The reason why some cardiomyocytes, when responding to stress, will undergo apoptosis while others survive and undergo hypertrophy has yet to be elucidated. We hypothesize that doxorubicin, a cancer chemotherapeutic agent, will produce oxidants that will stimulate increased expression of Hic-5/ARA55, which will then bind to Hsp27 preventing its anti-apoptotic effect and cells will die. After the treatment is removed, surviving cells will hypertrophy. Because doxorubicin's use is limited by its cardiotoxicity, elucidation of its mechanisms can help in the development of therapeutics to prevent this effect. To test this hypothesis, we will establish a dose-response curve using the MTT assay and then measure oxidant production with the fluorescent dye, 2',7'-dichlorofluorescein diacetate. We will evaluate changes in Hic-5/ARA 55 expression of Hsp27 expression and phosphorylation by Western blot and IEF analysis. Probucol, an antioxidant, will be used to determine if the toxicity can be ameliorated, helping to confirm the oxidative stress hypothesis. We will also truncate the Hic-5's/m-4 domain region where Hsp27 binds to determine if the protein's apoptotic is independent of Hsp27. Results of these studies are anticipated to show that toxicant induced increase in Hic-5 expression will prevent Hsp27's anti-apoptotic effect leading to cell death and surviving cells with hypertrophy.
