The goal of this grant is to provide critical information on the regulation of calcium handling and beta-adrenergic receptor signaling in a large mammalian model of severe left ventricular hypertrophy (LVH), that permits assessment of transmural differences in biochemical and molecular regulation as LVH develops. The first hypothesis to be tested is that the alterations in Ca2+ handling is an important component of altered beta-adrenergic contractile function and this defect precedes beta-adrenergic receptor desensitization, one of the hallmarks of heart failure. Specifically, our hypothesis is that the defect in Ca2+ handling is at the level of the sarcoplasmic reticulum (SR) involving the ryanodine receptor, as opposed to a major impairment in sarcolemmal Ca2+ influx. This will be studied transmurally in a large mammalian model, dogs, with pressure overload hypertrophy induced by chronic aortic banding. The second hypothesis is that in severe pressure overload LVH there is a change in myocardial Ca2+ spark properties, not simply a reduction in spark number, further supporting the concept indicating an alteration of the SR release apparatus in severe LVH. This will be studied by examining the properties of Ica-evoked Ca2+ sparks in relation to whole cell Ca2+ transients in the cardiac myocyte. The third hypothesis to be tested is that there are fundamental changes in the basic structure and molecular organization of the SR membranes in LVH. This will be accomplished using small-angle x-ray scattering approaches which will be used with tissue homogenates to directly examine the structure of SR from control and LVH samples. The fourth hypothesis to be tested is that changes in SR lipid bilayer structure associated with cardiac hypertrophy can be attributed to alterations in lipid composition and increased levels of oxy-radical damage. This will be studied by examining the biochemical composition (phospholipid headgroups, acyl chains) of SR samples as a function of disease progression and correlated with structure analyses. The extent of SR lipid and protein peroxidation will also be measured as part of this objective. In summary, this application involves examination of altered beta- adrenergic signalling and Ca2+ regulation at the cellular level in a unique large mammalian model of severe LVH, potentially more clinically relevant than murine models with more abrupt induction of hemodynamic overload and LVH.
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