We have previously demonstrated that protein kinase C (PKC)-alpha expressio
n is significantly elevated in failing human left ventricle, with immunosta
ining showing increased PKC-alpha localization at the intercalated disks of
cardiomyocytes. In the present study we sought to determine, in the failin
g heart, if PKC-alpha interacted with connexin-43 (Cx-43) both spatially an
d functionally, and to compare the association of PKC-alpha /Cx-43 with tha
t of PKC-epsilon, a PKC isozyme that does not significantly increase in fai
ling hearts. The possibility of a PKC-alpha or PKC-epsilon /Cx-43 associati
on in non-failing hearts was also investigated. Co-immunoprecipitation of P
KC-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 d
istribution in both cytosol and membrane, and colocalization of PKC-epsilon
with Cx-43. Recombinant PKC-alpha or epsilon increased PKC activity signif
icantly above endogenous levels in the co-immunoprecipitated Cx-43 complexe
s (P<0.05). However, phosphorylation of purified human Cx-43 (isolated from
failing human left ventricle) by recombinant PKC-<alpha> or PKC-epsilon re
sulted in only PKC-epsilon mediated Cx-43 phosphorylation. Thus, in the hum
an heart PKC-alpha, PKC-epsilon, and Cx-43 appear to form a closely associa
ted complex. Whereas only PKC-epsilon directly phosphorylates Cx-43, both P
KC isoforms result in increased phosphorylation within the Cx-43 co-immunop
recipitated complex. (C) 2001 Academic Press.