Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation

Antiarrhythmic peptides enhance gap junction current in pairs of cardiomyocytes and coupling in cardiac tissue. To elucidate the underlying mechanisms, we investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH2) on pairs of adult guinea pig ventricular cardiomyocytes and pairs of HeLa cells transfected with rat cardiac connexin 43 (Cx43). By using a double-cell voltage-clamp technique in pairs of cardiomyocytes, we found that under control conditions the gap junction conductance (gj) steadily decreased with time (by 0.292 +/- 0.130 nS/min). Use of 50 nmol/L AAP10 reversed this rundown and increased gj (by +0.290 +/- 0.231 nS/min, P<0.05). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM) and by the protein kinase C (PKC) subtype-specific inhibitor CGP54345 (PKC alpha). In HeLa-Cx43 cells, AAP10 exerted the same electrophysiological effect. In these cells, AAP10 activated PKC (determined by using ELISA) in CGP54345-sensitive manner and significantly enhanced incorporation of P-32 into Cx43 with dependence on PKC. If G-protein coupling was inhibited with 1 mM GDP-beta S, we found the effects of AAP10 on P-32 incorporation were also completely abolished. Next, we performed a radioligand binding study with C-14-AAP10 as radioligand and AAPnat as competitor. We found saturable binding of C-14-AAP10 to cardiac membrane preparations, which could be displaced with AAPnat. The K-d of AAP10 was 0.88 nmol/L. We conclude that 1) AAP10 increases gj both in adult cardiomyocytes and in transfected HeLa-Cx43 cells, 2) AAP10 exerts its effect via enhanced PKC-dependent phosphorylation of Cx43, 3) AAP10 activates PKC alpha, and 4) a membrane receptor exists for antiarrhythmic peptides in cardiomyocytes.