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Molecular heterogeneity of protein kinase C expression in human ventricle
H G Shin1, J V Barnett, P Chang
1Departments of Medicine, Pharmacology and Surgery, Vanderbilt University School of Medicine, 37232-6602, Nashville, TN, USA. hyeon-gyu.shin@mcmail.vanderbilt.edu
Insights
Human ventricular myocytes express diverse protein kinase C (PKC) isoforms, including conventional, novel, and atypical types. Proper sample preparation is crucial for accurate PKC expression studies in cardiac tissue.
Area of Science:
- Cardiovascular Biology
- Molecular Cardiology
- Signal Transduction
Background:
- Protein kinase C (PKC) activation influences cardiac myocyte function.
- PKC plays a role in cardiomyopathic disease pathogenesis.
- Detailed examination of PKC expression in the human ventricle is lacking.
Purpose of the Study:
- To investigate the molecular basis of protein kinase C (PKC) expression in human ventricular tissue.
- To identify which PKC isoforms are present in human ventricular myocytes.
Main Methods:
- Western analysis and immunohistochemistry on human cardiac explants.
- Isoform-specific antibodies were used to detect all known PKC isozymes.
- Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed expression and localized isoforms to cardiac myocytes.
Main Results:
- Most PKC isoforms (except gamma and theta) were detected in human ventricular homogenates.
- PKC-betaII, delta, and epsilon showed variable in vivo phosphorylation.
- RT-PCR confirmed expression of PKC-alpha, betaI, and zeta; all detected isoforms localized to cardiac myocytes.
- Sample degradation occurred with improper storage, emphasizing methodologic importance.
Conclusions:
- Human ventricular myocytes exhibit diverse expression of conventional, novel, and atypical PKC isoforms.
- Findings underscore the critical importance of meticulous sample preparation for comparative PKC isoform expression studies.
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.