Ischemic preconditioning: A potential role for constitutive low molecular weight stress protein translocation and phosphorylation?

We have investigated whet-her translocation of constitutive low molecular w eight stress proteins (alpha B-crystallin and HSP27) to the myofilament/cyt oskeletal compartment occurs during ischemic preconditioning and assessed i f this is causally associated with cardioprotection. Triton-insoluble prepa rations from fresh or aerobically perfused rat hearts (n=4/group) contained relatively little alpha B-crystallin (96 +/- 43 and 43 +/- 36 units respec tively) or HSP27 (177 +/- 32 and 101 +/- 26 units respectively). Three prec onditioning cycles of (5 min ischemia + 5 min reperfusion) increased the Tr iton-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 pre condi tioning protocol resulted the return of alpha B-crystallin and HSP27 to nea r control levels (189 +/- 14 units and 252 +/- 24 units, respectively). Str ess 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 alpha B-cr ystallin to 628 +/- 30 units (P<0.05) and HSP27 to 1353 +/- 53 units. In pa rallel Functional studies, the recovery of LVDP after 35 min ischemia and 6 0 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 grou p, Thus, translocation of alpha B-crystallin and/or is not of-itself suffic ient to induce cardioprotection. Using a phosphospecific antibody, we have demonstrated that preconditioning not only translocates alpha B-crystallin but also increases its phosphorylation at Ser-59 by 9.7-fold compared to ae robic controls (1616 +/- 402 v 166 +/- 28 units respectively). In contrast. hypercarbia while eliciting a comparable translocation, failed to alter th e phosphorylation state of alpha B-crystallin. Preconditioning-induced phos phorylation was significantly attenuated by 50 mu M genistein (by 61%), 10 mu M SB203580 (by 91%) and 10 mu M bisindolylmaleimide (by 68%), but not by 10 mu M PD98059 (by 4%). Our findings are consistent with the possibility that ischemic preconditioning may be mediated by phosphorylation and transl ocation of constitutive low molecular weight stress proteins. particularly alpha B-crystallin. (C) 2000 Academic Press.