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Title: The role of myosin regulatory light chain phosphorylation in cardiac health and disease
Author: Toepfer, Christopher
ISNI:       0000 0004 5920 6660
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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In this thesis we examined the effect of myosin associated regulatory light chain (RLC) phosphorylation level on cardiac muscle, ensembles and single molecules. We measured the ability of RLC phosphorylation change in muscle to alter force, power and unloaded shortening. The ATPase rate of full length cardiac myosin was determined with a novel protocol using gelsolin capped actin, which allowed novel measurements of myosin ATPase with full length (filamentous) myosin in low ionic strength. Actin gliding assays determined the effects of RLC phosphorylation level on actin gliding velocities under high and negligible load. The lifetime of strongly bound actomyosin states and the displacement of single myosin molecules were examined using an optical trapping three bead assay. A quantitative Phos-tag SDS-PAGE protocol was used to assess RLC phosphorylation level in inherited (mutation) and acquired (infarct and heart failure) human and rat diseases. Cardiac disorders in human and rat left ventricular myocardium correlated with increased RLC phosphorylation. RLC phosphorylation alters the ability of muscle to produce force, power and maximal unloaded shortening. Increased RLC phosphorylation accelerated the ATPase rate of cardiac myosin; reduced the lifetime of strongly bound actomyosin states and increased the displacement of actin by myosin. This data correlated with an increased ability of myosin with phosphorylated RLCs to translocate actin, under high and low load in the actin gliding assay. Cardiac myosin with raised RLC phosphorylation can produce more force and power during shortening due to changes in ATPase cycle, lifetime of the strongly bound states and power stroke size under load. Therefore myosin can perform work on actin faster and produce a longer actin displacement with each cycle. Thus proving that RLC phosphorylation level alteration impacts systolic myocardial performance in human health and disease by altering both myosin mechanics and kinetics.
Supervisor: Ferenczi, Michael Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral