusions

In summary, miR-208b was demonstrated to alleviate heart damage in a myocardial infarction rat model and to inhibit post-infarction myocardial fibrosis, which is possibly achieved via regulating an important transcription factor, GATA4. These findings reveal the potential use of miR-208b as a therapeutic target for treating post-infarction myocardial fibrosis.