Familial hypertrophic cardiomyopathy can be characterized by a specific pattern of orientation fluctuations of actin molecules

A single-point mutation in the gene encoding the ventricular myosin regulatory light chain (RLC) is sufficient to cause familial hypertrophic cardiomyopathy (FHC). Most likely, the underlying cause of this disease is an inefficient energy utilization by the mutated cardiac muscle. We set out to devise a simple method to characterize two FHC phenotypes caused by the R58Q and D166V mutations in RLC. The method is based on the ability to observe a few molecules of actin in working ex vivo heart myofibril. Actin is labeled with extremely diluted fluorescent dye, and a small volume within the I-band (10⁻¹⁶L), containing on average three actin molecules, is observed by confocal microscopy. During muscle contraction, myosin cross-bridges deliver cyclic impulses to actin. As a result, actin molecules undergo periodic fluctuations of orientation. We measured these fluctuations by recording the parallel and perpendicular components of fluorescent light emitted by an actin-bound fluorophore. The histograms of fluctuations of fluorescent actin molecules in wild-type (WT) hearts in rigor were represented by perfect Gaussian curves. In contrast, histograms of contracting heart muscle were peaked and asymmetric, suggesting that contraction occurred in at least two steps. Furthermore, the differences between histograms of contracting FHC R58Q and D166V hearts versus corresponding contracting WT hearts were statistically significant. On the basis of our results, we suggest a simple new method of distinguishing between healthy and FHC R58Q and D166V hearts by analyzing the probability distribution of polarized fluorescence intensity fluctuations of sparsely labeled actin molecules.9 page(s

The role of troponins in muscle contraction

Troponin (Tn) is the sarcomeric Ca2+ regulator for striated (skeletal and cardiac) muscle contraction. On binding Ca2+ Tn transmits information via structural changes throughout the actin-tropomyosin filaments, activating myosin ATPase activity and muscle contraction. Although the Tn-mediated regulation of striated muscle contraction is now well understood, the role of different Tn isoforms in these processes is the subject of intensive investigations. This review addresses the physiological significance of the multiple Tn isoforms in skeletal and cardiac muscles as well as their role in the regulation of contraction

E22K mutation of RLC that causes familial hypertrophic cardiomyopathy in heterozygous mouse myocardium: effect on cross-bridge kinetics

Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in sarcomeric proteins, including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca(2+)-binding site. We have studied transgenic (Tg) mouse cardiac myofibrils during single-turnover contraction to examine the influence of E22K mutation on 1) dissociation time (tau(1)) of myosin heads from thin filaments, 2) rebinding time (tau(2)) of the cross bridges to actin, and 3) dissociation time (tau(3)) of ADP from the active site of myosin. tau(1) was determined from the increase in the rate of rotation of actin monomer to which a cross bridge was bound. tau(2) was determined from the rate of anisotropy change of the recombinant essential light chain of myosin labeled with rhodamine exchanged for native light chain (LC1) in the cardiac myofibrils. tau(3) was determined from anisotropy of muscle preloaded with a stoichiometric amount of fluorescent ADP. Cross bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in Tg-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Tg wild-type (Tg-wt) and non-Tg muscles acted as controls. tau(1) was statistically greater in Tg-m than in controls. tau(2) was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. tau(3) was the same in Tg-m and controls. To determine whether the difference in tau(1) was due to intrinsic difference in myosin, we estimated binding of Tg-m and Tg-wt myosin to fluorescently labeled actin by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross bridges. The slight increase in tau(1) was probably caused by myofibrillar disarray. The decrease in tau(2) of Tg hearts was probably caused by replacement of the mouse RLC for the human isoform in the Tg mice

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

Regulatory light chain mutations associated with cardiomyopathy affect myosin mechanics and kinetics

The myosin regulatory light chain (RLC) wraps around the alpha-helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis, with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K show reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation

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.11 page(s