Upregulated miR-486 Induces Therapeutic Angiogenesis and Improves Infarction Recovery in Rat Myocardial Ischemia Model


 BackgroundBesides hematopoietic cells, miR-486 is also enriched in cardiac, skeletal, and smooth muscles. However, its roles in regulating the function of cardiomyocytes and tissue repair in myocardial infarction have not been explored yet.MethodsWe investigated the effects of miR-486 on the survival and hypoxic response of cardiomyocytes. Also, using adenovirus-mediated overexpression, we evaluated its therapeutic effects in myocardial repair in a rat acute myocardial infarct (AMI) model.ResultsHypoxia treatment upregulated miR-486 in cardiomyocytes. Moreover, adenovirus-mediated overexpression of miR-486 reduced cell injury, increased cell viability, and decreased apoptosis in hypoxic conditions. In a rat AMI model, administration of Ad-miR-486 reduced infarct size and collagen deposition, increased vessel density, and improved cardiac function. Furthermore, in vivo data suggest that the protective effects of miR-486 in cardiomyocytes were related to its anti-apoptotic function.Conclusion﻿miR-486 overexpression protects myocytes from hypoxia-induced apoptosis and has therapeutic potential in myocardial infarction.


Introduction
Acute myocardial infarction (AMI) is one of the critical cardiovascular diseases. Although many novel regenerative approaches such as stem cell transplantation and angiogenic gene therapy have received signi cant attention [1][2][3] , these have limited clinical effectiveness due to lower engraftment e cacy and impaired angiogenic ability [4,5] . Thus, it is essential to extensively elucidate the pathogenesis of AMI to develop novel approaches for the repair of infarct myocardium.
MicroRNAs (miRNAs), a kind of small non-coding regulatory RNAs, are associated with various diseases.
Accumulating evidence suggests that miRNAs might play critical roles in ischemia heart disease [6,7] regulating fundamental components of the disease pathogenesis and progression [8,9] . A multitude of miRNAs have been linked to vascular development, homeostasis, function, disease, and regeneration [10] . In addition to hematopoietic cells, miR-486, a multi-functional miRNA, is enriched in cardiac, skeletal, and smooth muscles. It was rst found in the human fetal liver cDNA library and has been located in the last intron of the Ankyrin-1 (Ank1) gene [11] . Several reports showed that miR-486 is an erythroid related miRNA that plays important regulatory roles in hematopoiesis and leukemogenesis [12][13][14] . Notably, miR-486 is also considered as a tumor suppressor and often down-regulated in non-small cell lung cancer (NSCLC) and hepatoma [15] . In several tumors including lung cancer, the expression of miR-486 is regulated by the methylation of its host gene Ank1 [16] .
Interestingly, a recent study showed that miR-486 is a hypoxia response miRNA and regulates the angiogenic activity of bone marrow-derived mesenchymal stem cells [17] . In myocytes, miR-486 is transcriptionally controlled by SRF and MRTF-A, as well as by MyoD, and regulates the phosphatase and tensin homolog (PTEN) and FOXO1a (FOXO1, Forkhead Box O1) to negatively affect the phosphoinositide-3-kinase (PI3K)/AKT signaling [18] . A rat study showed that miR-486 protects cardiomyocytes from apoptosis through targeting PTEN and activating the PI3K/AKT pathway [19] .
However, its role in regulating cardiomyocyte function and AMI pathogenesis has not been explored yet.
Given the importance of miR-486 in angiogenesis and hypoxia, we hypothesize that it might also modulate the functions of myocytes and affect AMI pathogenesis. In this study, we investigated the effects of miR-486 overexpression in cardiomyocytes to reveal its potential therapeutic effects in AMI.

Experimental animals
The Sprague-Dawley (SD) rats were obtained from the Animal Center of Academy of Military Medical Sciences, Beijing, China. A total of 120 healthy adult SD rats (age, 6-8 weeks; 200±20 g; both males and females) and 15 healthy neonatal rats (aged 24-48 h; 6.3±1.4 g; both males and females) were maintained under controlled conditions (20-25°C; relative humidity 40-60%; 12 h light/dark cycle with food and water ad libitum). Animals were handled as per the guidelines of the Institutional Animal Care and Use Committee of Capital Medical University. All experiments were performed following the National Institutes of Health guide for the care and use of laboratory animals and the guidelines of the International Association for the Study of Pain. The rats were anesthetized with iso urane (adult SD rats with 1.87%; neonatal rats with 1.6%) and euthanized by cervical dislocation.
Isolation and culture of cardiomyocytes Primary cultures of neonatal rat cardiac ventricular myocytes were performed as described previously [20,21] . Brie y, the hearts from 1-2 day(s) old SD rats were removed after hypothermia anesthesia by immersion in ice water and placed in ice-cold 1× phosphate-buffered saline solution. After repeated rinsing, the atria were cut off, and the ventricles were minced with scissors. The minced tissue and ventricular cells were dispersed by digestion with collagenase type IV (0.45 mg/ml), 0.1% trypsin, and 15 μg/ml DNase I. Cardiomyocytes were cultured in the cardiac myocyte culture medium containing Dulbecco's modi ed Eagle's medium supplemented with 10% serum, 4 μg/ml transferrin, 0.7 ng/ml sodium selenite, 2 g/L bovine serum albumin (fraction V), 3 mmol/L pyruvic acid, 15 mmol/L HEPES, 100 μmol/L ascorbic acid, 100 μg/ml ampicillin, 5 μg/ml linoleic acid, 1% penicillin and 1% streptomycin, and 100 μmol/L 5-bromo-2′deoxyuridine. The cells were seeded into six-well plates. The myocytes were passaged and used for in vitro studies.

Adenoviral vectors and cardiomyocyte gene transfection
The adenovirus vector carrying rat miR-486 gene was generated from Adeno-XTM Expression Systems For miR-486 inhibitor transfection, cardiac myocytes were cultured and transfected with siRNA by nucleofection. The transduced cardiac myocytes were transferred to the completed medium, cultured for 24 h, and then subjected to proliferation and apoptosis assays.

RNA Extraction and TaqMan miRNA assay
Total RNA was extracted from tissues/cells using the Trizol Reagent and cDNA synthesis was performed with the Taqman@ MicroRNA Reverse Transcription Kit (Thermo, USA). miR-486 and U6 (internal control) were ampli ed according to the TaqMan MicroRNA assay protocol (Applied Biosystems, USA). The assays were carried out in triplicates and the relative quantitative evaluation of the target gene levels was performed by comparing △Ct.

The rat AMI model and treatment
A schematic of our experimental approach is shown in Fig. 1. Experimental AMI was induced as described previously. Brie y, to establish the rat AMI model, adult SD rats were anesthetized and surgery was performed to ligate the left anterior descending coronary artery. The rats (number 120, weight 200±20 g) were divided into four groups, namely the sham group, AMI group, adenovirus group (Ad-ctr), and Ad-miR-486 group. In the last two groups, the AMI rats were treated with control virus and Ad-miR-486 respectively. The adenovirus was injected into the border zone surrounding the infarcts using a 27G needle.

Measurement of left ventricles ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)
Four weeks after establishing AMI in rats, echocardiographic examinations were performed after intraperitoneal injection with Chloral hydrate (10%, 3ml/100g) and using the Vevo770 ultrasound system (Visualsonics Inc., Toronto, Canada). The electrode of the Vevo770 ultrasound system was attached to the animal using adhesive plaster and the ECG was monitored. The hairs on the rat chest were removed with depilatory creams. Left ventricular end-diastolic diameters (LVEDD) and left ventricular end-systolic diameters (LVESD) were measured using the ultrasound coupler (Fig. 2). Based on the measurements, LVEF and LVFS were calculated. At least three successive records were used to calculate the average value.
Infarct size, collagen deposition, and vessel density The rats were sacri ced at 4 weeks after AMI. After hemodynamic studies, the animals were sacri ced to remove hearts. These were perfused with PBS for rinsing and frozen quickly. Myocardial infarct size was determined by pathological staining, a gold standard in AMI studies. The heart ventricles were sliced transversely into 2 mm thick slices and incubated in 1% triphenyltetrazolium chloride (TTC) at 37°C for 15 min to identify the noninfarcted and infarcted areas. TTC staining displayed a red color. TTC unstained area (white color) de ned the infarcted area and the size was expressed as a percentage of the ischemic area.
Microvessel density was evaluated based on CD-31 expression and calculated. The microvessel endothelial cell membranes were highlighted by staining using the anti-CD-31 antibodies. Any cluster of endothelial cells that was markedly separated from the adjacent microvessel was considered a vessel.
Then, ve random nonoverlapping 400x high-power elds around the MI area were selected and on average, 5 such records were counted on each slide.

Measurement of collagen volume fraction (CVF) by Masson's trichrome staining
Rats were sacri ced at 4 weeks after AMI. Hearts were excised and soaked immediately in saline to remove the excess blood from the ventricles. These were xed in 4% triformol. Para n-embedded samples were sectioned at 5 μm to perform the Masson's trichrome staining. Digital images of the stained sections were captured and CVF was analyzed using the Image Pro-Plus 6.0 software. CVF is expressed as a percentage of the collagen area in the whole eld and calculated as the average of 5 random AMI elds in each slide.

Measurement of myocardium apoptotic index (AI)
Rats were sacri ced at 4 weeks after AMI. The tissue sections from the heart were stained with TUNEL. Five consecutive nonoverlapping 400x high-power elds were selected and the average counts of total and apoptotic cells were calculated. The AI was determined using the formula: the number of apoptotic cells/total cells × 100%.
Cell proliferation and apoptosis assays The proliferation of cardiac myocytes was determined with CCK-8 assay. Cell apoptosis was determined by AnnexinV-APC staining. Brie y, the cardiac myocytes were cultured in a completed culture medium at 37°C for 48 h. The cells were treated with trypsin, washed with cold PBS, and resuspended in Annexin-V binding buffer. Then, the cells were stained with Annexin V-APC to detect apoptotic cells by ow cytometry.

Statistics
All data are presented as mean ± S.E. p-value < 0.05 was considered statistically signi cant.

Results
Hypoxia upregulated miR-486 in cardiomyocytes Cardiomyocytes were isolated from neonatal SD rats and cultured for expansion in 1% O 2 for different periods. Then, the expression of miR-486 was estimated using qRT-PCR. We observed that in a hypoxic environment, the cardiomyocytes exhibited upregulation of miR-486 at 6 h which reached to maximum at 24 h (Fig. 3A). This suggests that miR-486 is a hypoxia response miRNA in cardiomyocytes.
Upregulation of miR-486 protected cardiomyocytes from hypoxia-induced apoptosis Next, we evaluated the regulatory effect of miR-486 on cardiomyocytes. For this, the cardiomyocytes were transfected with miR-486 inhibitors or mimics and cultured in hypoxia for 6 h to estimate the cell viability, LDH activity, and apoptosis. We observed that miR-486 inhibitor transfected cardiomyocytes exhibited a signi cant increase in hypoxia-induced apoptosis and LDH release while the cell viability was decreased.
Whereas transfection with miR-486 led to decreased apoptosis and LDH release while increasing the cell viability, compared to the control group ( Fig. 3B-D). It indicates that miR-486 expression protected the cardiomyocytes from hypoxia-induced apoptosis.

Adenovirus-mediated expression of miR-486 improved heart function in the rat AMI model
The mortality rate in the Sham group was 6.67%. Unfortunately, the rat mortality rate after AMI increased to 20%. However, the differences were not signi cant enough (P 0.05).
To elucidate the role of miR-486 upregulation in cardiomyocyte functions, in vivo study using the adenovirus-mediated miR-486 overexpression was performed in the rat AMI model. The Ad-miR-486 and Ad-GFP control vector were injected into the myocardium and the expression of miR-486 was determined by qRT-PCR in the tissue lysate obtained from the injected area. Treatment with Ad-miR-486 resulted in high levels of miR-486 in the myocardium. However, after one month of treatment, the adenovirusmediated increase in miR-486 expression gradually returned to the basal level (Fig. 4A).
Then, four weeks post adenovirus administration, the heart functional parameters were determined in both Ad-miR-486 and control vector treated rat AMI models. We found that the treatment with Ad-miR-486 improved the cardiac function signi cantly. Fig. 4B shows the comparison of functional changes by echocardiography. We observed that compared to animals in the sham group, LVEF and LVFS were signi cantly lower in the MI and Ad-ctr group (P 0.05). However, upon Ad-miR-486 treatment, LVEF and LVFS were signi cantly increased in the rat AMI model (Fig. 4C-D). It suggests that Ad-miR-486 therapy can bene t in AMI. Although, we did not observe any signi cant differences in the survival curves of the four groups (P 0.05). (Fig. 5) Ad-miR-486 transduction reduced infarction sizeand brosis in the rat AMI model At 4 weeks after Ad-miR-486 therapy, compared to the control vector group, the heart infarcted size decreased and wall thickness increased in the Ad-miR-486 group. The heart infarcted size in the AMI group, Ad-ctr group, and Ad-miR-486 group were 35.81±1.51%, 35.05±1.75%, and 30.75±1.96% respectively (Fig. 6A-B).
Moreover, four weeks after AMI, the infarcted areas were subjected to histological evaluations using the hematoxylin-eosin staining. The collagen content in the noninfarcted area was stained with collagenspeci c Sirius red (Fig. 6C). We found that the collagen volume fraction was signi cantly lower in the Ad-miR-486 group compared to the Ad-ctr groups. Also, treatment with Ad-miR-486 markedly reduced the interstitial brosis in the non-infarcted myocardium. The pictures were analyzed using the Image-Pro Plus 6.0 image analysis system. The CVF in the AMI group, Ad-ctr group, and Ad-miR-486 group were signi cantly higher than the sham group (P 0.05). The CVF in the Ad-miR-486 group was signi cantly lower than the AMI group and Ad-ctr group (P 0.05). Notably, there was no signi cant difference between the AMI group and Ad-ctr group (P 0.05) ( Fig. 6D).
Ad-miR-486 treatment increased microvessel density (MVD) and the survival of myocardiocytes in the rat AMI model Meanwhile, we evaluated the vascular density in the infarcted areas. We found that compared to the sham group, AMI group, and Ad-ctr group, MVD was signi cantly higher (P<0.05, n = 6/group) in the Ad-miR-486 group (Fig. 7A-B). Moreover, we did not observe any signi cant difference between the AMI and Ad-ctr groups (P 0.05).
To examine the potential cellular mechanism of miR-486-mediated protection against myocardial infarction, we performed in vivo analysis of apoptosis in infarcted heart sections by immuno uorescence using TUNEL staining. The representative TUNEL-stained photomicrographs of heart sections from Ad-miR-486 treated rats are shown in Fig. 8A. Notably, we found that in the infarcted areas, the apoptosis of the cardiac cells signi cantly decreased in the Ad-miR-486-treated group (Fig. 8B). This indicates that the protective mechanism of miR-486 against myocardial infarction could be related to its anti-apoptotic effect in the cardiomyocytes. Discussion miRNA dysregulation has been linked to the initiation and pathogenesis of several diseases including cancer and cardiovascular ischemia [22][23][24] . Particularly, in myocardial infarction, several miRNAs are gaining huge interest as putative novel disease biomarkers and therapeutic targets [25] . miR-486, a hypoxia response miRNA, has multiple functions including angiogenic activities [26] . Notably, it was found downregulated in the muscles of dystrophin-de cient mice and DMD (Duchenne muscular dystrophy) patients [17] . Previous studies strongly suggest that miR-486 regulates the function of cardiomyocytes and plays a vital role in cardiovascular ischemia. In this study, we transduced rat cardiomyocytes with the miR-486 gene to investigate its protective effects in hypoxia-induced apoptosis. Furthermore, it was examined for potential therapeutic bene ts in a rat AMI model.
Our in vitro studies revealed that in hypoxic conditions, the levels of miR-486 were highly upregulated in the cultured cardiomyocytes. Therefore, we next investigated the protective effects of miR-486 on the survival of cardiomyocytes. Interestingly, results from LDH release and apoptosis assay suggest that upregulated miR-486 or transduction with its mimics signi cantly increased the survival of cardiomyocytes in hypoxia. On contrary, transduction with miR-486 inhibitor suppressed the growth and survival of cardiomyocytes. Meanwhile, multiple apoptosis and angiogenesis-related genes, such as PTEN, FOXO1, and Sirt1, identi ed as the targets of miR-486, were examined for their role in cardiomyocytes resistance against hypoxia-induced apoptosis. Notably, PTEN is a known negative regulator of H 2 O 2 -induced apoptosis in rat cardiomyocytes [27] . Interestingly, a recent report revealed that exosomal miR-486-5p carrying mesenchymal stromal cells suppresses cardiomyocyte apoptosis under ischemic and hypoxic conditions via targeting the PTEN/PI3K/AKT signaling pathway. It seems that miR-486 overexpression suppresses the PTEN molecules, leading to activation of the PI3K/AKT pathway, which enhances the cardiomyocyte survival. Importantly, it has also been reported that miR-486-5p targeting PTEN protects against coronary microembolization-induced apoptosis in rat cardiomyocytes by activating the PI3K/AKT pathway. FOXO1 regulates myocardial glucose oxidation via transcriptional control of pyruvate dehydrogenase kinase 4 [28] . Also, its inhibition signi cantly suppresses the myoblast proliferation and induces myoblast apoptosis [29] . Likewise, SIRT1 deactivation plays a key role in ischemia-reperfusion-induced apoptosis [30] . Besides, miR-486-5p is known to repress MRTF-A affecting a complex network of transcriptional and post-transcriptional regulatory mechanisms in myogenesis. Therefore, one can speculate that miR-486 mediated regulation of aforesaid targets could potentially contribute to its protective effects in cardiomyocytes.
We found that Ad-miR-486 administration showed therapeutic bene ts in a rat AMI model. Its overexpression improved the heart functions including a decrease in LVEDP, suggesting improvement in ventricular remodeling. Our results suggest that the potential mechanisms for the therapeutic effect of Ad-miR-486 in infarct myocardium include inhibition of apoptotic pathways and induced angiogenesis. Furthermore, our in vitro transfection studies veri ed that miR-486 expression improved the survival of myocardial myocytes. It was reported that exosomal miRNAs-486-5p mediates wound healing by promoting angiogenesis. Also, it is a familiar fact that therapeutic angiogenesis in stem cell/gene therapy largely depends on multiple angiogenic growth factors. For instance, miR-486 transduced bone marrow stromal cells secret increased levels of soluble angiogenic proteins such as VEGF and HGF, which induce therapeutic angiogenesis and has been shown to improve cardiac function in a rat AMI model [17] . Treatment with Ad-miR-486 also inhibited brosis in infarcted myocardium in AMI models. This phenomenon is in agreement with a recent report that showed anti-brotic effects of miR-486 in the pulmonary brosis model [31] . Notably, SMAD2, a crucial mediator of pulmonary brosis, is targeted by miR-486 [31] . In this study, both in vitro and in vivo investigations revealed that miR-486 overexpression protects cardiomyocytes from apoptosis and thereby improves infarction recovery.

Conclusion
Ad-miR-486 has the therapeutic effects in infarcted myocardium which involved increased angiogenesis, suppressed apoptosis, and reduction in myocardial brosis. Therefore, miR-486 gene therapy could be a potential therapeutic strategy in AMI. Datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval
This study was approved by the Institutional Laboratory Animal Care and Use Committee of Capital Medical University (Beijing, China).