Molecular heterogeneity of protein kinase C expression in human ventricle

Objective: Although activation of protein kinase C (PKC) modulates the func tion of normal cardiac myocytes and likely plays a role in the pathogenesis of cardiomyopathic disease states, the molecular basis of PKC expression i n human ventricle has not been examined in detail. Methods: We have perform ed Western analysis and immunohistochemistry on explanted human cardiac tis sue from nondiseased and diseased specimens using isoform-specific antibodi es directed against all known PKC isozymes. Results: In homogenates from le ft and right ventricle, all isoforms except PKC-gamma and theta were detect ed by immunoblotting, with confirmation using a second antibody directed ag ainst a different epitope when possible. For PKC-beta II, delta, and epsilo n, data indicated that these isoforms were variably phosphorylated in vivo, resulting in multiple bands during immunoblotting. Because of potential an tibody cross-reactivity, reverse transcriptase polymerase chain reaction (R T-PCR) was performed which confirmed expression of PKC-alpha, betaI, and ze ta. Immunohistochemistry demonstrated that all isoforms detected in ventric ular homogenate by Western analysis could be localized to cardiac myocytes. From a methodologic standpoint, significant degradation of PKC isoforms co uld be demonstrated when samples were either frozen or allowed to remain at room temperature, compared to immediate subcellular fractionation. Conclus ions: These findings indicate that the PKC expression in human ventricular myocytes is remarkably diverse, with multiple conventional, novel, and atyp ical isoforms present, and highlight the importance of sample preparation i n comparative studies of PKC isoform expression. (C) 2000 Elsevier Science B.V. All rights reserved.