Hyperbaric oxygen‐induced long non‐coding RNA MALAT1 exosomes suppress MicroRNA‐92a expression in a rat model of acute myocardial infarction

Abstract Hyperbaric oxygen (HBO) improves angiogenesis. The effect of HBO on metastasis‐associated lung adenocarcinoma transcript 1 (MALAT1), a pro‐angiogenic long non‐coding RNA, in cardiac myocyte‐derived exosomes and acute myocardial infarction (AMI) is unknown. We aimed to investigate whether MALAT1 is altered in cardiac myocyte‐derived exosomes in response to HBO as well as the molecular regulatory mechanisms of MALAT1 in cardiac myocytes treated with HBO. Cardiac myocytes were cultured, and HBO was applied at 2.5 atmosphere absolute in a hyperbaric chamber. Exosomes were extracted from the culture media. A rat model of AMI generated by the ligation of the left anterior descending artery was used. HBO significantly increased MALAT1 expression in cardiac myocytes and HBO‐induced MALAT1 and exosomes attenuated miR‐92a expression after myocardial infarction. Expression of krüppel‐like factor 2 (KLF2) and CD31 was significantly decreased after infarction and HBO‐induced exosomes significantly reversed the expression. Silencing of MALAT1 using MALAT1‐locked nucleic acid GapmeR significantly attenuated KLF2 and CD31 protein expression after infarction induced by HBO‐induced exosomes. HBO‐induced exosomes also decreased infarct size significantly. HBO‐induced exosomes from cardiac myocytes up‐regulate MALAT1 to suppress miR‐92a expression and counteract the inhibitory effect of miR‐92a on KLF2 and CD31 expression in left ventricular myocardium after myocardial infarction to enhance neovascularization.


| INTRODUC TI ON
Acute myocardial infarction (AMI) and the ensuing heart failure are major causes of morbidity and mortality worldwide. Hyperbaric oxygen (HBO) therapy significantly increases the oxygen content in hypoperfused tissues, and elevation of oxygen content in the hypoxic tissues promotes the ischaemic repair process. 2,3 One of the mechanisms through which HBO therapy improves wound healing is through the enhancement of neovascularization in the ischaemic tissue. 4 HBO was shown to induce cardioprotection through anti-apoptotic effect and decreased infarct size in a rat model of MI. 5,6 Several small clinical trials also demonstrated the beneficial effect of HBO in patients with acute coronary syndrome by reducing the infarct size, and the risk of major cardiovascular events and death. 7 Exosomes are 40-90 nm-sized extracellular vesicles of endosomal origin that play a crucial role in intercellular processes. 8,9 Exosomes are an emerging and promising class of diagnostic markers for cell-free therapeutic application in cardiovascular diseases. 9, 10 Giricz et al 11 13 MALAT1 plays a role in the promotion of angiogenesis by mesenchymal stem cells in thyroid tumours. 14,15 The effect of HBO on MALAT1 expression in MI remains unknown. It is also not known whether HBO-induced exosomes have a therapeutic potential post-MI. In this study, we used HBO-induced MALAT1 exosomes from cardiac myocytes to treat MI in an animal study.

| Rat model of acute myocardial infarction (AMI)
A rat model of AMI with left anterior descending (LAD) coronary artery ligation was used as previously described. 16 Wistar male rats weighing 280-330 g were used. Following the induction of anaesthesia with 2% isoflurane and confirmation of a fully anaesthetized state (no response to toe pinching), tracheotomy was performed, and the animal was ventilated on a Harvard rodent respirator. The heart was then rapidly exteriorized, and a 6-0 silk suture was tightened around the proximal LAD coronary artery (before the first branch of diagonal artery). Sham-operated control animals were prepared in a similar manner without LAD ligation. Following the surgical procedure, the wound and tracheotomy were closed to restore spontaneous respiration. For the AMI study, rats were randomly divided into nine groups (a) sham-operated, (b) AMI alone, (c) AMI and treatment with HBO, (d) AMI and treatment with HBO-induced exosomes, (e) AMI and treatment with MALAT1-locked nucleic acid (LNA) GapmeR (Cat. no. 339511 LG00157651-DDA, Qiagen), (f) AMI and treatment with MALAT1-scrambled LNA GapmeR (Cat. no. 339511 LG00199141-DDA, Qiagen), (g) AMI and treatment with antagomir-92a, (h) treatment with miR-92a in sham control and (i) treatment with mutant-miR-92a in sham control. After 60 minutes, the viable myocardium bordering the LV infarct zone was injected at three different sites with a total of 70 μg of MALAT1-containing exosomes (150 μL).
During the HBO sessions, the rats were kept in separate restrainers in the hyperbaric chamber. Oxygen flow (100%) through the chamber was set at 2.5 L/min to maintain the room temperature (RT). Rats in the AMI + HBO group received HBO treatment once a day for 60 minutes at 2.5 atmosphere absolute (ATA) for various time periods. The oxygen tension mentioned above were chosen based on the current human treatment protocols. 17 Rats in the sham group were treated with normobaric air at 1.0 ATA. At the end of experiment, the rats were killed by decapitation under anaesthesia with an overdose of isoflurane, and the heart was quickly removed and stored in liquid nitrogen. Left ventricular tissue was obtained for Western blot analysis and immunofluorescent staining. The infarct size was measured using the triphenyl-tetrazolium chloride method. Computerized morphometry (NIS Elements, Nikon) was used to calculate the infarct size (as the ratio of infarct size and total left ventricular area). All animal procedures were performed in accordance with institutional guidelines and conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health.

| Haemodynamic monitoring of rats
Haemodynamic monitoring of the rats was performed with polyethylene catheters for measurement through a Grass model tachograph pre-amplifier as previously described. 16

| Assessment of cardiac function
Cardiac function of the rats was evaluated non-invasively using echocardiography performed with an Acuson Sequoia 512 machine using a 15-MHz probe on the day of killing, and on days 7 and 14 following the surgery as described previously. 16 The sonographer was blinded to the randomization of the rats.

| Western blot analysis
Western blotting was performed as previously described. 19 The proteins of interest were identified through incubation with specific antibodies, as indicated (1:1000 dilution), for 1 hour at RT. Equal protein loading of the samples was further verified by staining with mouse anti-tubulin monoclonal antibody (Santa Cruz Biotechnology Inc). All Western blot signals were visualized using chemiluminescence and quantified using densitometry.

| Histological and immunofluorescence staining
Tissue samples were harvested and treated overnight in 4% paraformaldehyde and then embedded in paraffin. The tissue were cut into 5-μm-thick sections and incubated with the primary antibody at 4°C for 12 hours. The sections were then washed thrice with PBS, incubated with fluorescence-conjugated secondary antibody in PBS for 1-2 hours at RT in the dark and then stained with DAPI to visualize the nuclei. The sections were mounted with a coverslip and examined using fluorescence microscopy. Images were taken from at least three random fields for each sample.

| Transfection of siRNAs and MALAT1 LNA GapmeR
The LNA GapmeR was transfected into left ventricular myocardium using a low pressure-accelerated gene gun (Bioware Technologies) following the protocol from the manufacturer. In brief, 1 µmol/L of LNA GapmeR was suspended in 5 mL of PBS and placed in the loading hole near the nozzle. Pushing the trigger of the gene gun released the RNAcontaining solution, which was directly propelled by helium at a pressure of 15 psi into left ventricular myocardium of the MI rat. Scrambled siRNA or LNA GapmeR was transfected as negative controls.

| Culture of rat cardiac myocytes
Rat cardiac myocytes were obtained from ScienCell Research Laboratories (Cat. No. R6200; ScienCell). The cardiac myocytes were isolated from postnatal day 2 rat heart, cryopreserved at P0 and characterized by immunofluorescence using antibodies specific to smooth muscle actin, sarcomeric alpha actinin and tropomyosin. The cardiac myocytes were plated in a culture dish and cultured in cardiac myocyte medium (Cat. No. 6201; ScienCell) according to the manufacturer's instructions. After 3 days in culture, the cells were transferred to serum-free Dulbecco's modified Eagle's medium (DMEM, Cat. No. 31600-034; Invitrogen Corporation) and used for the experiments.

| Hypoxic stimulation
A humidified temperature-controlled incubator, Proox model 110 (BioSpherix) was used as the hypoxia chamber. For hypoxic conditions, the concentration of oxygen was reduced to 2.5% by replacement with N2, keeping CO 2 constant at 5%, and cells were incubated at 37°C for different time periods. Control condition was defined as 95% air and 5% CO 2 . To investigate the molecular regulatory mechanisms, cardiac myocytes were pre-treated with inhibitors for 30 minutes and then exposed to hypoxia without changing the medium. The samples were incubated at 4°C overnight and then centrifuged at 10 000 g for 1 hour at 4°C. The supernatant was aspirated and discarded, and the exosome pellet was resuspended in 1× PBS, and then stored at 4°C for short-term (1-7 days) or −20°C for long-term storage. The exosomes were quantitated using ExoQuant™ quantification assay kit (BioVision) according to the manufacturer's instructions.

| Assessment of the quality of exosomal-RNA
The quality of exosomal-RNA was assessed with an Agilent 2100 Bioanalyzer using an RNA Pico Chip (Agilent Technologies) following the manufacturer's protocol. For visualization and better interpretation, electropherograms and virtual gel images were generated. All chip analyses were performed in duplicate. The test plasmid (2 μg) and the control plasmid (pGL4-Renilla luciferase, 0.02 μg) were cotransfected with the gene gun in each well, and then, the medium was replaced with normal culture medium.

| Luciferase activity assay
Following 2 hours of exposure to hypoxic condition at 2.5% oxygen, cell extracts were prepared using the Nano-Glo dual-luciferase reporter assay system (Promega) and measured for luciferase activity by using a luminometer (Glomax Multi Detection System, Promega).

| Statistical analysis
The data are expressed as the mean ± standard deviation (SD).
Statistical significance was analysed using analysis of variance (ANOVA; GraphPad Software Inc). The Tukey-Kramer comparison test was used to conduct pairwise comparisons between multiple groups after ANOVA P < .05 was considered statistically significant.   Figure 4). These results indicate that MALAT1 and miR-92a mediate myocardial KLF2 and CD31 expression following AMI. Further, we confirmed that MALAT1 from the HBO-stimulated cardiac myocytes was incorporated into the cardiac myocytes in rat left ventricular myocardium using in situ hybridization ( Figure S2).

| HBO-induced exosomes decrease myocardial infarct size
The infarct size 14 days post-MI increased significantly compared with the sham group ( Figure 5). Treatment with HBO-induced  Table 1).
Transfection of MALAT1 LNA GapmeR did not reduce left ventricular end-systolic and end-diastolic dimension or improve fraction shortening.

| MiR-92a decreases KLF2 luciferase activity in cardiac myocytes under hypoxic conditions
To examine whether miR-92a-mediated reduction in KLF2 expression was regulated at the transcriptional level, luciferase activity of KLF2 was measured. The KLF2 3'UTR was found to contain a conserved miR-92a-3p-binding site (nucleotide 205-225; Figure 6A) using Clustal W method with the MegAlign software (DANSTAR, Madison, WI, USA). As shown in Figure 6B, miR-92a overexpression significantly decreased KLF2 luciferase activity in cardiac myocytes under hypoxic treatment for 2 hours when miR-92a bound to normal 3'UTR of KLF2 ( Figure 6). The overexpression of miR-92a in mutant KLF2 3'UTR did not affect the luciferase activity, indicating that KLF2 was the target gene of miR-92a.

| Effect of hypoxia on MALAT1, miR-92a and KLF2 expression in cardiac myocytes
To mimic hypoxia induced by the ligation of the LAD artery, cardiac myocyte cultures were subjected to in vitro hypoxia with 2.5% oxygen. Hypoxia (with 2.5% oxygen) and HBO (at 2.5 ATA) for 2 hours significantly increased MALAT1 expression in the cardiac myocytes ( Figure S3). HBO increased MALAT1 expression more than hypoxia.
The hypoxia time course experiment revealed that MALAT1 expression in the cardiac myocytes gradually increased under hypoxia between 1 and 2 hours, then gradually returned to the baseline level ( Figure S4A). In contrast to MALAT1 expression, miR-92a expression initially decreased between 1 and 2 hours, and then gradually increased from 4 to 8 hours ( Figure S4B). Hypoxia significantly increased KLF2 mRNA expression in the cardiac myocytes at 2 and 4 hours of hypoxia ( Figure S4C). Treatment of cardiac myocytes, under hypoxia for 8 hours, with HBO-induced exosomes significantly increased KLF2 mRNA expression while overexpression of miR-92a in hypoxic cardiac myocytes significantly decreased KLF2 mRNA expression compared with the control group ( Figure S5).

| D ISCUSS I ON
In our rat model of AMI, we found that the expression of MALAT1 factor receptor 2 to facilitate angiogenesis in human coronary artery endothelial cells. 22 The inhibition of miR-92a by antagomir-92a has been reported as a therapeutic strategy for promoting skin repair in healing-impaired diabetic mice. 25 Exosomes are secreted by cardiac and vascular cells in culture. 26 Exosomes were found to mediate communication between endothelial cells and smooth muscle cells, and fibroblasts and cardiac myocytes. 27,28 Boosting MALAT1-containing exosomes may be used to augment angiogenesis post-MI. Exosomes possess desirable properties such as stability, biocompatibility, biological barrier permeability, low toxicity and low immunogenicity, which make them an attractive vehicle for therapeutic delivery. MiR15a has been shown to inhibit angiogenesis. 29,30 We searched and found that rat MALAT1 has a matching site for miR-15a-5p and it is located from 4160 to 4178 bp of rat MALAT1 gene loci. Our study found that HBOinduced exosomes could significantly attenuate the miR15a expression induced by hypoxia in cardiac myocytes ( Figure S6).
MALAT1 LNA GapmeR significantly reversed miR15a expression that was attenuated by HBO-induced exosomes. Scrambled LNA GapmeR of MALAT1 did not reverse miR15a expression.
The role of MALAT1 in AMI is controversial. One study reported that the expression of MALAT1 increased in myocardial tissue post-MI in mice and the increased MALAT1 protected cardiac myocytes from apoptosis. 31 However, four other studies reported that the expression of MALAT1 increased post-MI, but the increased MALAT1 promoted apoptosis of cardiac myocytes in mice. [32][33][34][35] MALAT1 promoted cardiac myocytes apoptosis through different miR targets. [32][33][34][35] In human studies, the blood level of MALAT1 was higher in patients with AMI than in healthy controls. 36,37 The reason of the discrepancy among different studies is not known. Different animal studies may partially explain the discrepancy. As most lncRNAs are not conserved among species, 38 it is not surprising that MALAT1 expression decreases in myocardial tissue post-MI in rats. The biological function of MALAT1 in enhancing neovascularization is the same among species. Silencing of MALAT1 reduced capillary growth in a mouse model of hindlimb ischaemia 13 and rat model of diabetic retinopathy. 39 The biological function of MALAT1 in enhancing angiogenesis is consistent with our findings.
KLF2 has been reported to be a target gene of miR-92a 38 and plays a critical role in neovascularization. 40,41 In this study, we observed that miR-92a has a binding site in the 3'UTR of KLF2 and that it suppresses KLF2 luciferase activity by binding to the 3'UTR.

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N
Kou-Gi Shyu: Funding acquisition (lead); Writing-original draft (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.