Ischemic Preconditioning: a Potential Role for Constitutive Low Molecular Weight Stress Protein Translocation and Phosphorylation?

P. Eaton, W. I. Awad, J. I. A. Miller, D. J. Hearse and M. J. Shattock. Ischemic Preconditioning: a Potential Role for Constitutive Low Molecular Weight Stress Protein Translocation and Phosphorylation?Journal of Molecular and Cellular Cardiology (2000) 32, 961–971. We have investigated whether translocation of constitutive low molecular weight stress proteins (α B-crystallin and HSP27) to the myofilament/cytoskeletal compartment occurs during ischemic preconditioning and assessed if this is causally associated with cardioprotection. Triton-insoluble preparations from fresh or aerobically perfused rat hearts (n=4/group) contained relatively little α B-crystallin (96±43 and 43±36 units respectively) or HSP27 (177±32 and 101±26 units respectively). Three preconditioning cycles of (5 min ischemia+5 min reperfusion) increased the Triton-insoluble crystallin to 864±61 units (P<0.05) and HSP27 to 1353±53 units (P<0.05). Two hours of aerobic perfusion following the preconditioning protocol resulted the return of α B-crystallin and HSP27 to near control levels (189±14 units and 252±24 units, respectively). Stress protein translocation, comparable to that achieved by the IPC protocol was induced by aerobic perfusion with hypercarbic (pH 6.8) perfusion. Thus, three cycles of 5 min hypercarbia+5 min normocarbia increased α B-crystallin to 628 ± 30 units (P<0.05) and HSP27 to 1353±53 units. In parallel functional studies, the recovery of LVDP after 35 min ischemia and 60 min of reperfusion was 43±7% in the ischemic control group, 61±3% (P<0.05) in the preconditioned group and 42±6% in the hypercarbic group. Thus, translocation of α B-crystallin and/or is not of-itself sufficient to induce cardioprotection. Using a phospho-specific antibody, we have demonstrated that preconditioning not only translocates α B-crystallin but also increases its phosphorylation at Ser-59 by 9.7-fold compared to aerobic controls (1616±402 v 166±28 units respectively). In contrast, hypercarbia while eliciting a comparable translocation, failed to alter the phosphorylation state of α B-crystallin. Preconditioning-induced phosphorylation was significantly attenuated by 50 μ m genistein (by 61%), 10 μ m SB203580 (by 91%) and 10 μ m bisindolylmaleimide (by 68%), but not by 10 μ m PD98059 (by 4%). Our findings are consistent with the possibility that ischemic preconditioning may be mediated by phosphorylation and translocation of constitutive low molecular weight stress proteins, particularly α B-crystallin.