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. How ever, direct evidence for this hypothesis is lacking because of experi mental difficulty. As a preliminary approach to this problem, we appli ed an in-vitro force measurement technique to directly observe force i mpulse generated by a single cardiac myosin molecule. The force measur ement 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 s tiffness was increased, thus, isometric force measurement was made pos sible. We measured the force generated by the cardiac myosin V-3 isofo rm purified from hypothyroid rat ventricular muscle. With very low myo sin 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 wil l be able to gain a clear view of the molecular basis of cardiac mecha noenergetics.