In cardiac excitation-contraction coupling, the sarcoplasmic reticulum (SR) plays an essential role in the regulation of the cytosolic free Ca2+ concentration. There are three major functions of the SR: a) Ca2+ uptake from the cytosol into the SR lumen resulting in muscle relaxation; b) Ca2+ storage in the SR lumen; and c) Ca2+-release from the SR into the cytosol resulting in muscle contraction. The main SR proteins responsible for these functions are: the Ca2+-transport ATPase (SERCA2) with its regulator phospholamban (PLN); the Ca2+ storage protein calsequestrin and a histidine rich Ca2+-binding protein (HRC); and the Ca2+ release ensemble composed of the ryanodine receptor, junctin and triadin. Our overall hypothesis is that regulation of SR Ca2+-cycling plays a pivotal role in cardiac contractility and SR Ca2+-defects are crucial in heart failure progression. PLN represents a fundamental """"""""brake"""""""" in SR function and inhibition of PLN to normalize the depressed cardiomyocyte Ca2+ homeostasis has been suggested to be of therapeutic benefit. To further examine the validity and efficacy of PLN inhibition in restoring cardiac function and rescuing hypertrophy, we propose to generate a model, which will allow precise manipulation of PLN levels in a temporally regulated manner. This approach will allow assessment of the benefit of PLN inhibition in the settings of hypertrophy or heart failure, induced by pressure overload or genetic manipulation, and in ischemia/reperfusion injury. We also propose to elucidate the functional significance of the N27K-PLN mutant in vivo, since PLN is highly conserved across species with the only substitution of Lys for Asn at position 27 in human. Our preliminary findings indicate that K27-PLN is a super-inhibitor of cardiac function, compared to N27-PLN. Furthermore, since alterations in the levels or activity of PLN reflect alterations in SR Ca2+ load and release, we propose to elucidate the functional role of HRC, a newly discovered Ca2+ binding protein in SR. Animal models with alterations in the expression levels of this protein (overexpression and knockout) will be generated and their cardiac phenotypes will be analyzed at the subcellular, cellular, organ and intact animal levels. These studies will help reveal the pathophysiological role of HRC in vivo. Overall, our proposed studies will: a) advance our knowledge on the mechanisms underlying regulation of Ca2+ homeostasis by the SR function; and b) provide valuable insights into the crosstalk between the SR Ca2+ handling proteins and their regulatory effects on cardiac contractility in health and disease. ? ? ? ? ?
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