Regulatory Mechanisms of Cardiac Muscle

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DESCRIPTION (provided by applicant): Contraction of cardiac muscle is initiated by calcium binding to TnC, which is a member of the heterotrimeric troponin complex (TnC, Tnl, TnT). This binding results in structural changes that occur in several proteins that make up the muscle thin filament and alterations of the interactions among these proteins. These molecular changes define the transition of muscle from the inactive to the active state. A calcium switch controls these events during activation/deactivation. A mechanism that regulates cardiac function as a feed back control involves phosphorylation by protein kinase A (PKA) of 2 adjacent serine residues located in the unique N-terminal extension of Tnl. Alteration of the extent of calcium binding and phosphorylation can lead to abnormal cardiac functions. The molecular switch is located in the interface between the 2 subunits Tnl and TnC. How this switch operates in terms of structural alterations and changes in protein-protein interactions remains to be elucidated. A long-term goal of this program is to define these molecular events and changes of these events as related to normal cardiac function, using fluorescence spectroscopy/Forster resonance energy transfer (FRET) and rapid kinetics as major tools. The system to be used is the synthetic thin filament consisting of the troponin complex, tropomyosin and actin. Most of these proteins will be recombinant proteins with specific amino acids substitutions designed for FRET studies. Another long-term goal relates to the mechanisms by which the signal of PKA phosphorylation of Tnl is transmitted to distant parts of the molecule and to the other 2 troponin subunits. The third goal and fourth goals are construction of molecular models for the troponin complex using a large number FRET distances to study structural changes in response to calcium binding, particularly in regions of the complex known to be functionally important, but for which no high-resolution structural information is available. Elucidation of these mechanisms will contribute to the understanding of the structural basis of the diseased state of the heart.