The objective is to create animal models that are relevant to human cardiovascular disease. The immediate goals are to explore the role of phospholamban (PLB), the regulator of the Ca2+-pump in cardiac sarcoplasmic reticulum (SR), as a determinant of myocardial contractility in the rabbit heart. Dephosphorylated PLB inhibits the SR Ca2+-pump and phosphorylation of PLB relieves this inhibition. We hypothesize that altering PLB levels or its phosphorylation status will alter the SR Ca2+-ATPase activity (SERCA2), leading to changes in overall contractility and the heart's responses to (beta-adrenergic stimulation. Although this question has been studied in the mouse, the mouse heart is fundamentally different from the human heart in terms of its contractile cycle, basic motor proteins and handling of Ca 2+ flux.
In SPECIFIC AIM 1, the functional role of PLB/SERCA2 will be determined in vivo by cardiac over-expression of wild type PLB in transgenic (TG) rabbits or a dominant/negative mutant PLB in transgenic (TG) rabbits. We hypothesize that regulation of the relative PLB and SERCA2 levels is critical for maintaining proper cardiac function: increases in the PLB/SERCA2 levels will result in increased inhibition of the SERCA2 Ca2+ affinity and contractile parameters while decreases in the PLB inhibitory effects will have the opposite effect.
In SPECIFIC AIM 2, the functional significance of PLB phosphorylation will be examined in vivo by creating TG rabbits with increased levels of either non-phosphorylatable or """"""""chronically phosphorylated"""""""" cardiac PLB. Residues Ser 16 and Thr 17 are rapidly phosphorylated during beta-adrenergic stimulation; phosphorylation is associated with increases in the affinity of the SR Ca 2+pump, cardiac contractile parameters, and myocyte Ca 2+ kinetics. The hypothesis is that PLB phosphorylation is an important modulator of cardiac contractility, and that a) over-expression of a non-phosphorylatable PLB will mimic """"""""chronic inhibition"""""""" of a fraction of the SR Ca2+-pumps, with pathology developing over the animal's lifespan while; b) a """"""""phosphorylated"""""""" PLB mutant will result in reduced SERCA inhibition and conservation of cardiac function under stressed conditions.
In SPECIFIC AIM 3, the effects of the modified PLB complement on the time course of the heart's response to pacing-induced and pressure overload heart failure models will be studied. We hypothesize that PLB/SERCA ratios and relative phosphorylation status of PLB will affect the rate of progression, and overall severity of developing heart failure. Outcomes will be measured at the molecular, biochemical, cellular, whole organ and whole animal levels.
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