verbatim): Reactive oxygen species (ROS) are increased in the myocardium coincident with the progression to left ventricular (LV) systolic failure. In cardiac myocytes in vitro, we have found that ROS can mimic the cellular events observed in myocardial remodeling, and that the hypertrophic and apoptotic effects of adrenergic stimulation and mechanical strain are ROS-dependent. We will use a combination of in vitro, in vivo and ex vivo methods to test our central hypothesis that ROS play a critical role in mediating the effects of excessive adrenergic stimulation and mechanical overload on myocardial phenotype. We will use adult rat ventricular myocytes in vitro 1) to elucidate the roles of the quantity and specific types of ROS (e.g., O2, H2O2 and OH) in mediating the effects of adrenergic stimulation and mechanical strain on myocyte phenotype, and 2) to determine the source of ROS in response to adrenergic stimulation and mechanical strain. Building on these in vitro experiments, we will test our central hypothesis in vivo using transgenic mice that exhibit an apoptotic phenotype due to myocardial overexpression of beta1AR or a hypertrophic phenotype due to overexpression of Galphaq, the primary G protein coupled to alpha1AR and mechanical strain. To test the role of ROS in mediating the myocardial phenotypes in these models, ROS levels will be decreased by cross-breeding with mice that express increased levels of the antioxidant enzymes manganese superoxide dismutase (MnSOD) or Cu/ZnSOD. The primary endpoints for these experiments will be fundamental measures of remodeling at the structural and functional level - LV chamber dilation and contractile function. Secondary endpoints will be important cellular events that have been implicated in myocardial remodeling - hypertrophy, fetal program expression and apoptosis in cardiac myocytes, and activation of metalloproteinases in cardiac fibroblasts. The interpretation of these experiments will be aided by measurement of ROS by electroparamagnetic resonance and cellular redox state by the ratio of reduced to oxidized glutathione. These studies offer to provide new mechanistic understanding of the process of myocardial remodeling.
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