The ATPase cycle of human muscle myosin II isoforms: Adaptation of a single mechanochemical cycle for different physiological roles.

Striated muscle myosins are encoded by a large gene family in all mammals, including humans. These isoforms define several of the key characteristics of the different striated muscle fiber types, including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin·myosin cross-bridge cycle in solution. Here, we present data on an eighth isoform, the perinatal, which has not previously been characterized. The perinatal is distinct from the embryonic isoform, appearing to have features in common with the adult fast-muscle isoforms, including weak affinity of ADP for actin·myosin and fast ADP release. We go on to use a recently developed modeling approach, MUSICO, to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction, including duty ratio, shortening velocity, ATP economy, and load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.

Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

Dilated cardiomyopathy myosin mutants have reduced force-generating capacity.

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human ß-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M·D complex in the steady state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force-holding capacity due to the reduced occupancy of the force-holding state.

Modeling the Actin.myosin ATPase Cross-Bridge Cycle for Skeletal and Cardiac Muscle Myosin Isoforms.

Modeling the complete actin.myosin ATPase cycle has always been limited by the lack of experimental data concerning key steps of the cycle, because these steps can only be defined at very low ionic strength. Here, using human ß-cardiac myosin-S1, we combine published data from transient and steady-state kinetics to model a minimal eight-state ATPase cycle. The model illustrates the occupancy of each intermediate around the cycle and how the occupancy is altered by changes in actin concentration for [actin] = 1-20Km. The cycle can be used to predict the maximal velocity of contraction (by motility assay or sarcomeric shortening) at different actin concentrations (which is consistent with experimental velocity data) and predict the effect of a 5 pN load on a single motor. The same exercise was repeated for human α-cardiac myosin S1 and rabbit fast skeletal muscle S1. The data illustrates how the motor domain properties can alter the ATPase cycle and hence the occupancy of the key states in the cycle. These in turn alter the predicted mechanical response of the myosin independent of other factors present in a sarcomere, such as filament stiffness and regulatory proteins. We also explore the potential of this modeling approach for the study of mutations in human ß-cardiac myosin using the hypertrophic myopathy mutation R453C. Our modeling, using the transient kinetic data, predicts mechanical properties of the motor that are compatible with the single-molecule study. The modeling approach may therefore be of wide use for predicting the properties of myosin mutations.

Nebulin and titin modulate cross-bridge cycling and length-dependent calcium sensitivity.

Various mutations in the structural proteins nebulin and titin that are present in human disease are known to affect the contractility of striated muscle. Loss of nebulin is associated with reduced actin filament length and impairment of myosin binding to actin, whereas titin is thought to regulate muscle passive elasticity and is likely involved in length-dependent activation. Here, we sought to assess the modulation of muscle function by these sarcomeric proteins by using the computational platform muscle simulation code (MUSICO) to quantitatively separate the effects of structural changes, kinetics of cross-bridge cycling, and calcium sensitivity of the thin filaments. The simulations show that variation in thin filament length cannot by itself account for experimental observations of the contractility in nebulin-deficient muscle, but instead must be accompanied by a decreased myosin binding rate. Additionally, to match the observed calcium sensitivity, the rate of TnI detachment from actin needed to be increased. Simulations for cardiac muscle provided quantitative estimates of the effects of different titin-based passive elasticities on muscle force and activation in response to changes in sarcomere length and interfilament lattice spacing. Predicted force-pCa relations showed a decrease in both active tension and sensitivity to calcium with a decrease in passive tension and sarcomere length. We conclude that this behavior is caused by partial redistribution of the muscle load between active muscle force and titin-dependent passive force, and also by redistribution of stretch along the thin filament, which together modulate the release of TnI from actin. These data help advance understanding of how nebulin and titin mutations affect muscle function.