HORMONE REGULATION OF CARDIAC INJURY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The central hypothesis of this proposal is that HDL-associated sex hormones (estrogen and testosterone) regulate the activity of cardiac myocyte constitutive nitric oxide synthase which subsequently affects the extent of ischemic injury. Epidemiological studies demonstrate that women are less prone to ischemic heart disease than men. The mechanisms responsible the differences in ischemic injury observed between men and women are not know although the current focus is on sex hormones, in particular estrogen and testosterone. A large number of studies have implicated estrogen-induced stimulation of constitutive nitric oxide synthase (cNOS) as providing protection against ischemic injury of cardiac myocytes. The role of testosterone is unclear with reports of protection against ischemic injury and reports of testosterone promoting ischemic injury. The majority of the studies on estrogen and testosterone do not distinguish between effects mediated by the endothelium in hearts and cardiac myocytes. One of the novel aspects of the proposed studies is that the preliminary data indicate that estrogen and testosterone have direct effects on cardiac myocytes, independent of the endothelium. Another novel aspect of the proposed studies is that it is estrogen or testosterone associated with HDL that is responsible for the generation of nitric oxide in cardiac myocytes and protection/promotion of ischemic injury. The molecular mechanism whereby estrogen limits ischemic injury and testosterone increases ischemic injury will be tested in two Aims. Aim 1: To determine the mechanism whereby HDL-associated estrogen stimulates the production of nitric oxide and limits ischemic injury. The preliminary studies suggest that estrogen receptor alpha and caveolin-1 form a complex that stimulates cNOS via AMP kinase. Cardiac myocytes isolated from wild type mice, caveolin-1 null mice, or estrogen receptor alpha null mice will be used along with dominant negative adenoviral constructs, agonist/antagonists, and enzyme assays to dissect the mechanism(s). In addition, wild type mice, SR-BI null mice, caveolin-1 null mice, and estrogen receptor alpha null mice along with ovariectomies and ovariectomies/estrogen replacement will be used to determine the effect on ischemic injury. Aim 2: To determine the mechanism whereby HDL-associated testosterone decreases the generation of nitric oxide and increases ischemic injury. The preliminary studies demonstrate that HDL-associated testosterone decreases the generation of nitric oxide without affecting the cellular levels of cNOS. Isolated cardiac myocytes will be used to examine several possible mechanisms for the HDL-testosterone mediated inhibition of cNOS such as, altered caveolin binding to cNOS, depletion of caveolae cholesterol, re-localization of cNOS, and modification of cNOS and/or caveolin-1. Additional studies will determine if the androgen receptor and/or testosterone uptake are involved in this process. Wild type mice, SR-BI null mice, and caveolin-1 null mice along with orchidectomies and orchidectomies/testosterone replacement will be used to determine the effect on ischemic injury.