SpringerProtocols: Abstract: Myofilament Protein Phosphorylation by PKC in Genetically Engineered Adult Cardiac Myocytes

Myofilament Protein Phosphorylation by PKC in Genetically Engineered Adult Cardiac Myocytes

By: Margaret V. Westfall³

Abstract

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Virus-based gene transfer results in the rapid and efficient expression of individual contractile proteins within the myofilaments of adult cardiac myocytes (1–3). This ability to express genetically modified contractile proteins in myocytes now makes it possible to investigate the relative phosphorylation of a given contractile protein as it resides within the intact myofilament. Contractile proteins, including troponin I (TnI), troponin T (TnT), myosin light chain 2 (MLC₂), and myosin binding protein C (MyBPC) are in vitro targets for protein kinase C (PKC) phosphorylation (4). Several protocols have been developed to detect contractile protein phosphorylation using ³²P-ATP and PKC preparations (5–7). However, questions remain about whether these proteins are phosphorylated by agonist-activated PKC in intact adult cardiac myocytes (5,8,9). Thus, there is a need to identify the relative phosphorylation of myofilament proteins in response to PKC activation in adult cardiac myocytes. This goal is accomplished by studying myofilament protein phosphorylation in genetically modified myocytes expressing contractile proteins with and without modified phosphorylation sites.

Affiliation(s): (3) Cardiac Surgery Section, University of Michigan School of Medicine, Ann Arbor, MI

Book Title: Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications

Series: Methods in Molecular Biology | Volume: 219 | Pub. Date: Dec-19-2002 | Page Range: 159-166 | DOI: 10.1385/1-59259-350-X:159

Subject: Genetics/Genomics

References

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2.	Westfall, M. V., Albayya, F. P., Turner, I. I., and Metzger, J. M. (2000) Chimera analysis of troponin I domains that influence Ca²⁺-activated myofilament tension in adult cardiac myocytes. Circ. Res. 86, 470–477.

3.	Westfall, M. V., Turner, I. I., Albayya, F., and Metzger, J. M. (2001) Troponin I chimera analysis of the cardiac myofilament tension response to protein kinase A. Am. J. Physiol. 280, C324–C332.

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14.	Westfall, M. V., Rust, E. M., Albayya, F., and Metzger, J. M. (1997) Adenovirus-mediated myofilament gene transfer into adult cardiac myocytes. Methods Cell. Biol. 52, 307–322.

15.	Rust, E. M., Westfall, M. V., and Metzger, J. M. (1998) Stability of the contractile assembly and Ca²⁺-activated tension in adenovirus infected adult cardiac myocytes. Mol. Cell. Biochem. 181, 143–155.

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