Abstract 415: Molecular Mechanisms of Connexin43 Lateralization in Ischemic Ventricular Myocytes After Coronary Artery Occlusion in the Canine Heart
Following coronary occlusion (CO) there is a decrease in the gap junction protein Connexin43 (Cx43) in intercalated disks (ID) and an increase in Cx43 in non-disk lateral membranes in surviving epicardial border zone (EBZ) myocytes which is associated with an increased anisotropic conduction velocity ratio and ventricular arrhythmias. We examined the molecular mechanisms for these changes. We used yeast two hybrid (Y2H), surface plasmon resonance (SPR), and nuclear magnetic resonance (NMR) to analyze interactions of Cx43 with its principle anchoring protein in the ID, Zonula Occludens-1 (ZO-1) in vitro at low pH to mimic the ischemic environment. Y2H showed interactions between the receptor tyrosine kinase cSrc and the carboxyl terminal domain of Cx43 (Cx43CT) as well as an interaction between cSrc and ZO-1. SPR indicated that the cSrc interaction with ZO-1 was stronger than the interaction of cSrc with Cx43CT. NMR showed that upon binding of cSrc to Cx43CT/ZO-1 complex, the structure of Cx43CT was altered bringing cSrc in close proximity to the bound ZO-1. The cSrc bound then chaperoned ZO-1 off of Cx43CT. To determine if cSrc also was involved in unhooking Cx43 from ZO-1 in vivo, EBZ tissue was studied 3 hours after CO. Western Blot and co-immunoprecipitation studies showed activated cSrc at 3 hrs post CO and increased Cx43/cSrc binding with a concurrent loss of the normal ZO-1 levels. Immunostaining indicated that increased Cx43 on lateral membranes was primarily dephosphorylated, thus non-functional. Mapping electrical propagation in these tissues showed a decrease in conduction velocity primarily in the transverse direction (22.3%) with only minor decreases in the longitudinal direction (5.1%). The in vitro and in vivo data support the hypothesis that lateralization of Cx43 in myocardial infarction is due to alterations in interactions of cSrc with Cx43 and subsequently unhooking of Cx43 from ZO-1. The data also support the conclusion that loss of Cx43 at the ID combined with deposition of dephosphorylated, non-functional Cx43 on the lateral myocyte membranes may play a role in the excessive slowing of conduction in the transverse direction.