Prolonged myosin binding increases muscle stiffness in Drosophila models of Freeman-Sheldon syndrome.

Freeman-Sheldon syndrome (FSS) is characterized by congenital contractures resulting from dominant point mutations in the embryonic isoform of muscle myosin. To investigate its disease mechanism, we used Drosophila models expressing FSS myosin mutations Y583S or T178I in their flight and jump muscles. We isolated these muscles from heterozygous mutant Drosophila and performed skinned fiber mechanics. The most striking mechanical alteration was an increase in active muscle stiffness. Y583S/+ and T178I/+ fibers' elastic moduli increased 70 and 77%, respectively. Increased stiffness contributed to decreased power generation, 49 and 66%, as a result of increased work absorbed during the lengthening portion of the contractile cycle. Slower muscle kinetics also contributed to the mutant phenotype, as shown by 17 and 32% decreases in optimal frequency for power generation, and 27 and 41% slower muscle apparent rate constant 2πb. Combined with previous measurements of slower in vitro actin motility, our results suggest a rate reduction of at least one strongly bound cross-bridge cycle transition that increases the time myosin spends strongly bound to actin, ton. Increased ton was further supported by decreased ATP affinity and a 16% slowing of jump muscle relaxation rate in T178I heterozygotes. Impaired muscle function caused diminished flight and jump ability of Y583S/+ and T178I/+ Drosophila. Based on our results, assuming that our model system mimics human skeletal muscle, we propose that one mechanism driving FSS is elevated muscle stiffness arising from prolonged ton in developing muscle fibers.

Force-velocity and tension transient measurements from Drosophila jump muscle reveal the necessity of both weakly-bound cross-bridges and series elasticity in models of muscle contraction.

Muscle contraction is a fundamental biological process where molecular interactions between the myosin molecular motor and actin filaments result in contraction of a whole muscle, a process spanning size scales differing in eight orders of magnitude. Since unique behavior is observed at every scale in between these two extremes, to fully understand muscle function it is vital to develop multi-scale models. Based on simulations of classic measurements of muscle heat generation as a function of work, and shortening rate as a function of applied force, we hypothesize that a model based on molecular measurements must be modified to include a weakly-bound interaction between myosin and actin in order to fit measurements at the muscle fiber or whole muscle scales. This hypothesis is further supported by the model's need for a weakly-bound state in order to qualitatively reproduce the force response that occurs when a muscle fiber is rapidly stretched a small distance. We tested this hypothesis by measuring steady-state force as a function of shortening velocity, and the force transient caused by a rapid length step in Drosophila jump muscle fibers. Then, by performing global parameter optimization, we quantitatively compared the predictions of two mathematical models, one lacking a weakly-bound state and one with a weakly-bound state, to these measurements. Both models could reproduce our force-velocity measurements, but only the model with a weakly-bound state could reproduce our force transient measurements. However, neither model could concurrently fit both measurements. We find that only a model that includes weakly-bound cross-bridges with force-dependent detachment and an elastic element in series with the cross-bridges is able to fit both of our measurements. This result suggests that the force response after stretch is not a reflection of distinct steps in the cross-bridge cycle, but rather arises from the interaction of cross-bridges with a series elastic element. Additionally, the model suggests that the curvature of the force-velocity relationship arises from a combination of the force-dependence of weakly- and strongly-bound cross-bridges. Overall, this work presents a minimal cross-bridge model that has predictive power at the fiber level.

A myosin-based mechanism for stretch activation and its possible role revealed by varying phosphate concentration in fast and slow mouse skeletal muscle fibers.

Stretch activation (SA) is a delayed increase in force following a rapid muscle length increase. SA is best known for its role in asynchronous insect flight muscle, where it has replaced calcium's typical role of modulating muscle force levels during a contraction cycle. SA also occurs in mammalian skeletal muscle but has previously been thought to be too low in magnitude, relative to calcium-activated (CA) force, to be a significant contributor to force generation during locomotion. To test this supposition, we compared SA and CA force at different Pi concentrations (0-16 mM) in skinned mouse soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscle fibers. CA isometric force decreased similarly in both muscles with increasing Pi, as expected. SA force decreased with Pi in EDL (40%), leaving the SA to CA force ratio relatively constant across Pi concentrations (17-25%). In contrast, SA force increased in soleus (42%), causing a quadrupling of the SA to CA force ratio, from 11% at 0 mM Pi to 43% at 16 mM Pi, showing that SA is a significant force modulator in slow-twitch mammalian fibers. This modulation would be most prominent during prolonged muscle use, which increases Pi concentration and impairs calcium cycling. Based upon our previous Drosophila myosin isoform studies and this work, we propose that in slow-twitch fibers a rapid stretch in the presence of Pi reverses myosin's power stroke, enabling quick rebinding to actin and enhanced force production, while in fast-twitch fibers, stretch and Pi cause myosin to detach from actin.

The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production.

KEY POINTS: Hypertrophic cardiomyopathy (HCM) is a genetic disease that causes thickening of the heart's ventricular walls and is a leading cause of sudden cardiac death. HCM is caused by missense mutations in muscle proteins including myosin, but how these mutations alter muscle mechanical performance in largely unknown. We investigated the disease mechanism for HCM myosin mutation R249Q by expressing it in the indirect flight muscle of Drosophila melanogaster and measuring alterations to muscle and flight performance. Muscle mechanical analysis revealed R249Q decreased muscle power production due to slower muscle kinetics and decreased force production; force production was reduced because fewer mutant myosin cross-bridges were bound simultaneously to actin. This work does not support the commonly proposed hypothesis that myosin HCM mutations increase muscle contractility, or causes a gain in function; instead, it suggests that for some myosin HCM mutations, hypertrophy is a compensation for decreased contractility. ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an inherited disease that causes thickening of the heart's ventricular walls. A generally accepted hypothesis for this phenotype is that myosin heavy chain HCM mutations increase muscle contractility. To test this hypothesis, we expressed an HCM myosin mutation, R249Q, in Drosophila indirect flight muscle (IFM) and assessed myofibril structure, skinned fibre mechanical properties, and flight ability. Mechanics experiments were performed on fibres dissected from 2-h-old adult flies, prior to degradation of IFM myofilament structure, which started at 2 days old and increased with age. Homozygous and heterozygous R249Q fibres showed decreased maximum power generation by 67% and 44%, respectively. Decreases in force and work and slower overall muscle kinetics caused homozygous fibres to produce less power. While heterozygous fibres showed no overall slowing of muscle kinetics, active force and work production dropped by 68% and 47%, respectively, which hindered power production. The muscle apparent rate constant 2πb decreased 33% for homozygous but increased for heterozygous fibres. The apparent rate constant 2πc was greater for homozygous fibres. This indicates that R249Q myosin is slowing attachment while speeding up detachment from actin, resulting in less time bound. Decreased IFM power output caused 43% and 33% decreases in Drosophila flight ability and 19% and 6% drops in wing beat frequency for homozygous and heterozygous flies, respectively. Overall, our results do not support the increased contractility hypothesis. Instead, our results suggest the ventricular hypertrophy for human R249Q mutation is a compensatory response to decreases in heart muscle power output.

Drosophila myosin mutants model the disparate severity of type 1 and type 2B distal arthrogryposis and indicate an enhanced actin affinity mechanism.

BACKGROUND: Distal arthrogryposis (DA) is a group of autosomal dominant skeletal muscle diseases characterized by congenital contractures of distal limb joints. The most common cause of DA is a mutation of the embryonic myosin heavy chain gene, MYH3. Human phenotypes of DA are divided into the weakest form-DA1, a moderately severe form-DA2B (Sheldon-Hall Syndrome), and a severe DA disorder-DA2A (Freeman-Sheldon Syndrome). As models of DA1 and DA2B do not exist, their disease mechanisms are poorly understood. METHODS: We produced the first models of myosin-based DA1 (F437I) and DA2B (A234T) using transgenic Drosophila melanogaster and performed an integrative analysis of the effects of the mutations. Assessments included lifespan, locomotion, ultrastructural analysis, muscle mechanics, ATPase activity, in vitro motility, and protein modeling. RESULTS: We observed significant defects in DA1 and DA2B Drosophila flight and jump ability, as well as myofibril assembly and stability, with homozygotes displaying more severe phenotypes than heterozygotes. Notably, DA2B flies showed dramatically stronger phenotypic defects compared to DA1 flies, mirroring the human condition. Mechanical studies of indirect flight muscle fibers from DA1 heterozygotes revealed reduced power output along with increased stiffness and force production, compared to wild-type controls. Further, isolated DA1 myosin showed significantly reduced myosin ATPase activity and in vitro actin filament motility. These data in conjunction with our sinusoidal analysis of fibers suggest prolonged myosin binding to actin and a slowed step associated with Pi release and/or the power stroke. Our results are supported by molecular modeling studies, which indicate that the F437I and A234T mutations affect specific amino acid residue interactions within the myosin motor domain that may alter interaction with actin and nucleotide. CONCLUSIONS: The allele-specific ultrastructural and locomotory defects in our Drosophila DA1 and DA2B models are concordant with the differential severity of the human diseases. Further, the mechanical and biochemical defects engendered by the DA1 mutation reveal that power production, fiber stiffness, and nucleotide handling are aberrant in F437I muscle and myosin. The defects observed in our DA1 and DA2B Drosophila models provide insight into DA phenotypes in humans, suggesting that contractures arise from prolonged actomyosin interactions.

Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy.

K146N is a dominant mutation in human ß-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.