Ischaemic preconditioning blunted myocardial DNA methylation and regulated myocardial transcriptome in ischaemic reperfusion settings

Background. Ischemic preconditioning (IPC) reduces infarct size but may increase the extent of reversible cardiac dysfunction (myocardial stunning). DNA methylation is thought to regulate IPC-induced gene expression, impacting inflammation, oxidative stress, apoptosis, and DNA repair pathways. Aims. This study aimed to identify transcriptomic changes associated with IPC and elucidate the role of DNA methylation in regulating these changes, thereby distinguishing the mechanisms underlying IPC-mediated protection from necrosis and myocardial stunning. Methods. A rat model of cardiac ischemia-reperfusion (I/R) injury was used, with IPC induced by two 5-minute ischemia-reperfusion cycles followed by 13.5 minutes of ischemia, and a control group undergoing 13.5 minutes of ischemia without IPC. Myocardial samples were collected at early (T1) and 4-hour (T2) post reperfusion, representing stunned myocardium in the IPC group and necrosis in the control group. RNA sequencing, DNMT activity assay and promoter methylation analyses of differentially expressed genes (DEGs) were performed to assess transcriptomic and epigenetic changes. Results. IPC reprogrammed the cardiac transcriptome, with 53 DEGs at T1 and 166 at T2, including key regulators of inflammation (Nfkbia), DNA repair (Gadd45b), and stress responses (Cebpd, Jun). Long non-coding RNAs (lncRNAs) were also enriched. IPC reduced global DNMT activity, promoting hypomethylation of protective genes such as Cebpd, Nfkbia, Gadd45b, Jun, and Aplod1 at T1, while selectively hypermethylating maladaptive genes like Tmem200c and Fgfr4 despite persistent reduced global DNMT activity at T2. Conclusion. IPC reprograms the cardiac transcriptome through dynamic DNA methylation changes, potentially mediating protection against myocardial necrosis as an adaptive response. Overall design: RNA seq profilling of myocardial tissue of rats that underwent ischaemia-reperfusion injury with or without ischaemic preconditioning using temporary left anterior descending artery ligation (LAD) at the base line (T1) and 4 hours (T2) post reperfusion.