Fluorescence tools to identify Novel SERCA activators

University of Minnesota Ph.D. dissertation. August 2013. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: David D. Thomas. 1 computer file (PDF); x, 121 pages.One of the universal hallmarks of heart failure is defective calcium cycling. The calcium concentration in a muscle cell must be high to cause contraction and low to allow relaxation, and most of the calcium removal is accomplished by the intracellular membrane pump known as the sarco-endoplasmic reticulum calcium ATPase (SERCA). When SERCA activity is too low in cardiac muscle, the heart does not fully relax and fill with blood, so the next contraction cannot pump enough blood through the body. The ubiquity of calcium cycling dysfunction in heart failure and other muscle diseases has made SERCA a major target for novel heart failure therapeutics since the late 1990s. All of the work presented in this thesis focuses on methods to activate SERCA as a treatment for heart failure. SERCA is regulated by phospholamban (PLB) in heart muscle, preventing the enzyme from being fully active all the time but allowing maximal activity when the body demands. Some methods of activating SERCA seek to remove the inhibitory effects of PLB, either partially or fully. In this thesis, PLB mutants are investigated as potential gene therapy vectors. PLB mutants that are less inhibitory but still bind to SERCA could allow the enzyme to be more active if they displace endogenous PLB. A FRET assay using genetically engineered fluorescent fusions of SERCA and PLB expressed stably in a human cell line was used to measure the ability of different mutants to compete for SERCA binding. Fluorescently labeled SERCA and PLB were also reconstituted in an in vitro lipid bilayer system to screen for small-molecule compounds that activate SERCA. Several compounds were found to decrease SERCA-PLB FRET and many of these turned out to be SERCA activators that improved myocyte contractility. However, none of the compounds were specific to the SERCA-PLB interaction. Finally, an intramolecular FRET assay was developed to detect changes in the relative distance between cytoplasmic domains within SERCA in living cells. This assay was used to screen a small-scale compound library to show that FRET between SERCA domains is sensitive to both activators and inhibitors of SERCA function. All of these FRET assays are being followed up in the Thomas lab to identify potential SERCA activators for heart failure and other diseases