Interaction of α Carboxyl Terminus 1 Peptide With the Connexin 43 Carboxyl Terminus Preserves Left Ventricular Function After Ischemia-Reperfusion Injury

Background-alpha Carboxyl terminus 1 (alpha CT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that alpha CT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase CE phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of alpha CT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results-To study alpha CT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cs phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the alpha CT1 Asp-Asp-Leu-Glu-lso sequence and lysines (Lys345, Lys346) in an alpha -helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that alpha CT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of alpha CT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of alpha CT1 (alpha CT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions-Interaction of alpha CT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.