Cardioprotective MicroRNA (ProtectomiRs) identified in a porcine model of acute myocardial infarction and ischemic conditioning: in vitro validation of novel candidates

Abstract Acute myocardial infarction (AMI) is one of the leading causes of death worldwide, however an effective pharmacotherapy for ischemia-reperfusion (I/R) injury remains an unmet need. MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a key role in post-transcriptional gene regulation and represent promising therapeutic targets for cardioprotection in ischemic heart disease. In a previous study, we revealed a global miRNA expression network in a porcine model of AMI that underwent ischemic preconditioning (IPreC), postconditioning (IPostC), and remote perconditioning (RIPerC). We identified 220 differentially expressed miRNAs, of which 4 miRNAs showed upregulation and 10 showed downregulation across all conditioning protocols. We validated miRNAs which had homologous sequences to 12 rat miRNAs and tested them at 25, 50, and 100 nM concentrations in neonatal rat cardiomyocytes. Two miRNAs showed significant cardioprotective effects in this concentration range. We planned to test the remaining candidates over a broader concentration range. From the previously validated and tested miRNAs, we identified 6 miRNAs (PHmiR-SA201X, PHmiR-SA202X, PHmiR-SA203X, PHmiR-SA204X, PHmiR-SA205X, PHmiR-SM102X) whose cardioprotective effects had not been previously reported in the literature, suggesting their potential as novel candidates for cardioprotection. In the first set of experiment, we tested these protecomiR candidates in neonatal rat cardiomyocytes (NRCM) at 6.25, 12.5, and 25 nM concentrations using DharmaFECT1 transfection reagent (n=6). Cell viability was assessed after 6 hours of simulated ischemia and 2 hours of reperfusion (sI/R). For systematic analysis, in the second set of experiment, these miRNAs were tested at even lower concentrations, 0.75, 1.5, 3.125 nM using the same protocol (n=5). In the third set of experiments, 4 (PHmiR-SA201X, PHmiR-SA203X, PHmiR-SA204X, PHmiR-SA205X) out of the 6 identified miRNAs were considered novel candidates based on the literature and were subsequently tested in human cardiomyocytes (AC16) at 1.5, 3.125, 6.25, 12.5, 25, and 50 nM concentrations (n=4). Cell survival was evaluated after 16 hours of simulated ischemia and 2 hours of reperfusion. Treatment with PHmiR-SA203X antagomiR significantly improved cell viability at 6.25, 12.5, and 25 nM concentrations in neonatal rat cardiomyocytes compared to the negative control. However, no significant differences were observed among the candidates tested in AC16 cells.