Molecular heterogeneity of protein kinase C expression in human ventricle.
OBJECTIVE
Although activation of protein kinase C (PKC) modulates the function of normal cardiac myocytes and likely plays a role in the pathogenesis of cardiomyopathic disease states, the molecular basis of PKC expression in human ventricle has not been examined in detail.


METHODS
We have performed Western analysis and immunohistochemistry on explanted human cardiac tissue from nondiseased and diseased specimens using isoform-specific antibodies directed against all known PKC isozymes.


RESULTS
In homogenates from left and right ventricle, all isoforms except PKC-gamma and theta were detected by immunoblotting, with confirmation using a second antibody directed against a different epitope when possible. For PKC-betaII, delta, and epsilon, data indicated that these isoforms were variably phosphorylated in vivo, resulting in multiple bands during immunoblotting. Because of potential antibody cross-reactivity, reverse transcriptase polymerase chain reaction (RT-PCR) was performed which confirmed expression of PKC-alpha, betaI, and zeta. Immunohistochemistry demonstrated that all isoforms detected in ventricular homogenate by Western analysis could be localized to cardiac myocytes. From a methodologic standpoint, significant degradation of PKC isoforms could be demonstrated when samples were either frozen or allowed to remain at room temperature, compared to immediate subcellular fractionation.


CONCLUSIONS
These findings indicate that the PKC expression in human ventricular myocytes is remarkably diverse, with multiple conventional, novel, and atypical isoforms present, and highlight the importance of sample preparation in comparative studies of PKC isoform expression.
Shin
H G
HG
Departments of Medicine, Pharmacology and Surgery, Vanderbilt University School of Medicine, 37232-6602, Nashville, TN, USA. hyeon-gyu.shin@mcmail.vanderbilt.edu
Barnett
J V
JV
Chang
P
P
Reddy
S
S
Drinkwater
D C
DC
Pierson
R N
RN
Wiley
R G
RG
Murray
K T
KT
D000293:Adolescent; D000328:Adult; D000368:Aged; D015153:Blotting, Western; D005260:Female; D006333:Heart Failure / Q000201:enzymology*; D006352:Heart Ventricles / Q000201:enzymology; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000032:analysis*; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007527:Isoenzymes; D011493:Protein Kinase C