Small molecule drugs to improve sarcomere function in those with acquired and inherited myopathies.

Muscle weakness is a hallmark of inherited or acquired myopathies. It is a major cause of functional impairment and can advance to life-threatening respiratory insufficiency. During the past decade, several small-molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small-molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin. We also discuss their use in the treatment of skeletal myopathies. The first of three classes of drugs discussed here increase contractility by decreasing the dissociation rate of calcium from troponin and thereby sensitizing the muscle to calcium. The second two classes of drugs directly act on myosin and stimulate or inhibit the kinetics of myosin-actin interactions, which may be useful in patients with muscle weakness or stiffness.NEW & NOTEWORTHY During the past decade, several small molecule drugs that improve the contractility of skeletal muscle fibers have been developed. In this review, we provide an overview of the available literature and the mechanisms of action of small molecule drugs that modulate the contractility of sarcomeres, the smallest contractile units in striated muscle, by acting on myosin and troponin.

Acute and chronic tirasemtiv treatment improves in vivo and in vitro muscle performance in actin-based nemaline myopathy mice.

Nemaline myopathy, a disease of the actin-based thin filament, is one of the most frequent congenital myopathies. To date, no specific therapy is available to treat muscle weakness in nemaline myopathy. We tested the ability of tirasemtiv, a fast skeletal troponin activator that targets the thin filament, to augment muscle force-both in vivo and in vitro-in a nemaline myopathy mouse model with a mutation (H40Y) in Acta1. In Acta1H40Y mice, treatment with tirasemtiv increased the force response of muscles to submaximal stimulation frequencies. This resulted in a reduced energetic cost of force generation, which increases the force production during a fatigue protocol. The inotropic effects of tirasemtiv were present in locomotor muscles and, albeit to a lesser extent, in respiratory muscles, and they persisted during chronic treatment, an important finding as respiratory failure is the main cause of death in patients with congenital myopathy. Finally, translational studies on permeabilized muscle fibers isolated from a biopsy of a patient with the ACTA1H40Y mutation revealed that at physiological Ca2+ concentrations, tirasemtiv increased force generation to values that were close to those generated in muscle fibers of healthy subjects. These findings indicate the therapeutic potential of fast skeletal muscle troponin activators to improve muscle function in nemaline myopathy due to the ACTA1H40Y mutation, and future studies should assess their merit for other forms of nemaline myopathy and for other congenital myopathies.

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-Pérot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

Recessive MYH7-related myopathy in two families.

Myopathies due to recessive MYH7 mutations are exceedingly rare, reported in only two families to date. We describe three patients from two families (from Australia and the UK) with a myopathy caused by recessive mutations in MYH7. The Australian family was homozygous for a c.5134C > T, p.Arg1712Trp mutation, whilst the UK patient was compound heterozygous for a truncating (c.4699C > T; p.Gln1567*) and a missense variant (c.4664A > G; p.Glu1555Gly). All three patients shared key clinical features, including infancy/childhood onset, pronounced axial/proximal weakness, spinal rigidity, severe scoliosis, and normal cardiac function. There was progressive respiratory impairment necessitating non-invasive ventilation despite preserved ambulation, a combination of features often seen in SEPN1- or NEB-related myopathies. On biopsy, the Australian proband showed classical myosin storage myopathy features, while the UK patient showed multi-minicore like areas. To establish pathogenicity of the Arg1712Trp mutation, we expressed mutant MYH7 protein in COS-7 cells, observing abnormal mutant myosin aggregation compared to wild-type. We describe skinned myofiber studies of patient muscle and hypertrophy of type II myofibers, which may be a compensatory mechanism. In summary, we have expanded the phenotype of ultra-rare recessive MYH7 disease, and provide novel insights into associated changes in muscle physiology.

Muscle weakness in TPM3-myopathy is due to reduced Ca2+-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres.

Dominant mutations in TPM3, encoding α-tropomyosinslow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients. We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant α-tropomyosinslow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant α-tropomyosinslow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant α-tropomyosinslow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca(2+)] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca(2+)-sensitivity, at sub-saturating [Ca(2+)] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca(2+)], and impaired acto-myosin cross-bridge cycling kinetics. Fast myofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca(2+)-sensitivity in TPM3-myopathy patients suggests Ca(2+)-sensitizing drugs may represent a useful treatment for this condition.

Muscle histopathology in nebulin-related nemaline myopathy: ultrastrastructural findings correlated to disease severity and genotype.

Nemaline myopathy (NM) is a rare congenital myopathy characterised by hypotonia, muscle weakness, and often skeletal muscle deformities with the presence of nemaline bodies (rods) in the muscle biopsy. The nebulin (NEB) gene is the most commonly mutated and is thought to account for approximately 50% of genetically diagnosed cases of NM. We undertook a detailed muscle morphological analysis of 14 NEB-mutated NM patients with different clinical forms to define muscle pathological patterns and correlate them with clinical course and genotype. Three groups were identified according to clinical severity. Group 1 (n = 5) comprises severe/lethal NM and biopsy in the first days of life. Group 2 (n = 4) includes intermediate NM and biopsy in infancy. Group 3 (n = 5) comprises typical/mild NM and biopsy in childhood or early adult life. Biopsies underwent histoenzymological, immunohistochemical and ultrastructural analysis. Fibre type distribution patterns, rod characteristics, distribution and localization were investigated. Contractile performance was studied in muscle fibre preparations isolated from seven muscle biopsies from each of the three groups. G1 showed significant myofibrillar dissociation and smallness with scattered globular rods in one third of fibres; there was no type 1 predominance. G2 presented milder sarcomeric dissociation, dispersed or clustered nemaline bodies, and type 1 predominance/uniformity. In contrast, G3 had well-delimited clusters of subsarcolemmal elongated rods and type 1 uniformity without sarcomeric alterations. In accordance with the clinical and morphological data, functional studies revealed markedly low forces in muscle bundles from G1 and a better contractile performance in muscle bundles from biopsies of patients from G2, and G3.In conclusion NEB-mutated NM patients present a wide spectrum of morphological features. It is difficult to establish firm genotype phenotype correlation. Interestingly, there was a correlation between clinical severity on the one hand and the degree of sarcomeric dissociation and contractility efficiency on the other. By contrast the percentage of fibres occupied by rods, as well as the quantity and the sub sarcolemmal position of rods, appears to inversely correlate with severity. Based on our observations, we propose myofibrillar dissociation and changes in contractility as an important cause of muscle weakness in NEB-mutated NM patients.