© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Molecular heterogeneity of protein kinase C expression in human ventricle
Departments of Medicine, Pharmacology and Surgery, Vanderbilt University School of Medicine, Nashville, TN 37232-6602, USA
* Corresponding author. Tel.: +1-615-936-1514; fax: +1-615-322-4707 hyeon-gyu.shin{at}mcmail.vanderbilt.edu
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-
and 
were detected by immunoblotting, with confirmation using a second antibody directed against a different epitope when possible. For PKC-βII, 
, and 
, 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-
, βI, and 
. 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.
KEYWORDS Signal transduction; Heart failure; Cardiomyopathy; Protein phosphorylation; Protein kinases; Myocytes
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