Cross-bridge kinetics in myofibrils containing familial hypertrophic cardiomyopathy R58Q mutation in the regulatory light chain of myosin.

Familial hypertrophic cardiomyopathy (FHC) is a heritable form of cardiac hypertrophy caused by single-point mutations in genes encoding sarcomeric proteins including ventricular myosin regulatory light chain (RLC). FHC often leads to malignant outcomes and sudden cardiac death. The FHC mutations are believed to alter the kinetics of the interaction between actin and myosin resulting in inefficient energy utilization and compromised function of the heart. We studied the effect of the FHC-linked R58Q-RLC mutation on the kinetics of transgenic (Tg)-R58Q cardiac myofibrils. Kinetics was determined from the rate of change of orientation of actin monomers during muscle contraction. Actin monomers change orientation because myosin cross-bridges deliver periodic force impulses to it. An individual impulse (but not time average of impulses) carries the information about the kinetics of actomyosin interaction. To observe individual impulses it was necessary to scale down the experiments to the level of a few molecules. A small population (∼4 molecules) was selected by using (deliberately) inefficient fluorescence labeling and observing fluorescent molecules by a confocal microscope. We show that the kinetic rates are significantly smaller in the contracting cardiac myofibrils from Tg-R58Q mice then in control Tg-wild type (WT). We also demonstrate a lower force per cross-section of muscle fiber in Tg-R58Q versus Tg-WT mice. We conclude that the R58Q mutation-induced decrease in cross-bridge kinetics underlines the mechanism by which Tg-R58Q fibers develop low force and thus compromise the ability of the mutated heart to efficiently pump blood.

Observing cycling of a few cross-bridges during isometric contraction of skeletal muscle.

During muscle contraction a myosin cross-bridge imparts periodic force impulses to actin. It is possible to visualize those impulses by observing a few molecules of actin or myosin. We have followed the time course of orientation change of a few actin molecules during isometric contraction by measuring parallel polarized intensity of its fluorescence. The orientation of actin reflects local bending of a thin filament and is different when a cross-bridge binds to, or is detached from, F-actin. The changes in orientation were characterized by periods of activity during which myosin cross-bridges interacted normally with actin, interspersed with periods of inactivity during which actin and myosin were unable to interact. The periods of activity lasted on average 1.2 +/- 0.4 s and were separated on average by 2.3 +/- 1.0 s. During active period, actin orientation oscillated between the two extreme values with the ON and OFF times of 0.4 +/- 0.2 and 0.7 +/- 0.4 s, respectively. When the contraction was induced by a low concentration of ATP both active and inactive times were longer and approximately equal. These results imply that cross-bridges interact with actin in bursts and suggest that during active period, on average 36% of cross-bridges are involved in force generation.

Fluorescence lifetime of actin in the familial hypertrophic cardiomyopathy transgenic heart.

Clinical studies have revealed that the D166V mutation in the ventricular myosin regulatory light chain (RLC) can cause a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). It has been proposed that RLC induced FHC in the heart originates at the level of the myosin cross-bridge due to alterations in the rates of cross-bridge cycling. In this report, we examine whether the environment of an active cross-bridge in cardiac myofibrils from transgenic (Tg) mice is altered by the D166V mutation in RLC. The cross-bridge environment was monitored by tracking the fluorescence lifetime (tau) of Alexa488-phalloidin-labeled actin. The fluorescence lifetime is the average rate of decay of a fluorescent species from the excited state, which strongly depends on various environmental factors. We observed that the lifetime was high when cross-bridges were bound to actin and low when they were dissociated from it. The lifetime was measured every 50 ms from the center half of the I-band during 60 s of rigor, relaxation and contraction of muscle. We found no differences between lifetimes of Tg-WT and Tg-D166V muscle during rigor, relaxation and contraction. The duty ratio expressed as a fraction of time that cross-bridges spend attached to the thin filaments during isometric contraction was similar in Tg-WT and Tg-D166V muscles. Since independent measurements showed a large decrease in the cross-bridge turnover rate in Tg-D166V muscle compared to Tg-WT, the fact that the duty cycle remains constant suggests that the D166V mutation of RLC causes a decrease in the rate of cross-bridge attachment to actin.