Abstract 16380: Phosphorylation of Connexin43 at Serine368 is Necessary for Induction of Cardioprotection by a Connexin43 Carboxyl-Terminal Mimetic Peptide
Background: Remodeling of Cx43, the main cardiac gap junction (GJ) protein, is a pathogenic hallmark of myocardial infarction. αCT1, a peptide mimetic of the carboxyl terminus (CT) 9 amino acids of Cx43, reduced GJ remodeling and arrhythmias and improved function following ventricular injury-PMID:24527326. These effects were associated with reduced Cx43 interaction with the scaffolding protein ZO-1 and increased PKC-ε phosphorylation of Cx43 at a serine at position 368 (pS368). Here, we address whether cardioprotection induced by αCT1 results mechanistically from effects on Cx43 interaction with ZO-1 or phosphorylation.
Methods & Results: αCT1 prompted increased pS368 phosphorylation of Cx43 CT in an in vitro assay. Analysis of interacting proteins in this reaction by tandem mass spectroscopy, Surface Plasmon Resonance (SPR) and molecular modeling indicated that pS368 induction involved a novel interaction between a cluster of 3 negatively charged residues in αCT1 and a pair of positively charged lysines (K344, K345) in a CT α-helical domain of Cx43. We generated αCT1 mutant peptides (pS368-/PDZ2+) in which negatively charged aspartic and glutamic acids were substituted by alanines. A mutant αCT1 (pS368+/PDZ2-) missing the CT isoleucine required for interaction with ZO-1 was also made. Using the phosphorylation assay, together with immunoprecipitation, SPR and thermal shift assays, we confirmed that αCT1 pS368-/PDZ2+ peptides neither interacted with Cx43 CT nor increased pS368, but were able to bind ZO-1 PDZ2. Conversely, αCT1 pS368+/PDZ2- peptide was unable to bind PDZ2, but retained interaction with Cx43 CT and induction of pS368. αCT1 and mutant peptides were tested in a mouse heart ischemia-reperfusion model. αCT1 reduced ischemic injury and improved functional recovery in association with upregulated pS368. By contrast, αCT1 pS368-/PDZ2+ peptides showed no effects on cardioprotection or pS368.
Conclusion: αCT1 binding to Cx43 and induction of pS368 phosphorylation explains its cardioprotective effects. The Cx43 CT has key roles in the ischemic injury response.
Author Disclosures: J. Jiang: None. J.A. Palatinus: None. H. He: None. J. Iyyathuraia: None. J. Jordan: None. F.X. McGowan Jr: None. K. Schey: None. G. Bultynck: None. Z. Zhang: None. R.G. Gourdie: None.
- © 2016 by American Heart Association, Inc.