Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

Abstract

ABSTRACTBackgroundαCT1 is a 25 amino acid therapeutic peptide incorporating the Zonula Occludens-1 (ZO-1)-binding domain of connexin43 (Cx43) that is currently in Phase III clinical testing for healing chronic skin wounds. In preclinical studies in mice, we reported that αCT1 reduces arrhythmias and improves ventricular function following cardiac injury, effects that were accompanied by increases in PKCε phosphorylation of Cx43 at serine 368 (pS368). In this study, we undertake a systematic characterization of the molecular mode-of-action of αCT1 in mitigating the effects of ischemia reperfusion injury on ventricular contractile function.Methods and ResultsTo determine the basis of αCT1-mediated increases in pS368 we undertook tandem mass spectrometry of reactants in an in vitro assay of PKCε phosphorylation, identifying an interaction between negatively charged amino acids in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and positively charged lysines (Lys345, Lys346) in a short α-helical sequence (H2) within the Cx43 CT domain. In silico modeling provided further support of the specificity of this interaction, leading us to conclude that αCT1 has potential to directly interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift and phosphorylation assays, we characterized a series of αCT1 variant peptides, identifying sequences competent to interact with either ZO-1 PDZ2 or the Cx43 CT, but with limited or no ability to bind both polypeptides. Based on this analysis, it was found that only those peptides competent to interact with Cx43, but not ZO-1 alone, resulted in increased pS368 phosphorylation in vitro and in vivo. Moreover, in a mouse model of global ischemia reperfusion injury we determined that pre-ischemic infusion only with those peptides competent to bind Cx43 preserved left ventricular (LV) contractile function following injury. Interestingly, a short 9 amino acid (MW=1110) Cx43-binding variant of the original 25 amino acid αCT1 sequence demonstrated potent LV-protecting effects when infused either before or after ischemic injury.ConclusionsInteraction of αCT1 with the Cx43 CT, but not ZO-1 PDZ2, explains cardioprotection mediated by this therapeutic peptide. Pharmacophores targeting the Cx43 carboxyl terminus could provide a novel translational approach to preservation of ventricular function following ischemic injury.