The power output of the heart is a key determinant that differentiates ventricular performance in patients with or without heart failure. Normal and abnormal cardiac function depends, in part, on the force and motion generating capacity of the myosin motor. We plan to study how myosin's molecular structure contributes to its enzymatic and mechanical function and how this then translates into the mechanical performance of the myocardium. We exploit the fact that there are two isoforms of myosin in heart muscle, V1 and V3, which are 95% homologous and exhibit marked differences in mechanical and enzymatic behavior (ATPase, shortening velocity, Average Force). The differing amino acids fall into clusters that are situated on the myosin molecule at functionally important sites (based on the S1 crystal structure). Two such sites of divergence are found in the surface loops that span the nucleotide binding pocket (loop 1) and the actin binding region (loop2). It has been hypothesized (Spudich, 1994) that loop 1 controls the rate of ATP binding and ADP release and thus shortening velocity while loop2 controls the rate of myosin binding to actin and thus the ATPase activity, while the combination of the two contributes to the average force. We will use transgenic techniques to produce mouse hearts with a series of chimeric myosins consisting of a V1 backbone with either a V3 loop 1, V3 loop2, or both V3 loops (1 + 2). The chimeric myosin will then be compared to myosin from wild type V1 hearts an transgenic V3 preparations. State-of-the-art in vitro motility and laser trap techniques will be used to determine the performance of these myosins at the level of a single myosin molecule (e.g. actin filament velocity, average force, unitary displacement, unitary force, attachment time). At a more organized level, skinned fiber and whole heart mechanics will provide detailed mechanical information regarding the translation of molecular mechanics to whole organ mechanics. These studies should provide important information about myosin's molecular structure and function and how alterations to myosin's molecular performance is mechanically expressed in the ventricular power output.
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