Presenter Information

Sean ClearyFollow

Submission Type

Oral/Paper Presentation

Degree Type

PhD

Discipline

Sciences

Department

Biology

Access Type

Open Access

Abstract or Description

Cardiac muscle cells contract in response to elevations in cytosolic calcium released from an intracellular calcium store in the sarcoplasmic reticulum (SR). The SR calcium pump (SERCA) relaxes the heart between beats by sequestering calcium back into the SR lumen. SERCA function in the heart is regulated by its interaction with the transmembrane peptide, phospholamban (PLB), which reduces SERCA activity to maintain resting heart rate. We have previously shown that although PLB largely remains bound to SERCA during calcium elevations, SERCA slightly loses affinity for PLB when elevated calcium concentrations are sustained. Here we used fluorescence resonance energy transfer (FRET) to measure how the PLB-SERCA binding equilibrium changes in a model system that mimics cardiac calcium oscillations. Our results indicate that a small fraction of PLB-SERCA complexes dissociate during calcium elevations on a beat-to-beat basis. When unbound, PLB monomers homo-oligomerize into PLB pentamers. Slow dissociation of PLB from pentamers delays PLB re-association with SERCA. This kinetic trapping of PLB in pentamers delays the resumption of basal PLB inhibition by about 5s – much slower than the 1-second timescale of the cardiac cycle. Modeling the kinetics of PLB exchange between SERCA- and pentamer-bound pools revealed that increasing cardiac pacing to high frequencies associated with exercise reduces the equilibrium level of PLB inhibition. Therefore, we propose a new mechanism where the level of PLB inhibition of SERCA is dynamically adjusted by cardiac pacing frequency to meet changing physiological demands.

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Dynamic Exchange of Phospholamban from SERCA During Elevations in Intracellular Calcium

Cardiac muscle cells contract in response to elevations in cytosolic calcium released from an intracellular calcium store in the sarcoplasmic reticulum (SR). The SR calcium pump (SERCA) relaxes the heart between beats by sequestering calcium back into the SR lumen. SERCA function in the heart is regulated by its interaction with the transmembrane peptide, phospholamban (PLB), which reduces SERCA activity to maintain resting heart rate. We have previously shown that although PLB largely remains bound to SERCA during calcium elevations, SERCA slightly loses affinity for PLB when elevated calcium concentrations are sustained. Here we used fluorescence resonance energy transfer (FRET) to measure how the PLB-SERCA binding equilibrium changes in a model system that mimics cardiac calcium oscillations. Our results indicate that a small fraction of PLB-SERCA complexes dissociate during calcium elevations on a beat-to-beat basis. When unbound, PLB monomers homo-oligomerize into PLB pentamers. Slow dissociation of PLB from pentamers delays PLB re-association with SERCA. This kinetic trapping of PLB in pentamers delays the resumption of basal PLB inhibition by about 5s – much slower than the 1-second timescale of the cardiac cycle. Modeling the kinetics of PLB exchange between SERCA- and pentamer-bound pools revealed that increasing cardiac pacing to high frequencies associated with exercise reduces the equilibrium level of PLB inhibition. Therefore, we propose a new mechanism where the level of PLB inhibition of SERCA is dynamically adjusted by cardiac pacing frequency to meet changing physiological demands.