Effects of the Connexin43 CT on Gap Junction Sub-Domain Organization and Myocyte-Fibroblast Interactions in the Injury Border Zone

Phosphorylation of connexin43 (Cx43) has been shown to regulate gap junction (GJ) intercellular communication. A Cx43 carboxyl-terminus(CT) mimetic peptide (aCT1) increases GJ size via reduced Cx43/ZO-1 interaction in vitro and increases phosphorylation of Cx43 at aa S368 (Cx43pS368) in vitro and in vivo. We previously reported the effects of aCT1-treatment in a left ventricular (LV) cryoinjury model in vivo (Circ Res 2011;108(6):704-15). At 8-weeks after cryoinjury, histological assessment showed decreases in scar size and increases in collagen fibril uniformity in aCT1-treated hearts versus controls. Interdigitation of collagen and fibroblasts between myocytes in the injury border zone (IBZ) was reduced in treated hearts versus controls. Treated hearts exhibited a decreased propensity for induced arrhythmias (p<0.02) and an increase in Cx43pS368 in the IBZ which correlated with a tendency for Cx43 to be maintained at intercalated discs.To study the mechanistic effect of aCT1 on myocyte-fibroblast interactions via Cx43, we created a 3D heterocellular system to model the injury border zone (IBZ) in vitro. Neonatal rat ventricular fibroblasts were seeded to form a shell around a core of aggregated neonatal rat ventricular myocytes (NRVMs) in 96-well micromolds. After 72 hours, aCT1-treatment of aggregates induced an increase in heterocellular interactions via total Cx43 (p=0.03) and Cx43pS368 (p=0.01). Studies of an in vivo LV cryoinjury model were used to validate these data. Changes in myocyte-fibroblast interactions, GJ plaque size, and GJ sub-domain organization after aCT1 treatment were detected with confocal and super-resolution microscopy (both gSTED and STORM). Super-resolution microscopy enabled resolution of discrete phospho-S368 non-phosphorylated subdomains of Cx43 within GJ plaques. In sum, our results indicate the potential for Cx43 CT effects on heterocellular interactions via Cx43 in the IBZ and scar following myocardial infarction.