PROJECT SUMMARY/ABSTRACT The goal of this two aim project is to explore dynamic mechanisms that regulate the cardiac calcium transporter, SERCA. SERCA plays a central role in the cardiac cycle; therefore, its regulation is critical for both survival and adapting to changing physiological demands. This regulation is primarily mediated by two transmembrane micropeptides: the inhibitory peptide, Phospholamban (PLB), and the stimulatory peptide, dwarf open reading frame (DWORF). These regulators compete to bind SERCA and control its function. We have found that intracellular Ca2+ elevations that drive contractions in cardiac muscle also cause dynamic shifts the binding equilibria of SERCA with PLB and DWORF. Specifically, Ca2+ elevations simultaneously lower the affinity of SERCA for PLB and increase SERCA affinity for DWORF. This is expected to lower inhibition and increase stimulation of SERCA during the peak of Ca2+ transients. Additionally, our preliminary results revealed that a dynamic fraction of PLB monomers that unbind from SERCA during Ca2+ elevations are dynamically sequestered in PLB pentamers. Slow unbinding of PLB pentamers causes PLB to accumulate in pentamers during rapid cardiac pacing, sequestering PLB away from SERCA to lower inhibition at exercising heart rates. Aim 1 will explore how this frequency-dependent accumulation of the PLB pentamer may mediate a critical role for PLB in the Bowditch effect, a positive force-frequency relationship in which a faster heart rate causes more forceful contractions of the heart. This phenomenon is a critical mechanism that adjusts cardiac output for exercise and, importantly, it is lacking in heart failure. Thus, experiments will examine how this novel mechanism is altered physiologically during the heart’s response to adrenaline and pathologically by PLB mutations linked to heart failure. These insights may reveal why patients with these mutations are more susceptible to arrythmias/heart failure. Additionally, our preliminary data revealed that Ca2+-dependent changes in PLB and DWORF affinity occur because these regulators prefer to bind different intermediate conformations of the SERCA enzymatic cycle. Aim 2 will investigate how distinct changes in the energetics of SERCA-micropeptide binding during the SERCA enzymatic cycle may underly the distinct inhibitory and stimulatory effects of PLB and DWORF, respectively. Specifically, experiments will explore how tight binding of PLB to the ATP- bound state of SERCA deters Ca2+ binding to mediate PLB inhibition. On the other hand, DWORF prefers to bind to states of SERCA that predominate when the pump is cycling due to rate-limiting steps. We will determine if DWORF stabilizes high energy intermediate states of the enzymatic cycle to lower an energy barrier and increase SERCA enzyme turnover. The proposed experiments will shift classic paradigms of SERCA-micropeptide regulation and inform the development of small molecules to treat heart failure.