Spectroscopic Design of Phospholamban Mutants to Treat Heart Failure

Ca2+ cycling through the SR in muscle cells is largely controlled by the Ca-pump (SERCA). SERCA transports Ca2+ into the SR and is inhibited by phospholamban (PLB) at submicromolar [Ca2+], and this inhibition can be relieved by adrenergic stimulation. Contraction takes place when the Ca-release channel opens and the intracellular [Ca2+] is high. One of the most common symptoms of heart failure (HF) is impaired calcium handling, frequently resulting from decreased SERCA activity. We are using EPR and NMR to study the relationships among structure, dynamics, and function of PLB, with the goal of designing LOF-PLB mutants (PLBM) that can compete with WT-PLB and thus relieve SERCA inhibition. Several studies have shown that a pseudophosphorylated PLB (S16E-PLB) is effective for gene therapy in rodents and sheep, and we are using spectroscopic methods to refine this approach. We have developed a system for examining the function and interactions of SERCA and PLB in HEK cells. Active SERCA is expressed at a level high above basal ATPase activity, and cells are co-transfected with WT-PLB and/or PLBM to measure SERCA inhibition in living cells. Unlike S16E-PLB, these mutants are able to respond to adrenergic stimulation. In addition to quantifying SERCA activity in the presence of PLBM, we measure the ability of each mutant to compete with WT-PLB for binding to SERCA. This is done by measuring fluorescence resonance energy transfer (FRET) between labeled SERCA and WT-PLB. If PLBM displaces WT-PLB, less energy is transferred between fluorophores and a decrease in FRET is observed. Based on these results, rAAV is used to test PLBM in rodent and porcine models of HF for efficacy in vivo and ability to respond to adrenergic stimulation, with the goal of developing a novel, gene therapy based treatment for HF.