THE EXPRESSION OF CONSTITUTIVELY ACTIVE ISOTYPES OF PROTEIN-KINASE-C TO INVESTIGATE PRECONDITIONING

The role of protein kinase C (PKC) in ischemic preconditioning remains controversial because of difficulties with both its measurement and p harmacological manipulation. We investigated preconditioning in isolat ed neonatal rat cardiocytes by expressing constitutively active isotyp es of PKC. Observations at differing durations of simulated ischemia s uggested beta-galactosidase (beta-gal) activity reflected viability wi thin transfected myocytes, Preconditioning with 90 min of ischemia sig nificantly increased beta-gal activity and myocyte survival after 6 h of ischemia; an effect abolished by PKC inhibitors. After co-transfect ion with plasmids encoding beta-gal and either constitutively active m utants of PKC-delta, PKC-alpha, wild type PKC-delta, or empty vector, cardiocytes were subjected to 6 h of ischemia, Only PKC-delta, rendere d constitutively active by a limited deletion within the pseudosubstra te domain, consistently increased resistance to simulated ischemia (be ta-gal activity was 85.6 +/- 11.9% versus 53.7 +/- 6.5% (p less than o r equal to 0.01) and dead myocytes 46.8 +/- 3.4% versus 68.7 +/- 2.8% (p less than or equal to 0.01)). Since transfection was apparent in on ly 5-12% of cells, the results suggested a protective bystander effect that was confirmed by co-culture of transfected myocytes with untrans fected myocytes, In neonatal cardiocytes expression of active PKC-delt a increases resistance to simulated ischemia. This observation may pro vide further insight into the mechanism and possible avenues for thera peutic exploitation of preconditioning.