Myofibre Hyper-Contractility in Horses Expressing the Myosin Heavy Chain Myopathy Mutation, MYH1E321G

Myosinopathies are defined as a group of muscle disorders characterized by mutations in genes encoding myosin heavy chains. Their exact molecular and cellular mechanisms remain unclear. In the present study, we have focused our attention on a MYH1-related E321G amino acid substitution within the head region of the type IIx skeletal myosin heavy chain, associated with clinical signs of atrophy, inflammation and/or profound rhabdomyolysis, known as equine myosin heavy chain myopathy. We performed Mant-ATP chase experiments together with force measurements on isolated IIx myofibres from control horses (MYH1E321G−/−) and Quarter Horses homozygous (MYH1E321G+/+) or heterozygous (MYH1E321G+/−) for the E321G mutation. The single residue replacement did not affect the relaxed conformations of myosin molecules. Nevertheless, it significantly increased its active behaviour as proven by the higher maximal force production and Ca2+ sensitivity for MYH1E321G+/+ in comparison with MYH1E321G+/− and MYH1E321G−/− horses. Altogether, these findings indicate that, in the presence of the E321G mutation, a molecular and cellular hyper-contractile phenotype occurs which could contribute to the development of the myosin heavy chain myopathy.

A reverse stroke characterizes the force generation of cardiac myofilaments, leading to an understanding of heart function

Changes in the molecular properties of cardiac myosin strongly affect the interactions of myosin with actin that result in cardiac contraction and relaxation. However, it remains unclear how myosin molecules work together in cardiac myofilaments and which properties of the individual myosin molecules impact force production to drive cardiac contractility. Here, we measured the force production of cardiac myofilaments using optical tweezers. The measurements revealed that stepwise force generation was associated with a higher frequency of backward steps at lower loads and higher stall forces than those of fast skeletal myofilaments. To understand these unique collective behaviors of cardiac myosin, the dynamic responses of single cardiac and fast skeletal myosin molecules, interacting with actin filaments, were evaluated under load. The cardiac myosin molecules switched among three distinct conformational positions, ranging from pre– to post–power stroke positions, in 1 mM ADP and 0 to 10 mM phosphate solution. In contrast to cardiac myosin, fast skeletal myosin stayed primarily in the post–power stroke position, suggesting that cardiac myosin executes the reverse stroke more frequently than fast skeletal myosin. To elucidate how the reverse stroke affects the force production of myofilaments and possibly heart function, a simulation model was developed that combines the results from the single-molecule and myofilament experiments. The results of this model suggest that the reversal of the cardiac myosin power stroke may be key to characterizing the force output of cardiac myosin ensembles and possibly to facilitating heart contractions.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2−/−) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2−/− mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2−/− mice. Small-angle X-ray diffraction of C2−/− EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2−/− EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2−/− mice. Finally, C2−/− muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

Procoagulant activities of skeletal and cardiac muscle myosin depend on contaminating phospholipid

Recent reports indicate that suspended skeletal and cardiac myosin, such as might be released during injury, can act as procoagulants by providing membrane-like support for factors Xa and Va in the prothrombinase complex. Further, skeletal myosin provides membrane-like support for activated protein C. This raises the question of whether purified muscle myosins retain procoagulant phospholipid through purification. We found that lactadherin, a phosphatidyl-l-serine–binding protein, blocked >99% of prothrombinase activity supported by rabbit skeletal and by bovine cardiac myosin. Similarly, annexin A5 and phospholipase A2 blocked >95% of myosin-supported activity, confirming that contaminating phospholipid is required to support myosin-related prothrombinase activity. We asked whether contaminating phospholipid in myosin preparations may also contain tissue factor (TF). Skeletal myosin supported factor VIIa cleavage of factor X equivalent to contamination by ∼1:100 000 TF/myosin, whereas cardiac myosin had TF-like activity >10-fold higher. TF pathway inhibitor inhibited the TF-like activity similar to control TF. These results indicate that purified skeletal muscle and cardiac myosins support the prothrombinase complex indirectly through contaminating phospholipid and also support factor X activation through TF-like activity. Our findings suggest a previously unstudied affinity of skeletal and cardiac myosin for phospholipid membranes.

Abstract MP102: Skeletal Muscle Alpha Actin Acetylation Enhances Myosin Binding and Increases Calcium Sensitivity in vitro

Actin and myosin are key proteins for muscle contraction/relaxation, while tropomyosin regulates actin-myosin interactions in the presence or absence of calcium. Mutations or dysregulation of any of these proteins results in cardiomyopathy. We previously showed that skeletal muscle alpha actin (ACTA1) was significantly deacetylated on lysine residues, K52, K317, and K328 in remodeled left ventricular tissue of obese mice. Computational modeling suggests that the positively charged lysine residues K328 and K317 of ACTA1 can interact electrostatically with the negatively charged glutamic acid residue E181 of tropomyosin and E286 of myosin. As acetylation is predicted to neutralize the positively charged lysine, ACTA1 acetylation would be postulated to decrease actin-myosin or actin-tropomyosin electrostatic interactions. To test this hypothesis, we used an in vitro actin motility assay to determine myosin sliding velocity, calcium sensitivity, and attachment/detachment kinetics of acetylated/deacetylated ACTA1. In addition, an actin binding protein spin-down assay was used to determine actin-myosin binding affinity using skeletal and cardiac myosin. In these assays, ACTA1 was chemically acetylated with acetic anhydride. In vitro actin motility analysis showed a significant decrease in sliding velocity with acetylated ACTA1 and skeletal myosin (1.709±0.210 μm/s) compared with deacetylated ACTA1 (4.427±0.275 μm/s). A similar significant decrease was also noted with cardiac myosin. Further analysis showed a significant increase in calcium sensitivity with ACTA1 acetylation (3.197x10-7 Kd compared to deacetylated ACTA1 1.191x10-6 Kd) and a loss of tropomyosin regulation with increasing ACTA1 acetylation. Lastly, ACTA1 acetylation enhanced actin binding affinity to cardiac and skeletal myosin. Investigation of attachment/detachment kinetics are currently underway. These data suggest that ACTA1 acetylation disrupts tropomyosin’s ability to inhibit myosin binding in the absence of calcium and further regulates actin-myosin interactions. Lastly, these data highlight acetylation as an additional post-translational modification outside of phosphorylation in the regulation of muscle contraction.

Discovery of a Novel, Selective and Short-Acting Skeletal Myosin II Inhibitor

Myosin IIs, actin-based motors that utilize the chemical energy of ATP to generate force, have potential as therapeutic targets. Their heavy chains differentiate the family into muscle (skeletal [SkMII], cardiac, smooth) and nonmuscle myosin IIs. Despite therapeutic potential for muscle disorders, no SkMII-specific inhibitor has been reported and characterized. Here we present the discovery, synthesis and characterization of “skeletostatins”, novel derivatives of the pan-myosin II inhibitor blebbistatin, with selectivity within the myosin IIs for SkMII. In addition, the skeletostatins bear improved potency, solubility and photostability, without cytotoxicity. Based on its optimal in vitro profile, Skeletostatin 1’s in vivo tolerability, efficacy and pharmacokinetics were determined. Skeletostatin 1 was well-tolerated in mice, impaired motor performance, and had an excellent muscle to plasma ratio. Skeletostatins are useful probes for basic research and a strong starting point for drug development.

Single-molecule analysis reveals that regulatory light chains fine-tune skeletal myosin II function

Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by ∼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.