Electrical Pacing Inhibits Gap Junction-Mediated Cardiac Cell-Cell Communication by Promoting Cx43-Acetylation

Background: Communication among cardiomyocytes depends upon Gap Junction (GJ) protein expression and conductance. Previous studies demonstrated that electrical stimulation can induce GJ remodeling and evidences from neurons also indicate that electrical pacing modifies Lysine acetylase (KAT) and deacetylases (KDAC) activities. Objectives: Aim of the present work was to establish whether electrical stimulation modulates GJ-mediated cardiac cell-cell communication by acetylation dependent mechanisms. Methods and Results: Neonatal rat cardiomyocytes (NRCM; n=3) and in HL-1 atrial cells (n=20) were exposed to electrical field stimulation for 24 hours (Ionoptix C-Pace®; 0.5 Hz, 20 V, 0.5 msec pulses). Connexin 43 (Cx43) expression decreased almost 50% in NRCM and 40 % in HL-1; in contrast Cx40 and Cx45 expression was unchanged. Further, confocal microscopy revealed that electrical stimulation induced Cx43 accumulation in the cytoplasm of HL-1 cells. Electrical stimulation significatly down-regulated KDAC activity up to the 30% (n=3), whereas KAT activity was not modified; the net effect was a general increase of cell protein acetylation, confirmed by western blot analysis. Specifically, the pacing-dependent acetylation of Cx43 was proven by immunoprecipitation assay (n=5). Interestingly, our model mimicked the action of the KDAC pan-inhibitors TSA and SAHA on Cx43 expression and intracellular distribution, although we did not observe Cx43 mRNA significant reduction in electrically stimulated cells. In agreement, MG132 proteasome inhibitor (10 μM) restored Cx43 expression level. Finally, also the treatment of paced cells with the KAT inhibitor Anacardic Acid (0.5 μM) was able to rescue Cx43 level (n=4). Intriguingly, preliminary results also indicate lateralization and increased acetylation of Cx43 in the left ventricles of dogs with pacing-induced dilated cardiomyopathy (n=2). Conclusions: In vitro electrical stimulation of cardiac cells promotes Cx43 acetylation, which results in Cx43 down-modulation and intracellular relocalization. These findings suggest that electrical activity-dependent increase in acetylation may represent a novel mechanism for the regulation of cardiomyocyte communication.