HSP72, the inducible form of HSP70, is known to protect cells against a variety of injuries, but the exact mechanisms underlying this protection are only poorly defined. To further investigate the protective effects of HSP72, multiple clones expressing HSP72 wild type (WT) and two mutants with defective nucleolar and nuclear localization, respectively (M45 and 985A) were made using the tet-off system in C2C12 cells ( a muscle cell line). Four different parameters of cell function/injury were examined after simulated ischemia: protein synthesis, polysome formation, DNA synthesis and LDH release. Overexpression of WT HSP72 was also compared to nontransfected C2C12 cells. As expected, overexpression of HSP72 protected against simulated ischemia and reoxygenation for all four parameters. In contrast, both M45 and 985A clones showed abnormal protein synthesis and polysome formation not only after simulated ischemia, but also, under normal, control culture conditions. Total RNA, which reflects rRNA, was slightly reduced in M45 and 985A at baseline, but 1 h after hypoxia, RNA levels were protected in all clones, but significantly decreased in nontransfected C2C12 cells. Clones expressing the mutant 985A (no nuclear localization) had nuclear retention of mRNA, suggesting that HSP72 is needed for nuclear export of RNA. All clones, both WT and mutant had protection of DNA synthesis (BrDu incorporation) compared to C2C12 cells, but 985A had much greater release of LDH following injury than any of the other groups. These results support a multi-factoral protective effect of HSP72, some dependent on nuclear localization with stress and some not. The protection of protein synthesis and polysome formation, and abnormalities in these with the mutants, support a role for HSP72 in these processes both in the normal cell as well as with injury.