Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Tirasemtiv enhances submaximal muscle tension in an Acta1:p.Asp286Gly mouse model of nemaline myopathy.

Nemaline myopathies are the most common form of congenital myopathies. Variants in ACTA1 (NEM3) comprise 15-25% of all nemaline myopathy cases. Patients harboring variants in ACTA1 present with a heterogeneous disease course characterized by stable or progressive muscle weakness and, in severe cases, respiratory failure and death. To date, no specific treatments are available. Since NEM3 is an actin-based thin filament disease, we tested the ability of tirasemtiv, a fast skeletal muscle troponin activator, to improve skeletal muscle function in a mouse model of NEM3, harboring the patient-based p.Asp286Gly variant in Acta1. Acute and long-term tirasemtiv treatment significantly increased muscle contractile capacity at submaximal stimulation frequencies in both fast-twitch extensor digitorum longus and gastrocnemius muscle, and intermediate-twitch diaphragm muscle in vitro and in vivo. Additionally, long-term tirasemtiv treatment in NEM3 mice resulted in a decreased respiratory rate with preserved minute volume, suggesting more efficient respiration. Altogether, our data support the therapeutic potential of fast skeletal muscle troponin activators in alleviating skeletal muscle weakness in a mouse model of NEM3 caused by the Acta1:p.Asp286Gly variant.

L-tyrosine for treatment of an infant with nemaline rod myopathy.

Nemaline rod myopathy is an extremely rare muscle disease responsible for hypotonia and poor muscle strength in infants. The disease has variable phenotypic presentations across different ages, ranging from neonatal to the adult onset and from severe to asymptomatic varieties. Clinical features, muscle biopsy and genetic testing help in diagnosis. The histopathological examination shows the presence of rod-like structures or nemaline bodies in muscles. Management remains mainly supportive, and currently, there is no available curative treatment. This case report describes an infant presenting with gross hypotonia, poor handling of secretions and multiple extubation failures who was diagnosed by clinical exome sequencing. The patient harboured compound heterozygous variants in the NEB gene suggestive of nemaline rod myopathy. The newborn showed significant improvement in muscle strength after he was started on dietary L-tyrosine supplementation. This case highlights the emerging role of L-tyrosine in the supportive care of infants with nemaline rod myopathy.