This is a proposal to study mechanisms of ischemic preconditioning using isolated cardiac myocytes from rabbits, as an example of an animal with a fast heart rate, and from pigs, as an example of an animal with a slow heart rate. Preliminary data show 1) that rabbits precondition by mechanisms independent of K-ATP channels and ATP conservation, while both of these factors are required for protection of pig cardiomyocytes. 2) Ca+2-free ischemia protects in the pig but not in the rabbit. 3) Fostriecin, a water soluble, non-toxic protein phosphatase 2A-selective inhibitor, mimics ischemic preconditioning in both species, but protection can be blocked by K-ATP channel inhibitors in pig but not rabbit. Fostriecin is prototypic of ischemia-selective protein phosphatase inhibitors that could protect by either a) maintaining the phosphorylated state of K-ATP channels which would keep these channels open at higher ATP concentration or b) maintaining the phosphorylated state of cytoskeletal proteins during ischemia. 4) Protein phosphatase inhibitors protect rabbit cells even when added during ischemia, after the onset of rigor. This type of protection which occurs after the myocytes are already severely ATP depleted is new and of potential clinical importance. The basic hypothesis to be tested is that the fundamental protective effect of preconditioning is due to better maintenance of phosphorylation of critical proteins, and ultimately to phosphorylation dependent cytoskeletal integrity. In the rabbit, the hypothesis is that protection is due to a direct effect on cytoskeletal proteins whereas in the pig, the protective effect is an energy sparing effect which indirectly preserves cytoskeletal phosphorylation. The first Specific Aim is to compare the effects of protein phosphatase inhibitors and preconditioning in rabbit and pig cardiac myocytes. The applicant presents preliminary data that preconditioning does not affect protein phosphatase activity per se but if the endpoint of preconditioning is increased phosphorylation of critical proteins, then there should be considerable similarity between the two interventions, and where differences appear, they should be related to differences in the ultimate level of specific protein phosphorylation. The second specific aim is to compare the response of rabbit and pig myocytes to ATP-sparing interventions. The rationale is that ATP seems to be irrelevant in the rabbit myocytes whereas it seems to be very important in the pig myocytes. In the rabbit myocytes, a similar protective effect is obtained with preconditioning and with addition of protein phosphatase inhibitors after 75 minutes of ischemia, at which time the ATP is extremely low. In the pig myocytes, the hypothesis is that preconditioning is protective because of phosphorylation of the K-ATP channel which has an ATP-sparing effect, and that ultimately, the pig myocytes differ from rabbit in that ATP is necessary to maintain cytoskeletal protein phosphorylation. The proposal culminates with the third specific aim which is to show how specific cytoskeletal protein phosphorylation relates to lethal injury. Ultimately, if the hypothesis is correct, protection should be related to enhanced phosphorylation of specific proteins, regardless of whether the intervention is preconditioning or addition of a phosphatase inhibitor, and since he will be using phosphatase inhibitors with different specificities, some of which are protective and some of which are not, he has the opportunity to separate which protein phosphorylation is most important.
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