Heat shock preconditioning reduces ischemic tissue necrosis by heat shock protein (HSP)-32-mediated improvement of the microcirculation rather than induction of ischemic tolerance

Introduction: Supraphysiologic stress induces a heat shock response, which may exert protection against ischemic necrosis. Herein we analyzed in vivo whether the induction of heat shock protein (HSP) 32 improves survival of chronically ischemic myocutaneous tissue. and whether this is based on amelioration of microvascular perfusion or induction of ischemic tolerance. Methods: The dorsal skin of mice was subjected to local heat preconditioning (n = 8) 24 hours before surgery. In additional heat-preconditioned animals (n = 8), HSP-32 was inhibited by tin-protoporphyrin-IX. Unconditioned animals served as controls (n = 8). A random-pattem myocutaneous flap was elevated in the back of the animals and fixed into a dorsal skinfold chamber. The microcirculation, edema formation, apoptotic cell death, and tissue necrosis were analyzed over a 10-day period using intravital fluorescence microscopy. Results: HSP-32 protein expression was observed only in heat-preconditioned but not in unconditioned flaps. Heat preconditioning induced arteriolar dilation, which was associated with a significant improvement of both arteriolar blood flow and capillary perfusion in the distal part of the flap. Further, heat shock reduced interstitial edema formation, attenuated apoptotic cell death, and almost completely abrogated the development of flap necrosis (4% +/- 1% versus controls: 53% +/- 5%; P[r] < 0.001). Most strikingly, inhibition of HSP-32 by tin-protoporphyrin-IX completely blunted the preconditioning-induced improvement of microcirculation and resulted in manifestation of 72% +/- 4% necrosis. Conclusion: Local heat preconditioning of myocutaneous tissue markedly increases flap survival by maintaining adequate nutritive perfusion rather than inducing ischemic tolerance. The protection is caused by the increased arteriolar blood flow due to significant arteriolar dilation, which is mediated through the carbon monoxide-associated vasoactive properties of HSP-32.