Inhibition of GRK4 reduces arrhythmia susceptibility and alleviates connexin43 dysregulation after myocardial infarction.

G protein-coupled receptors (GPCRs) can transduce a variety of extracellular signals into cellular responses and serve as crucial targets for the drug treatment of cardiovascular diseases. G protein-coupled receptor kinases (GRKs) regulate receptor desensitization and downregulation by phosphorylating the intracellular region of GPCRs. Despite the reported involvement of GRKs in cardiac injury and failure, G protein-coupled receptor kinases 4 (GRK4) in ischemic arrhythmias are not fully understood. Therefore, we discovered that GRK4 exerts a pro-arrhythmic effect, with its mRNA and protein levels upregulated in ischemic hearts. Our studies revealed that knockdown of GRK4 effectively improves cardiac electrical conduction, reduced susceptibility to ventricular arrhythmias, and attenuated the abnormal distribution of connexin43 (Cx43). Similar results were observed upon knockdown or overexpression of GRK4 in cardiomyocytes under hypoxic conditions using its siRNA or an adenovirus carrying GRK4. Mechanistically, our study showed that GRK4 exacerbates arrhythmia susceptibility by binding to the M3 subtype of the muscarinic acetylcholine receptor (M3-mAChR) and increases M3-mAChR phosphorylation, which in turn impeded the association between M3-mAChR and Cx43, leading to downregulation of Cx43, redistribution, and subsequent arrhythmia. Meanwhile, methyltransferase-like 3 (METTL3) recruited YTH domain family, member 1 (YTHDF1) to increase GRK4 stability via an N6-methyladenosine (m6A)-dependent mechanism, which contributed to abnormally increased GRK4 expression in ischemic hearts and served as a major factor in triggering ventricular arrhythmias after acute myocardial infarction (AMI). In summary, GRK4 increases susceptibility to arrhythmias after AMI via modulation of the M3-mAChR/Cx43 axis, providing new insights into the clinical therapy of ventricular arrhythmias.