Direct characterization of single molecular kinetics of cardiac myosin in vitro

Distinct crossbridge kinetics among cardiac myosin isoforms have been proposed as the basis of differences in energetic characteristics. However, direct evidence for this hypothesis is lacking because of experimental difficulty. As a preliminary approach to this problem, we applied an in-vitro force measurement technique to directly observe force impulse generated by a single cardiac myosin molecule. The force measurement system was constructed with an inverted microscope coupled with a laser optical trap. With the feedback of the position signal to the driving circuit for a galvanomirror that steers the laser beam, trap stiffness was increased; thus, isometric force measurement was made possible. We measured the force generated by the cardiac myosin V3 isoform purified from hypothyroid rat ventricular muscle. With very low myosin density and low adenosine triphosphate (ATP) concentration of the assay buffer, we successfully observed a single force impulse similar in shape to that of skeletal muscle myosin. With this approach, we will be able to gain a clear view of the molecular basis of cardiac mechanoenergetics.