Comparison of Unitary Displacements and Forces Between 2 Cardiac Myosin Isoforms by the Optical Trap Technique

Abstract

Abstract-To provide information on the mechanism of cardiac adaptation at the molecular level, we compared the unitary displacements and forces between the 2 rat cardiac myosin isoforms, V 1 and V 3 . A fluorescently labeled actin filament, with a polystyrene bead attached, was caught by an optical trap and brought close to a glass surface sparsely coated with either of the 2 isoforms, so that the actin-myosin interaction took place in the presence of a low concentration of ATP (0.5 mol/L). Discrete displacement events were recorded with a low trap stiffness (0.03 to 0.06 pN/nm). Frequency distribution of the amplitude of the displacements consisted of 2 gaussian curves with peaks at 9 to 10 and 18 to 20 nm for both V 1 and V 3 , suggesting that 9 to 10 nm is the unitary displacement for both isoforms. The duration of the displacement events was longer for V 3 than for V 1 . On the other hand, discrete force transients were recorded with a high trap stiffness (2.1 pN/nm), and their amplitude showed a broad distribution with mean values between 1 and 2 pN for V 1 and V 3 . The durations of the force transients were also longer for V 3 than for V 1 . These results indicate that both the unitary displacements and forces are similar in amplitude but different in duration between the 2 cardiac myosin isoforms, being consistent with the reports that the tension cost is higher in muscles consisting mainly of V 1 than those consisting mainly of V 3 . (Circ Res. 1998;82:1029-1034.)Key Words: cardiac myosin Ⅲ unitary displacement Ⅲ unitary force Ⅲ laser optical trap M ammalian ventricular muscle myosin is divided into 3 different isoforms, V 1 , V 2 , and V 3 . 1 Actin-activated MgATPase activity is highest for V 1 and lowest for V 3 . Contractile properties of these isoforms have been studied in muscle preparations, in which myosin isoform composition was modified either hormonally 2 or by imposing overload. 3,4 All these studies indicated that the maximum unloaded shortening velocity (V max ) of muscle preparations correlated well with their V 1 isoform content, suggesting the fast crossbridge cycling rate in this isoform. In these experiments with muscle preparations, however, it was difficult to preclude the possible influence of concomitant changes of other cellular components on V max . In addition, it was difficult to estimate the number of crossbridges generating contractile force.In vitro motility assay systems are effective in studying the kinetics of the ATP-dependent interaction between purified actin and myosin molecules. In accordance with the results for V max in muscle preparations, the unloaded velocity of actin-myosin sliding in vitro has been shown to be much faster for V 1 than for V 3 isoforms. 5,6 Using the centrifuge microscope, with which constant centrifugal forces are applied as loads on in vitro actin-myosin sliding, we showed that the shape of force-velocity curves was markedly different between V 1 and V 3 isoforms, reflecting their different interaction kinetics with actin.7 Recent developmen...

Impaired myocardial performance in a normotensive rat model of intrauterine growth restriction

“…For example, the expression of MHCs reflects compensatory mechanisms under increased myocardial load (17) as well as in manifest cardiomyopathy (18). Compared with α-MHC, β-MHC is considered as the MHC isoform with a higher degree of efficiency (19). Assessing the expressional ratio of α-MHC/β-MHC, we were able to show at least a tendency toward an overexpression of β-MHC in 70-d-old LP rats.…”

Forced Expression of α-Myosin Heavy Chain in the Rabbit Ventricle Results in Cardioprotection Under Cardiomyopathic Conditions

“…22,25 Singlemolecule studies in the laser trap suggest that differences in the kinetics of the cross-bridge cycle for V1 and V3 myosins are the major determinant of the mechanical performance of cardiac myosin. 26,27 Clearly, there also are relationships between MHC isoform content and whole-organ function. In rats, low-level expression (12% replacement) of ␤-MHC on the ␣-MHC background resulted in altered systolic and diastolic function.…”

Factors controlling cardiac myosin-isoform shift during hypertrophy and heart failure

“…Warshaw's group has shown in an in vitro motility assay, that the actin sliding velocity (V actin) of the myosin mixture was highest at 100% αMHC and gradually decreased as the proportion of the βMHC isoform was raised, reaching the lowest level at 100% βMHC isoform, thus confirming the linear correlation between V actin and α:βMHC isoforms ratio. Single molecule studies have also demonstrated that the higher actin sliding velocity of the αMHC isoform is related to a shorter attachment time of myosin after the power stroke and not to the displacement generated by the myosin power stroke [29,30]. These studies have suggested that the kinetics (attachment time) rather than the mechanics (unitary displacement) of the myosin molecule accounts for the difference in the actin-sliding velocity of the α and βMHC isoforms [23,[28][29][30].…”

“…Single molecule studies have also demonstrated that the higher actin sliding velocity of the αMHC isoform is related to a shorter attachment time of myosin after the power stroke and not to the displacement generated by the myosin power stroke [29,30]. These studies have suggested that the kinetics (attachment time) rather than the mechanics (unitary displacement) of the myosin molecule accounts for the difference in the actin-sliding velocity of the α and βMHC isoforms [23,[28][29][30]. Based on these observations, it is generally believed that hearts expressing primarily αMHC isoform have a significantly higher velocity of muscle shortening; whereas hearts expressing mostly βMHC, which has low ATPase activity, have the ability to generate force with greater economy.…”