This is an application to study agents which may be protective against myocardial ischemic injury. The applicants have found a novel compound, fostriecin, which has been purified from the fermentation broth of Streptomyces pulveraceus, and which has a pronounced cardioprotective effect in ischemic rabbit hearts. At concentrations that have no apparent toxicity, fostriecin reduces the size of a myocardial infarct induced by a controlled coronary occlusion from approximately 33% to approximately 8% of the risk zone. On the molecular level, fostriecin is a potent inhibitor of type 2A, a strong inhibitor of type 5, and a weak inhibitor of type 1 serine/threonine protein phosphatases, suggesting that the cardioprotective properties of fostriecin arise from its inhibitory activity against these or related phosphatases. The goal of the proposal is to test the hypothesis that the inhibition of certain serine/threonine protein phosphatases is the biochemical basis for the cardioprotective properties of fostriecin observed in ischemic heart tissue.
The aims are 1) to characterize fostriecin-sensitive protein phosphatases in heart; 2) to characterize the high affinity fostriecin binding domain of PP2A and PP5; and 3) to determine the role of fostriecin sensitive protein phosphatases in ischemic cardiac tissue. Through a better understanding of the molecular events by which the inhibition of these enzymes limits myocardial tissue damage, insight into the mechanisms that produce protection will be gained. Such insight should aid in the development of new and improved methods for the medical management of an acute MI. 1) Significance: The proposal is novel. PKC activation is thought to play an important role in ischemic preconditioning, and it is logical to investigate the role of protein phosphatases in the protective effect of ischemic preconditioning since inhibition of protein phosphatases could modulate or amplify the effects to PKC stimulation. Previous work to understand the importance of protein phosphatases has been hampered by the lack of selective inhibitors; non-selective inhibition of protein phosphatases could have offsetting effects which would make it very difficult to draw definitive conclusions. For example, early work by Ganote and co-workers showed that the protein phosphatase inhibitor calyculin A had conflicting results on ischemic injury. It accelerated the onset of contracture but slowed the development of lethal injury. Calyculin is a non-selective protein phosphatase inhibitor and the results could reflect opposing effects of sustained phosphorylation of different proteins. The proposed approach to develop more selective inhibitors will help to resolve this issue. 2) Approach: The first specific aim is to characterize the fostriecin-sensitive PPs in heart. This involves purification followed by dose-inhibition studies with fostriecin. The applicant published a 1998 paper in JBC reporting the cloning, expression, and characterization of PP7 in human retina, and he has provided more detail of the proposed PP purification methods in the revised application. The second specific aim is to characterize the fostriecin binding domains of the relevant PPs. Preliminary data are presented on the generation of PP1/PP2A chimeras to identify PP inhibitor binding sites, and the data suggest that okadaic acid and fostriecin bind to different sites.
This specific aim has been modified to include a collaboration to generate crystal structures of PP/fostriecin complexes. The third specific aim is to characterize the role of PPs in ischemic preconditioning. The PI and his collaborators have already looked to see if PP1 or PP2A activity is affected by ischemic preconditioning and has found that there was no difference between non-preconditioned and preconditioned myocardium at any time during a subsequent sustained period of ischemia, and therefore, he proposes to pursue the hypothesis that it could be PP5 activity which is decreased by preconditioning. This is a reasonable hypothesis that would explain the similarity between the protective effect of ischemic preconditioning and fostriecin. An alternative hypothesis to explain the similarity between the effects of preconditioning and fostriecin is that preconditioning could stimulate PKC and achieve a relatively high level of phosphorylation of a PKC substrate whereas fostriecin treatment results in a comparably high level of phosphorylation of the same substrate by slowing the dephosphorylation. The latter hypothesis will be tested by observing whether the protective effect of fostriecin is abolished in the presence of a PKC inhibitor. Likewise, it is possible that one of the stress-activated protein kinase pathways could be involved in phosphorylation of a substrate that could be dephosphorylated by a fostriecin-sensitive PP; this possibility will be tested by pretreating with various MAP kinase inhibitors. Finally, they will explore the possibility that HSP27 phosphorylation is involved in the protective effect of ischemic preconditioning and fostriecin by measuring the effect of fostriecin on HSP27 phosphorylation A minor weakness is that the proposed method for assessing changes in PP5 activity may not be reliable. Measurement of PP5 activity in the myocardium will be performed using a subtraction procedure, and if PP1 and PP2A activities are much higher than PP5 activity, it may be difficult to detect changes in PP5 activity by subtracting PP1 and PP2A activity from the total PP activity. 3) Innovation: The possibility that PPs are involved in ischemic hypothesis that is difficult to test because of the lack of specific PP inhibitors. Most of the currently available PP inhibitors are toxic and inhibit multiple PPs. Better understanding of the interaction between various inhibitors and different PPs may aid in the development of better inhibitors that are selective for only one class of PP. A recently developed PP inhibitor has been found to be remarkably protective in a model of ischemia/reperfusion injury. Further study of the effects of this PP inhibitor may lead to greater insight concerning downstream effectors of preconditioning.
