Protein Kinase C- α and - ε Modulate Connexin-43 Phosphorylation in Human Heart

We have previously demonstrated that protein kinase C (PKC)- α expression is significantly elevated in failing human left ventricle, with immunostaining showing increased PKC- α localization at the intercalated disks of cardiomyocytes. In the present study we sought to determine, in the failing heart, if PKC- α interacted with connexin-43 (Cx-43) both spatially and functionally, and to compare the association of PKC-α /Cx-43 with that of PKC- ε, a PKC isozyme that does not significantly increase in failing hearts. The possibility of a PKC- α or PKC- ε/Cx-43 association in non-failing hearts was also investigated. Co-immunoprecipitation of PKC- α or PKC- ε and Cx-43 in non-failing and failing left ventricle was achieved using antibodies to PKC- α or Cx-43. Confocal microscopy confirmed that PKC- α distribution within the cardiomyocyte included co-localization with connexin-43 in both failing and non-failing myocardium. In a similar manner, confocal imaging of PKC- ε showed cardiomyocyte distribution in both cytosol and membrane, and colocalization of PKC-ε with Cx-43. Recombinant PKC- α or - ε increased PKC activity significantly above endogenous levels in the co-immunoprecipitated Cx-43 complexes (P<0.05). However, phosphorylation of purified human Cx-43 (isolated from failing human left ventricle) by recombinant PKC- α or PKC- ε resulted in only PKC-ε mediated Cx-43 phosphorylation. Thus, in the human heart PKC- α, PKC- ε, and Cx-43 appear to form a closely associated complex. Whereas only PKC- ε directly phosphorylates Cx-43, both PKC isoforms result in increased phosphorylation within the Cx-43 co-immunoprecipitated complex.