A potential therapeutic target at Connexin 43 serine phosphorylation against ischaemia/reperfusion arrhythmia

Dephosphorylation of several phosphor residues in connexin 43 (Cx43) is critically involved in ischaemia/reperfusion (I/R) arrhythmias. Here, we sought to identify constitutive Cx43-serine 282 (S282) dephosphorylation as an independent and targetable substrate to delineate reperfusion arrhythmogenesis.

Reperfusion arrhythmias were induced by rat I/R (15/45 min), identified in heterozygous knock-in mice with S282 to alanine (Cx43-S282A/+) and rescued by LB100, a specific protein phosphatase 2A (PP2A) inhibitor, via surface electrocardiogram and epicardial mapping detections. TAT-HA-L2, a mimic of Cx43-cytoplasmic-loop domain (L2), was assessed in mitigating Cx43-S282 dephosphorylation-associated arrhythmogenicity.

Upon I/R, rats exeperienced myocardium injury, ventricular tachycardia/fibrillation, increased PP2A activity and Cx43-S282 hypophosphorylation, whereas Cx43-S282A/+ mice had spontaneous ventricular arrhythmias with normal cardiac function/morphology. LB100 significantly attenuated these arrhythmias by restoring PP2A-C and Cx43-S282 phosphorylation. Slow and irregular electrical conduction, premature Ca²⁺ transients and afterdepolarisations were found and normalised by LB100, or Gap19 peptide treatment (Cx43 hemichannel inhibitor), in Cx43-S282A/+ ventricles. Mechanistically, serine 282 locates within SH3-binding domain of the Cx43-carboxyl-terminus. TAT-HA-L2 bound with Cx43-S282 dephosphorylated proteins in S282A-expressed HeLa cells, as well as in S282A/+ and I/R isolated cardiomyocytes, more than in their counterpart controls. LB100 can prevent their enhanced binding and ATP release because of S282 dephosphorylation.

Cx43-S282 dephosphorylation can trigger reperfusion arrhythmias by impairing gap junction intercellular communication while favouring hemichannel permeability. An up-regulated intramolecular interaction between L2 and Cx43-carboxyl-terminus is associated with this arrhythmogenicity, providing a novel and targetable mechanism to preserve the heart from ischaemia/reperfusion arrhythmias.