Browsing by Author "Stroik, Daniel"

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    A Study of the SERCA-Phospholamban Regulatory Interaction Using Time-Resolved Fluorescence
    (2018-08) Stroik, Daniel
    Cardiac muscle contraction and relaxation is controlled by changes in intracellular Ca, indicating that Ca transport is a fundamental regulator of proper muscle function in the heart. The primary cardiac Ca transporter is the sarcoendoplasmic reticulum Ca-ATPase 2a (SERCA2a), a transmembrane protein pump embedded in the sarcoplasmic reticulum (SR). SERCA2a translocates two Ca ions from the cytosol into the SR at the cost of one ATP molecule, effectively lowering the cytosolic Ca concentration and inducing cardiomyocyte relaxation. Ca transport is regulated by a second transmembrane protein, phospholamban (PLB), which binds to SERCA2a and inhibits its activity by reducing the Ca affinity of the Ca pump. The significance of PLB-dependent regulation in muscle function is highlighted by the existence of hereditary mutations in PLB linked to cardiomyopathy. Further, the membrane protein complex between SERCA2a and PLB is a validated therapeutic target for reversing cardiac contractile dysfunction in more common diseases (e.g., heart failure) caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. The work presented in this thesis focuses on the structure and function of the SERCA2a-PLB complex and its connection to several chronic cardiac diseases. In the first study (Chapter 4), we developed a structure-based high-throughput screening (HTS) method to discover compounds that disrupt the SERCA2a-PLB interaction. We identified ten compounds that reproducibly alter SERCA2a-PLB structure and function, including one compound that increases SERCA2a calcium affinity in cardiac SR membranes but not in skeletal. These results suggest that the compound is acting specifically on SERCA2a-PLB, as needed for a drug to mitigate deficient Ca transport in heart failure. In the second study (Chapter 5), we studied the effects of the disease-causing R9C-PLB mutation in a human induced pluripotent stem cell (hiPSC) line to understand the mechanistic details of hereditary cardiomyopathy progression. In addition to blunted lusitropic response to β-adrenergic stimulation, we found that the R9C mutation results in an altered metabolic state and significant gene expression changes. Ongoing studies (Chapter 6) are presented, focusing on recent work to develop PLB-based gene therapy constructs and elucidate the structural mechanism by which a third transmembrane protein, dwarf opening reading frame (DWORF), regulates Ca transport in the heart.