Abstract 14224: Inhibition of Calmodulin Increases Intercellular Coupling of Ventricular Myocardium by Enhancement of Connexin43 Localization in Intercalated Disk.
Background: Intercellular communication through gap junctions enables coordinated electrical activation and contraction in the heart, and connexin43 (Cx43) is the major constituent of gap junctions in the ventricular muscle. We previously showed that calmodulin inhibiters, such as W7 and bepridil, increase conduction velocity, without affecting excitability, in the ventricular myocardium of perfused rabbit hearts, and hypothesized that inhibition of calmodulin increases intercellular coupling through modifying the localization of Cx43 in the intercalated disk.
Methods: Intercellular communication through gap junctions was assessed by the measurement of space constant by high resolution optical mapping on perfused rabbit hearts as well as a dye transfer assay using Lucifer yellow(LY) on cultured neonatal rat cardiomyocyte (CM) monolayers . The subcellular distribution of Cx43 was evaluated by Western blot analysis on triton X-100-based fractionation of soluble (non-junctional) and insoluble (junctional) proteins from Langendorff-perfused rabbit ventricles. We also performed fluorescence confocal microscopy of Cx43-immunostained ventricular tissues of 1-hour perfused rabbit hearts.
Results: Space constant in longitudinal and transverse direction (λL and λT, respectively) was significantly increased by 5 μM of W7 (λL; from 1.12±0.08 to 1.38±0.15 mm, λT; from 0.96±0.16 to 1.24±0.17 mm, n=5, p<0.01). Dye transfer assay revealed that the number of dye-coupled CMs was significantly increased in the presence of W7 (from 27±3 to 41±5, n=7, p<0.05). In the Western blot assay, the amount of Cx43 in the Triton X-insoluble protein fraction (junctional Cx43) was significantly increased by W7 (by 10%, n=5, p<0.05), whereas that in the Triton X-soluble fraction (non-junctional Cx43) was significantly decreased (by 8%, n=5, p<0.05). Confocal images showed that the cluster size of immunolabled Cx43 in the intercalated disk was substantially enlarged in ventricular tissues after treatment with W7.
Conclusion: Inhibition of calmodulin increases intercellular coupling of ventricular myocardium through gap junctions by the enhancement of Cx43 localization in the intercalated disk.
- © 2010 by American Heart Association, Inc.