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J Biol Chem, Vol. 273, Issue 36, 23072-23079, September 4, 1998

The Expression of Constitutively Active Isotypes of Protein Kinase C to Investigate Preconditioning

Jing Zhao, Oliver Renner, Lionel Wightman, Peter H. SugdenDagger , Luisa Stewart§, Andrew D. Miller§, David S. Latchman, and Michael S. Marber

From the Department of Cardiology, United Medical and Dental Schools of Guy's and St Thomas' Hospitals, London, SE1 7EH the Dagger  Division of Cardiac Medicine and the § Department of Chemistry, Imperial College of Science, Technology, and Medicine, London, SW3 6LY and the  Department of Molecular Pathology, University College London Medical School, London WIP 6DB, United Kingdom

The role of protein kinase C (PKC) in ischemic preconditioning remains controversial because of difficulties with both its measurement and pharmacological manipulation. We investigated preconditioning in isolated neonatal rat cardiocytes by expressing constitutively active isotypes of PKC. Observations at differing durations of simulated ischemia suggested beta -galactosidase (beta -gal) activity reflected viability within transfected myocytes. Preconditioning with 90 min of ischemia significantly increased beta -gal activity and myocyte survival after 6 h of ischemia; an effect abolished by PKC inhibitors. After co-transfection with plasmids encoding beta -gal and either constitutively active mutants of PKC-delta , PKC-alpha , wild type PKC-delta , or empty vector, cardiocytes were subjected to 6 h of ischemia. Only PKC-delta , rendered constitutively active by a limited deletion within the pseudosubstrate domain, consistently increased resistance to simulated ischemia (beta -gal activity was 85.6 ± 11.9% versus 53.7 ± 6.5% (p <=  0.01) and dead myocytes 46.8 ± 3.4% versus 68.7 ± 2.8% (p <=  0.01)). Since transfection was apparent in only 5-12% of cells, the results suggested a protective bystander effect that was confirmed by co-culture of transfected myocytes with untransfected myocytes. In neonatal cardiocytes expression of active PKC-delta increases resistance to simulated ischemia. This observation may provide further insight into the mechanism and possible avenues for therapeutic exploitation of preconditioning.

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.


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