Impairing N-cadherin-mediated Adhesion Affects Cardiac Gap Junctions And Arrhythmic Susceptibility In Isolated Rat Hearts

Impairing N-cadherin-mediated adhesion affects cardiac gap junctions and arrhythmic susceptibility in isolated ratheartsBack ground Gap junctions, adherens junctions and desmosomes are collocated in the intercalated disc build up a specialized constructure to keep up the mechanical coupling as well as electrical coupling in the myocardium. The different junctional complexes must be properly organized within the intercalated disc to mediate normal mechanical and electrical coupling between the individual cardiomyocytes in the heart. Increasing evidences from diseased myocardium and animal models indicates that alteration in electrical coupling via gap junctions is a critical determinant in the development of an arrhythmogenic substrate. It has been reportated that several diseases and animal model with abnormal mechanical joint proterins linked with high risk of ventricular arrhythmias and sudden cardiac death. Remodeling of gap junctions is common in these conditions. But the initiate effects of impaired cell-cell adhesion on the gap junctions and arrhythmias are to be clarified.Objective In the present study, a synthetic peptide AHAVD was introduced as a selective inhibitor of N-cad-mediated adhesion. We set out to observe the acute effects of impairing adherent junctions on the gap junctions in cultured cardiomyocytes as well as in isolated perfused rat hearts. The susceptibility of induced ventricular arrhythmias in isolated-perfused rat hearts was also observed. Methods and results According to the aims mentioned above, this study was divided in to three parts. First part AHAVD (0.2 mM) or IPPINL (A nonsense peptidase contains the high conservative sequence in cadherins, 0.2 mM) was incubated with cultured neonatal rat cardiomyocytes for 1 hour. With immunofluorescence and laser confocal microscopy, we revealed that AHAVD can rapidly disrupt cell-cell adhesion, accompanied with redistribution of Cx43. Lucifer yellow transfer test show significant decreased gap junction communication function between AHAVD handled cells (diffuse distance of lucifer yellow are 0.25±0.09mm, 0.74±0.23mm and 0.83±0.26mm, respectively, P<0.05).Second part Fifty-six isolated adult male rat hearts were randomly divided in to three groups, and perfused with oxygenated K-H buffer containing 0.2 mM AHAVD (n=18), 0.2 mM IPPINL (n=18) or oxygenated K-H buffer as control (n=20) for 1hour with a Langendorff device respectively. Detached adherens junctions can be observed in the left ventricular myocardium of AHAVD-perfused hearts by electron microscope. With a quantitive immunofluorescence microscopy, both reduction and side distribution of Cx43 was detected in the AHAVD-perfused hearts, the relative quantity of Cx43 signals decreased significantly in AHAVD-perfused hearts(46±11.3%, 69±19.5% and 72±18.2%, respectively, P<0.01), no disturb in the N-cadherin/catenin complex was observed. Lucifer yellow transfer test shows significant decreased communication between cardiomyocytes of AHAVD-perfused hearts (the distinction of lucifer yellow and rhodamine dextran are 1.37±0.33, 2.84±0.78 and 2.83±0.65, respectively, P<0.05).Third part By electrophysiological method and monophasic action potential recording, the reduced conduction ability of left ventricular myocardium was observed in AHAVD-perfused hearts (53.5±6.3cm/s, 65.2±6.2cm/s and 66.3±8.8cm/s, respectively, P<0.05), accompanied with increased incidence of inducible ventricular arrhythmias (50%, 22.2%, and 15%, respectively, P<0.05). However, the ERP and APD90 show no significant discrimination between the AHAVD-, IPPINL-perfused and control groups.Conclusion Impair N-cadherin-mediated adhesion may result in rapid redistribution without decreasing total Cx43 in cultured cardiomyocytes. AHAVD-perfusion can also reduce Cx43 in the IC discs of isolated rat hearts, decrease the communication between cardiomyocytes, as well as slow down conduction velocity of left ventricular myocardium. An increased incidence of inducible ventricular arrhythmias was also observed in AHAVD-perfused hearts. Collecting all the results, impair N-cadherin-medianted adhesion increasing the susceptibility of isolated rat hearts probably through affecting the gap junctions in the intercalated discs.