Cardiac myosin velocity and force are dramatically improved with an alternative triphosphate substrate.

Abstract

Cardiac myosin is the molecular motor that drives the pumping action of the heart; therefore, there is strong interest in gaining control over its function in an effort to develop more effective treatments for various forms of heart disease. We sought to determine if the force and motion generating capacity of β-cardiac myosin could be altered in vitro by replacing ATP with different isomers of an alternative energy source, azobenzene triphosphate (AzoTP). Using the ortho-isomer of AzoTP, cardiac myosin moved actin filaments 62% faster than ATP in a motility assay (p<0.001). Subsequent experiments suggested that the increased velocity may be the result of an accelerated rate of ortho-AzoDP-release from myosin. Cardiac myosin also generated 10% more force when using ortho-AzoTP compared to ATP in a laser trap assay (p<0.001), which was likely due to an increased rate of attachment to actin because the frequency of single actomyosin binding events observed in a three-bead laser trap assay was double that observed for ortho-AzoTP vs. ATP (p<0.001). In contrast to ortho-AzoTP, with the meta isomer of AzoTP cardiac myosin translocated actin filaments 90% slower than with ATP (p<0.001). This finding is likely do to a slowed rate of detachment from actin, based on preliminary single molecule findings. With the para-isomer of AzoTP no movement was detected in the motility assay, suggesting it may not bind to the nucleotide-binding site. Thus, these findings demonstrate that the function of beta-cardiac myosin can be manipulated using structural isomers of an alternative energy substrate (AzoTP). These findings therefore may provide important new insights into how to alter the function of cardiac myosin in vivo, which could help in the development of novel therapies to different forms of heart disease.