M6A-modified circArhgap26 attenuates cardiac ischemia‒reperfusion injury by suppressing plakophilin-1 palmitoylation.

circArhgap26 expression was significantly decreased in ischemia-reperfusion myocardium and in plasma of patients undergoing percutaneous coronary intervention.

• Expression of circArhgap26 was significantly decreased in the I/R myocardium in mouse models.
• circArhgap26 expression in the plasma of patients undergoing PCI was decreased compared to controls.
• The decrease in expression was identified through transcriptomic or circRNA profiling approaches.

circArhgap26 expression is regulated by m6A modification.

• The study identified m6A modification as a regulatory mechanism controlling circArhgap26 levels.
• m6A modification represents one of two dual regulatory mechanisms (the other being palmitoylation) described for circArhgap26 in I/R injury.
• The m6A regulation of circArhgap26 is described as a novel upstream regulatory layer for this circRNA.

Cardiac-specific overexpression of circArhgap26 ameliorated cardiac dysfunction and reduced infarct area and cardiomyocyte apoptosis in I/R model mice.

• Cardiac-specific overexpression of circArhgap26 was used to assess functional outcomes in I/R model mice.
• Overexpression resulted in ameliorated cardiac dysfunction as measured by functional parameters.
• The infarct area was reduced in mice with cardiac-specific circArhgap26 overexpression.
• Cardiomyocyte apoptosis was reduced following circArhgap26 overexpression in the I/R model.

circArhgap26 directly bound to plakophilin-1 (PKP1) and inhibited its interaction with the palmitoyltransferase ZDHHC1.

• Direct binding between circArhgap26 and PKP1 was demonstrated mechanistically.
• The binding of circArhgap26 to PKP1 prevented PKP1 from interacting with ZDHHC1, a palmitoyltransferase.
• This interaction represents a novel RNA-protein regulatory axis in the context of cardiac I/R injury.

Inhibition of PKP1 palmitoylation by circArhgap26 reduced PKP1 protein stability and subsequently decreased APAF1 protein synthesis.

• Palmitoylation of PKP1 by ZDHHC1 was shown to stabilize PKP1 protein.
• When circArhgap26 inhibited PKP1-ZDHHC1 interaction, PKP1 palmitoylation was diminished, leading to reduced PKP1 protein stability.
• Reduced PKP1 stability led to a reduction in APAF1 protein synthesis.
• This represents a posttranslational modification (palmitoylation) regulatory pathway downstream of circArhgap26.

The reduction in APAF1 protein synthesis downstream of circArhgap26 led to inhibition of the Caspase-9/Caspase-3 signaling pathway and mitigation of cardiomyocyte apoptosis.

• APAF1 reduction downstream of PKP1 destabilization suppressed Caspase-9/Caspase-3 signaling.
• Inhibition of the Caspase-9/Caspase-3 pathway is the mechanistic basis for reduced cardiomyocyte apoptosis.
• This pathway connects the circRNA-PKP1-palmitoylation axis to intrinsic apoptotic signaling in cardiomyocytes.

The study elucidates dual regulatory mechanisms of circArhgap26 involving m6A modification and posttranslational modification (palmitoylation) in combating I/R injury.

• m6A modification regulates circArhgap26 expression at the RNA level.
• Palmitoylation of PKP1 is regulated by circArhgap26 at the protein level.
• These two mechanisms together constitute a dual regulatory framework described by the authors as novel in the context of cardiac I/R injury.
• The authors propose circArhgap26 holds dual value as both a prognostic biomarker and therapeutic target for I/R-related diseases.