Effect Of HIF-1α Mediated HSP70on Thermotolerance Against Cardiomyocytes Apoptosis Induced By Hyperthemia

Objective：To observe HIF-1ɑ’ s influence on the expression of HSP70and its effect on thethermotolerance induced by nonfatal high temperature. To clarify whether theexpression of HSP70in thermotolerance depends on HIF-1ɑ, whether HIF-1ɑmediated HSP70participates in myocardial cells’ thermotolerance protection throughits resistance to apoptosis.Methods：(1) H9c2cells were randomly divided into12groups,(n=3each): normaltemperature Control group，Thermotolerance group，Thermotolerance/Hyperthermiagroup, Hyperthermia group; DMSO+normal temperature group,YC-1+Ther-motolerance group, YC-1+Thermotolerance/Hyperthermia group,YC-1; Lipo+Nor-mal temperature group, RNAi+Thermotolerance group, RNAi+Thermotoleran-ce/Hyperthermia group, RNAi+Hyperthermia group (2) H9c2myocardial cells werecultured to establish the thermotolerance (40℃,3h) and heat stress (43℃,2h)models.(3) Before heated for thermotolerance or heat stress, H9c2cells were treatedby YC-1, the specific inhibitor of HIF-1ɑ, to inhibit the expression of HIF-1ɑafter thetranscription level.(4) HSP70-siRNA was transferred into H9c2cells byLipofectamine2000, then these cells were heated for thermotolerance or heat stress.(4) The cell apoptotic rates were assessed by flow cytometry. Western blotting wasused to detect the expression of HIF-1α, HSP70and caspase3.Result:(1) Flow cytometry results showed that cell apoptosis rate of Thermoto-lerance/Hyperthermia group was significantly lower than Hyperthermia group(p<0.01). Cell apoptosis rate of YC-1+Thermotolerance/Hyperthermia group wassignificantly higher than Thermotolerance/Hyperthermia group (p<0.01), meanwhileYC-1+Hyperthermia group was significantly higher than Hyperthermia group(p<0.01). What’s more, there is no significant difference in apoptosis rate between YC-1+Thermotolerance/Hyperthermia group and YC-1+Hyperthermia group.(2)Western blotting analysis found that the thermotolerant treatment can significantlyimproved the HIF-1ɑ and HSP70expressions’ level (p<0.05), compared with thenormal temperature control group. In Thermotolerance/Hyperthermia cells, HIF-1ɑand HSP70expressions were both obviously increased (p<0.01), compared withHyperthermia cells. After treatment with YC-1, HIF-1ɑexpressions ofThermotolerance group (p<0.01), Thermotolerance/Hyperthermia group (p<0.01) andHyperthermia group (p<0.01) were decreased evidently. After suppressing the HIF-1ɑexpression, HSP70levels of Thermotolerance group (p<0.01), Thermotoleran-ce/Hyperthermia group (p<0.01) and Hyperthermia group (p<0.01) were alsosignificantly decreased. After suppressing the HSP70expression by HSP70-siRNA,HIF-1ɑ level of Thermotolerance/Hyperthermia group was still obviously higher thanHyperthermia group (p<0.01).(3) Caspase3expression of Thermotolerance/Hyperthermia group was significantly lower than Hyperthermia group. Aftersuppressing the HIF-1ɑ expression, caspase3levels of Thermotoler-ance/Hyperthermia group (p<0.01) and Hyperthermia group (p <0.01) weresignificantly higher, and there is no significantly difference between YC-1+Th-ermotolerance/Hyperthermia group and YC-1+Hyperthermia group.Conclusion：(1) HIF-1ɑ plays a protective role in the process of thermotolerance againstapoptosis induced by heat stress.(2) The expression of HSP70in thermotolerance andheat stress depends on HIF-1ɑ.(3) HIF-1ɑ mediated HSP70participates inmyocardial cells’ thermotolerance protection through its resistance to apoptosis.