PDE4D Mediates the Cardiac Ischemia Reperfusion Injury and Is m6A Modified by METTL14.

Abstract

Background and objectives: Cardiac ischemia-reperfusion (I/R) injury is a major contributor to morbidity following myocardial infarction. While PDE4D has been implicated in cerebral I/R injury, its role and regulatory mechanisms in the cardiac context remain unclear.

Methods: PDE4D expression was analyzed using quantitative polymerase chain reaction and Western blot. Apoptosis was assessed by TUNEL staining, flow cytometry, and detection of caspase-3, Bax, and Bcl-2 levels. N6-methyladenosine (m6A) modification of PDE4D was examined via methylated RNA immunoprecipitation, luciferase reporter assays, and RNA stability measurements. The interaction between IGF2BP1 and PDE4D was confirmed by RNA immunoprecipitation.

Results: PDE4D was significantly upregulated in I/R-injured myocardium and in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation. Knockdown of PDE4D reduced infarct size, suppressed apoptosis, and improved cell viability. METTL14 mediated m6A methylation at 2 conserved sites within PDE4D mRNA, thereby enhancing its RNA stability. Silencing METTL14 decreased PDE4D expression and reduced its transcript stability. IGF2BP1 recognized m6A-modified PDE4D and promoted its RNA stability; ablation of IGF2BP1 abolished the effects of METTL14. Overexpression of PDE4D reversed the cardioprotective effects resulting from METTL14 silencing.

Conclusions: METTL14 stabilizes PDE4D mRNA through m6A methylation, which is recognized by IGF2BP1, thereby exacerbating cardiac I/R injury.