The long-term objective of this research is to understand the molecular events regulating contraction in cardiac and skeletal muscle. It is hypothesized that in the interaction of actin and myosin to produce contraction, regulation is achieved through interactions within the thin filament modulating the thin filament structure. Although there are some cases where modulation at the thick filament level is important (such as phosphorylation of the myosin light chains) most evidence supports the thin filament as the major site of control. Using probes on TnC to measure Ca2+ binding, we will investigate whether this binding is uniform along the thin filament and whether crossbridges affect this binding. By using probes of the thin filament structure and measurements of crossbridge (XBr) force and kinetics, we will test to what extent Ca binding to TnC per se and XBr attachment regulate thin filament activation leading to force and XBr kinetics. With major differences in regulation between cardiac and skeletal muscle, we will investigate a major mechanism by which Ca2+ binding to TnC initiates contraction, through the modification of the TnC-TnI and TnC-TnI-TnT interactions. Specifically we will investigate why the interaction between the cardiac isoforms of TnC-TnI are more electrostatic while that of the skeletal isoforms more hydrophobic. We will investigate whether this is maintained in the presence of TnT and which charged amino acids are responsible for this electrostatic interaction and the differing effects of phosphorylation. This will clarify the interactions between TnC-TnI- TnT that occur during the control of contraction.
These aims will be achieved using skinned muscle preparations, fluorescent indicators on proteins exchanged into these preparations, protein binding studies using mutant regulatory proteins, and caged Ca to obtain rapid activation. Achieving these aims will help us understand how differences in molecular isoforms combined with a similar architecture of regulation can give rise to such large differences of regulation between cardiac and skeletal muscle.
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