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

ABSTRACT: 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.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE284606 | GEO | 2025/12/02

REPOSITORIES: GEO

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Project description:Myocardial ischemic preconditioning (IPC) enhances myocardial resilience to ischemic injury. Myocardial stunning is a transient, reversible dysfunction, while necrosis involves irreversible cell death. The relationship between IPC, stunning, and necrosis is not well understood, requiring further molecular investigation. This study aimed to investigate the proteomic changes associated with IPC, focusing on its relationship with myocardial stunning and necrosis. A novel 13.5-minute ischemia-reperfusion (I/R) rat model was specifically chosen to induce myocardial stunning, providing a unique approach to assess IPC effects in this context. Rats underwent either IPC with two 5-minute ischemia/reperfusion cycles followed by a 13.5-minute ischemic period or the procedure without IPC (no ischemic preconditioning, NIPC). Myocardial samples were collected at early (T1) and 4-hour post-reperfusion (T2) time points for proteomic analysis. Protein levels were quantified by differential labeling using TMTpro reagents, and subsequent liquid chromatography-mass spectrometry. IPC induced upregulation of proteins involved in endocytosis and Fc gamma R-mediated phagocytosis pathways at T1, while downregulating proteins related to tissue remodeling, immune response, and coagulation at T2. Conversely, NIPC exhibited upregulation of proteins associated with tissue damage and inflammation. IPC rats demonstrated enhanced leukocyte migration, complement activation, and immune response between T1 and T2. Consistent proteomic changes were observed between T1 and T2 in IPC vs. NIPC groups, and common alterations between IPC T2 vs. T1 and NIPC T2 vs. T1 comparisons underline shared pathways in cardiac complement and coagulation cascades. Our study reveals distinct proteomic changes induced by IPC in the context of myocardial stunning and necrosis. IPC activates early protective pathways, attenuates tissue damage and inflammation, and preserves myocardial function. These findings underscore IPC's reparative potential and identify myocardial stunning as an important, transient adaptation, which may have implications for supportive clinical management in I/R.

Project description:Background: Ischemic preconditioning (IPC), i.e., brief periods of ischemia, protect the heart from subsequent prolonged ischemic injury, and reduces infarction size. Myocardial stunning refers to transient loss of contractility in the heart after myocardial ischemia that recovers without causing permanent damage. The relationship between IPC and myocardial stunning remains incompletely understood. Purpose: The primary aim of this study was to examine the effects of IPC on the relationship between ischemia duration, stunning, and infarct size in an ischemia-reperfusion injury model. The secondary aim of the study was to examine to which extent the phosphoproteomic changes induced by IPC relate to myocardial contractile function. Methods: Rats were subjected to different durations of left anterior descending artery (LAD) occlusion, with or without preceding IPC. Echocardiograms were acquired at 4 and 48 hours to assess cardiac contraction in the affected myocardial segment. Reversible akinesia was defined as the presence of myocardial akinesia at 4 hours that resolved by 48 hours; and was considered to represent myocardial stunning. Infarction size was evaluated using triphenyl tetrazolium chloride staining. Phosphoproteomic analysis was performed in heart tissue from preconditioned and non-preconditioned animals using nano-liquid chromatography-mass spectrometry. Results: Reversible akinesia was observed in a majority of the rats that were subjected to IPC and subsequently exposed to ischemia of 13.5 or 15 minutes of ischemia (83.3% [n/N] and 66.6% [n/N] respectively). Among rats without IPC, who were exposed to either 10, 11, 12 or 13.5 minutes of ischemia, reversible akinesia was observed in 0% (n/N), 17% (2/12), 0% (n/N) and 0% (n/N) of rats (p<0.001). Phosphoproteomic analysis revealed significant differential regulation of 809 phosphopeptides between IPC and non-IPC groups, with significant associations with the sarcomere, Z-disc, and actin binding. Conclusion: Our study shows that IPC preferentially induces changes in phosphosites of proteins involved in myocardial contraction, and both increases the incidence of reversible post-ischemic myocardial stunning after ischemia-reperfusion injury and reduces infarction size.

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

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