Abstract 14854: HIF-1α/HSP70 Interaction is a Critical Determinant of Stem Cell Survival During Ischemic Preconditioning
Background: The cellular and sub-cellular strategies of preconditioning significantly improve stem cell survival under ischemic stress. We have already reported that ischemically preconditioned stem cells showed improved survival post transplantation in an infarcted heart. While elucidating the underlying mechanism using skeletal myoblasts (SM) as cellular model, we report activation of Akt/HIF-1α signaling in preconditioned SM (PCSM) as a critical determinant of their survival upon subsequent exposure to lethal anoxia.
Methods and Results: SM were preconditioned using our optimized protocol of two 30 minute cycles of intermittent anoxia/re-oxygenation treatment followed by exposure to lethal anoxia for 8 hours. PCSM showed significantly higher survival as compared to non-preconditioned SM (non-PCSM) as determined by LDH release assay, TUNEL and Annexin-V staining/flow cytometry. Preconditioning also preserved mitochondrial membrane potential and reduced caspase-3 activity. HIF-1α and HSP70 which were significantly upregulated in PCSM, were abrogated upon pretreatment with HIF-1α specific siRNA and 50µM YC1 (HIF-1 inhibitor) with concomitant reduction in cell survival under lethal anoxia. Computational studies showed the presence of putative hypoxic response element (HRE) consensus sequence specific for HIF-1α binding located at 674-667bp in HSP70 promoter region. While elucidating the mechanism of cell survival, we observed HIF-1α dependent transcriptional regulation of HSP70 by direct and specific binding of HIF-1α to HRE in the promoter region of HSP70 as revealed by HIF-1α binding and chromatin immunoprecipitation assays. Loss-of-function studies showed that abrogation of HSP70 with specific siRNA and 100µM quercetin, an HSP70 specific blocker, significantly reduced SM survival under lethal anoxia.
Conclusion. PCSM showed improved survival under lethal anoxia through activation of HIF-1α during preconditioning via its interaction with HRE1 in the HSP70-2 promoter.
- © 2012 by American Heart Association, Inc.