Protein kinase C-α and -ε modulate connexin-43 phosphorylation in human heart

We have previously demonstrated that protein kinase C (PKC)-alpha expression is significantly elevated in failing human left ventricle, with immunostaining showing increased PKC-alpha localization at the intercalated disks of cardiomyocytes. In the present study we sought to determine, in the failing heart, if PKC-alpha interacted with connexin-43 (Cx-43) both spatially and functionally, and to compare the association of PKC-alpha /Cx-43 with that of PKC-epsilon, a PKC isozyme that does not significantly increase in failing hearts. The possibility of a PKC-alpha or PKC-epsilon /Cx-43 association in non-failing hearts was also investigated. Co-immunoprecipitation of PKC-alpha or PKC-epsilon and Cx-43 in non-fairing and failing left ventricle was achieved using antibodies to PKC-epsilon or Cx-43, Confocal microscopy confirmed that PKC-alpha 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-epsilon showed cardiomyocyte distribution in both cytosol and membrane, and colocalization of PKC-epsilon with Cx-43. Recombinant PKC-alpha or epsilon 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-<alpha> or PKC-epsilon resulted in only PKC-epsilon mediated Cx-43 phosphorylation. Thus, in the human heart PKC-alpha, PKC-epsilon, and Cx-43 appear to form a closely associated complex. Whereas only PKC-epsilon directly phosphorylates Cx-43, both PKC isoforms result in increased phosphorylation within the Cx-43 co-immunoprecipitated complex. (C) 2001 Academic Press.