Weikang Ma

Mavacamten stabilizes a folded-back sequestered super-relaxed state of β-cardiac myosin

Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity and ATPase activityof myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative modelpositsthat mutations in myosin affect the stability ofa sequestered, super-relaxed state (SRX) of the proteinwith very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here, using a combination of biochemical and structural approaches, we show that purified myosin enters aSRX thatcorresponds to a folded-back conformation, which in muscle fibersresults insequestration of heads around the thick filament backbone. Mutations that cause HCM destabilize this state, while the small molecule mavacamtenpromotes it. These findings provide a biochemical and structural link between the genetics and physiology ofcardiomyopathywith implications for therapeutic strategies.

Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers

Significance Cardiac muscle contraction is powered by ATP hydrolysis during cycles of interaction between myosin-containing thick filaments and actin-containing thin filaments. This generates force in the cardiac muscle necessary for pumping blood through the body. Mutations in myosin alter this force generation leading to hypercontractility and hypertrophic cardiomyopathy (HCM). An energy-conserving, super relaxed state (SRX) of myosin, which has a very low ATPase activity, has previously been described in muscle fibers. Destabilization of the SRX has been proposed to be a chief cause of HCM. This work sheds light on the biochemical and molecular nature of SRX and demonstrates the mechanism of action of mavacamten, a cardiac inhibitor in phase 2 clinical trials. Mavacamten exerts its effects primarily by stabilizing the SRX of β-cardiac myosin. Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

Nebulin stiffens the thin filament and augments cross-bridge interaction in skeletal muscle

Significance Nebulin is a giant actin-binding protein in skeletal muscle which localizes along most of the length of the thin filament. Genetic alterations or reduction in the expression level of nebulin are accompanied by dramatic loss in muscle force, resulting in muscle weakness and severe skeletal muscle myopathy. Using an inducible and tissue-specific nebulin-knockout mouse model in which nebulin is not expressed in skeletal muscle, we investigated the ultrastructure of thin filaments in passive and contracting muscle under physiological conditions using X-ray diffraction. Thin filaments were found to be threefold less stiff in nebulin-knockout muscle, and thin filament regulatory protein and cross-bridge behavior was impaired. Nebulin stiffens the thin filaments and is responsible for generating physiological levels of force. Nebulin is a giant sarcomeric protein that spans along the actin filament in skeletal muscle, from the Z-disk to near the thin filament pointed end. Mutations in nebulin cause muscle weakness in nemaline myopathy patients, suggesting that nebulin plays important roles in force generation, yet little is known about nebulin’s influence on thin filament structure and function. Here, we used small-angle X-ray diffraction and compared intact muscle deficient in nebulin (using a conditional nebulin-knockout, Neb cKO) with control (Ctrl) muscle. When muscles were activated, the spacing of the actin subunit repeat (27 Å) increased in both genotypes; when converted to thin filament stiffness, the obtained value was 30 pN/nm in Ctrl muscle and 10 pN/nm in Neb cKO muscle; that is, the thin filament was approximately threefold stiffer when nebulin was present. In contrast, the thick filament stiffness was not different between the genotypes. A significantly shorter left-handed (59 Å) thin filament helical pitch was found in passive and contracting Neb cKO muscles, as well as impaired tropomyosin and troponin movement. Additionally, a reduced myosin mass transfer toward the thin filament in contracting Neb cKO muscle was found, suggesting reduced cross-bridge interaction. We conclude that nebulin is critically important for physiological force levels, as it greatly stiffens the skeletal muscle thin filament and contributes to thin filament activation and cross-bridge recruitment.

Elastic proteins in the flight muscle of Manduca sexta

The flight muscles (DLM1) of the Hawkmoth, Manduca sexta are synchronous, requiring a neural spike for each contraction. Stress/strain curves of skinned DLM1 showed hysteresis indicating the presence of titin-like elastic proteins. Projectin and kettin are titin-like proteins previously identified in Lethocerus and Drosophila flight muscles. Analysis of Manduca muscles with 1% SDS-agarose gels and western blots showed two bands near 1 MDa that cross-reacted with antibodies to Drosophila projectin. Antibodies to Drosophila kettin cross-reacted to bands at ∼500 and ∼700 kDa, but also to bands at ∼1.6 and ∼2.1 MDa, that had not been previously observed in insect flight muscles. Mass spectrometry identified the 2.1 MDa protein as a product of the Sallimus (sls) gene. Analysis of the gene sequence showed that all 4 putative Sallimus and kettin isoforms could be explained as products of alternative splicing of the single sls gene. Both projectin and sallimus isoforms were expressed to higher levels in ventrally located DLM1 subunits, primarily responsible for active work production, as compared to dorsally located subunits, which may act as damped springs. The different expression levels of the 2 projectin isoforms and 4 sallimus/kettin isoforms may be adaptations to the specific requirements of individual muscle subunits.

Slow-twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain

Myosin light chain 2 (MYL2) gene encodes the myosin regulatory light chain (RLC) simultaneously in heart ventricles and in slow‐twitch skeletal muscle. Using transgenic mice with cardiac‐specific expression of the human R58Q‐RLC mutant, we sought to determine whether the hypertrophic cardiomyopathy phenotype observed in papillary muscles (PMs) of R58Q mice is also manifested in slow‐twitch soleus (SOL) muscles. Skinned SOL muscles and ventricular PMs of R58Q animals exhibited lower contractile force that was not observed in the fast‐twitch extensor digitorum longus muscles of R58Q vs. wild‐type‐RLC mice, but mutant animals did not display gross muscle weakness in vivo. Consistent with SOL muscle abnormalities in R58Q vs. wild‐type mice, myosin ATPase staining revealed a decreased proportion of fiber type I/type II only in SOL muscles but not in the extensor digitorum longus muscles. The similarities between SOL muscles and PMs of R58Q mice were further supported by quantitative proteomics. Differential regulation of proteins involved in energy metabolism, cell‐cell interactions, and protein‐protein signaling was concurrently observed in the hearts and SOL muscles of R58Q mice. In summary, even though R58Q expression was restricted to the heart of mice, functional similarities were clearly observed between the hearts and slow‐twitch skeletal muscle, suggesting that MYL2 mutated models of hypertrophic cardiomyopathy may be useful research tools to study the molecular, structural, and energetic mechanisms of cardioskeletal myopathy associated with myosin RLC.—Kazmierczak, K., Liang, J., Yuan, C.‐C, Yadav, S., Sitbon, Y. H., Walz, K., Ma, W., Irving, T. C., Cheah, J. X., Gomes, A. V., Szczesna‐Cordary, D. Slow‐twitch skeletal muscle defects accompany cardiac dysfunction in transgenic mice with a mutation in the myosin regulatory light chain. FASEB J. 33, 3152–3166 (2019). www.fasebj.org

Structural and functional impact of troponin C-mediated Ca2+ sensitization on myofilament lattice spacing and cross-bridge mechanics in mouse cardiac muscle

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca2+-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (kTR); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I1,1/I1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca2+ sensitization through this cTnC mutation or bepridil accelerates kTR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in kTR, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca2+ sensitization may act through stabilization of cTnCs D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.

Myosin Head Configurations in Resting and Contracting Murine Skeletal Muscle

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases, including those of skeletal muscle. We show that mouse skeletal muscle can produce high quality X-ray diffraction patterns establishing the mouse intact skeletal muscle X-ray preparation as a potentially powerful tool to test structural hypotheses in health and disease. A notable feature of the mouse model system is the presence of residual myosin layer line intensities in contracting mouse muscle patterns. This provides an additional tool, along with the I1,1/I1,0 intensity ratio, for estimating the proportions of active versus relaxed myosin heads under a given set of conditions that can be used to characterize a given physiological condition or mutant muscle type. We also show that analysis of the myosin layer line intensity distribution, including derivation of the myosin head radius, Rm, may be used to study the role of the super-relaxed state in myosin regulation. When the myosin inhibitor blebbistatin is used to inhibit force production, there is a shift towards a highly quasi-helically ordered configuration that is distinct from the normal resting state, indicating there are more than one helically ordered configuration for resting crossbridges.

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3): ACTA1-Related Myopathy

Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha‐actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients.