Protein kinase C-alpha and -epsilon 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-failing and failing left ventricle was achieved using antibodies to PKC- alpha 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.
Cardiovascular Research, Discovery Research, Eli Lilly and Company, Indianapolis, IN 46285, USA.
N
N
Bowling
X
X
Huang
G E
GE
Sandusky
R L
RL
Fouts
K
K
Mintze
M
M
Esterman
P D
PD
Allen
R
R
Maddi
E
E
McCall
C J
CJ
Vlahos
D015153:Blotting, Western / Q000379:methods; D018031:Connexin 43 / Q000378:metabolism*; D005260:Female; D006321:Heart; D006333:Heart Failure / Q000378:metabolism* / Q000473:pathology; D006352:Heart Ventricles / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D008875:Middle Aged; D010766:Phosphorylation; D011233:Precipitin Tests / Q000379:methods; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D018487:Ventricular Dysfunction, Left / Q000378:metabolism* / Q000473:pathology
D018031:Connexin 43; D007527:Isoenzymes; C496628:PRKCA protein, human; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon