Structure-Based Design of Phospholamban Mutants for Treatment of Heart Failure

Ca2+ cycling through the SR in muscle cells is largely controlled by the Ca-pump (SERCA). SERCA transports Ca2+ into the SR, allowing muscles to relax, and is inhibited by phospholamban (PLB) at submicromolar [Ca2+]. PLB inhibition can be relieved by adrenergic stimulation, leading to PLB phosphorylation. Heart Failure (HF), which contributes to 12% of US deaths, can be caused by a variety of genetic or environmental factors, but a common symptom is 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 and cells are co-transfected with WT-PLB and/or PLBM to measure SERCA inhibition in living cells. Effects of PLB/PLBM on SERCA specific activity (s−1), as well as PLB oligomeric states are characterized in stable cells lines. We can also measure a mutant's ability to compete with WT-PLB 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. Preliminary results have led to rAAV-mediated expression of a PLBM (P21G) in cardiomyocytes of a rat HF model, increasing contractility to near WT levels.