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.