Evidence from in vitro and in vivo studies indicates that phospholamban is an important regulatory phosphoprotein in the mammalian heart. Dephosphorylated phospholamban is an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase and phosphorylation of phospholamban relieves in inhibitory effects. Phospholamban is phosphorylated at distinct amino acids by three different protein kinases, namely serine 10 by protein kinase C, serine 16 by cAMP-dependent protein kinase, and threonine 17 by Ca2+-calmodulin-dependent protein kinase. Phospholamban has also been shown to be phosphorylated in vivo by both cAMP-dependent and Ca2+- calmodulin-dependent protein kinases during isoproterenol stimulation of intact hearts. Phosphorylation of phospholamban and the accompanied increases in the Ca2+-uptake rates by the SR have been postulated to the responsible for the increases in the rate of myocardial relaxation during beta-adrenergic stimulation of the heart. However, the exact role of phospholamban in the regulation of contractility and whether phosphorylation of this protein alone is directly responsible for and sufficient to cause the alterations in myocardial relaxation observed during beta-adrenergic stimulation is not presently known. Thus, our goal is to design animal models, which will elucidate the role of phospholamban in the regulation of SR function, myocardial contractility and the responses of the heart to beta-agonists. Specifically, we propose to target the phospholamban gene locus in murine embryonic stem cells. The targeting will be directed towards ablation of the protein or introduction of site specific mutations in the coding region. Unlike other transgenic systems, the chromosomal context will be maintained and other systems will be used to generate mice, which will be analyzed at the molecular genetic, biochemical and physiological levels. Ablation of phospholamban will allow definition of the basic function of this protein in the contractile performance of control hearts, their responses to beta-agonists and the role of phospholamban in heart disease, where evidence indicates that its mRNA expression levels are significantly reduced. Generation of phospholamban mutants will provide information on structure/function relationships in phospholamban in the intact animal. This genetic approach carried out via gene targeting technology will provide fundamental information on the basic function of phospholamban and the physiologic and pathophysiologic significance of alterations in its content or its primary structure.
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