This application is a natural extension of the parent grant (HL26057), with the specific aims of producing a phospholamban (PLB) knockout mouse. The phospholamban knockout mouse has been generated and is being characterized. The availability of this model offers a unique opportunity to address the role of phospholamban in the altered contractile responses of the heart in both hetero- and homozygous mice. . Compensatory mechanisms in the PLB knockout mouse will also be defined and their functional significance determined. Additional structure-function studies of PLB, such as point mutations at critical amino acids, in vivo are planned. The hypothesis that will be tested in this FIRCA application is: altered basal left ventricular function and responses to b-adrenergic stimulation of hypo- and hyperthyroid mammalian hearts may be mediated by alterations in the protein expression levels of PLB. The studies proposed will elucidate the specific contribution of PLB in hyper-and hypothyroid cardiac contractility. PLB is located in the sarcoplasmic reticulum (SR), the major Ca2+-sequestering membrane system. Myocardial relaxation is determined by the rate of Ca2+ transport from the cytosol into the SR, and is regulated by PLB. Non-phosphorylated PLB inhibits the SR Ca2+ pump and phosphorylation of PLB relieves the inhibitory effects. The role of PLB in the altered contractile parameters observed in various thyroid states will be studied. Specifically the investigators propose to: a) characterize the effects of altered thyroid state on SR Ca2+ uptake; b) determine alterations in mRNA and protein levels of key Ca2+-cycling proteins under various thyroid conditions; c) determine the basal myocardial contractile parameters in hypothyroid, euthyroid and hyperthyroid mouse hearts and correlate functional changes with alterations in the mRNA or protein levels of the Ca2+-cycling proteins; d) examine the effects of b-adrenergic agonists on the contractile parameters and the phosphorylation pattern of key cardiac regulatory proteins. Correlate the changes observed in myocardial contractility with alterations in the degree of phosphorylation of specific phosphoproteins. The proposed study will provide knowledge of the role of PLB and its phosphorylation on the altered SR function under various thyroid conditions. This may offer new insights into designing pharmacological interventions for this relatively common disease in humans.
