Scientific Report: Analysis of NEB Pathogenic Variants Reveals Novel Mechanisms of Nemaline Myopathy and the Mechanical Effects of Omecamtiv Mecarbil

Background and Motivation

Nemaline Myopathy (NEM) is a rare and heterogeneous genetic disorder primarily characterized by hypotonia and muscle weakness. Pathologically, the disease is caused by the disorganization of the sarcomeric Z-disc and the accumulation of nemaline bodies, which are aggregates of Z-disc and thin filament proteins. It is estimated that the incidence rate of NEM is about two cases per 100,000 live births, accounting for 17% of congenital myopathies. Currently, there is no effective treatment available for this disease.

NEM involves pathogenic variants of at least 13 genes, among which mutations in the NEB gene are a major cause, accounting for 35% of pathological variants. The NEB gene encodes a giant thin filament protein called nebulin, which plays a crucial role in various important physiological processes in skeletal muscle, such as regulating thin filament length (TFL), sarcomere alignment, and cross-bridge cycling. Therefore, this paper conducted a detailed study on the pathogenic variants of the NEB gene and their effects on mRNA, protein, and mechanical properties, aiming to reveal new pathogenic mechanisms and to validate the potential of the drug Omecamtiv Mecarbil in improving muscle function in patients with NEM.

Research Sources

This research was jointly completed by Esmat Karimi, Jochen Gohlke, Mila Van Der Borgh, and others at the University of Arizona, Medical College of Wisconsin, Amsterdam UMC, and other institutions. It was published in the 2024 issue of Acta Neuropathologica.

Research Process

Study Subjects and Sample Processing

The study included 10 diagnosed NEM2 patients with various pathogenic variant types, and 3 healthy individuals without muscle-related symptoms as a control group. All muscle biopsy samples were obtained from the Congenital Muscle Disease Tissue Repository (CMD-TR) at the Medical College of Wisconsin, freeze-preserved to -80°C, and subsequently subjected to RNA sequencing and protein expression identification experiments.

RNA Sequencing

Frozen muscle tissues were processed for RNA extraction and RNA sequencing. Post-sequencing, specific software was used to analyze the RNA data to examine the impact of variants on NEB gene transcription and RNA splicing. The results showed that most truncating variants affected the stability of NEB mRNA, leading to nonsense-mediated decay (NMD). Furthermore, a high proportion of cryptic splice site activation was observed in patient samples, suggesting these cryptic splice sites are typically activated only under the influence of variants.

Protein Expression Identification

SDS-agarose gel electrophoresis and Western blotting were used to evaluate nebulin protein expression. The detected nebulin protein levels were normalized against myosin heavy chain (MHC) in skeletal muscle. The results indicated that some patients had nearly normal levels of nebulin protein, whereas others had significantly reduced levels. Additionally, the α-helical segment of nebulin predicted by AlphaFold showed that insertions caused by cryptic splice sites might disrupt actin-binding sites.

Mechanical Properties of Muscle Fibers

Mechanical analysis was conducted on permeabilized single fibers to measure the force exerted by muscles at different calcium levels, including maximum and submaximal forces before and after treatment with the drug Omecamtiv Mecarbil (OM). The results indicated that OM significantly increased submaximal force in patients, especially those with the lowest levels of nebulin protein.

Thin Filament Length Measurement

Muscle tissues were stained using fluorescently labeled phalloidin to measure thin filament length. Some patients showed shortened thin filament lengths, whereas one patient with extended nebulin protein displayed longer thin filament lengths.

Key Findings and Conclusions

Genetic Variants and Their Effects on Transcription and Protein Levels

The study found that truncating variants generally led to the degradation of mRNA due to NMD. This mechanism explains why truncated nebulin protein is undetected at the protein level. Additionally, the activation of cryptic splice sites led to aberrant RNA splicing and atypical insertions, impacting NEB transcription and protein levels to varying extents.

Relationship between Protein Expression Levels and Muscle Function

Reduced nebulin protein levels were negatively correlated with shortened thin filament length (TFL) and decreased muscle tension. This phenomenon was most pronounced in patients with the lowest protein levels. Furthermore, the novel finding that patient 180, with extended protein structure due to NEB expansion variant, exhibited longer TFL suggests that NEM may involve both short and long thin filaments in its pathology.

Potential for Drug Therapy

OM, a small molecule activator, was shown to significantly enhance submaximal force in NEM2 patients, particularly those with the lowest levels of nebulin. OM increased calcium sensitivity and prolonged the duration of strong-binding myosin heads, thus significantly enhancing force at submaximal activation levels. This finding lays a foundation for the potential application of OM in treating NEM patients in the future.

Significance and Application Value of the Study

This research is the first to reveal new mechanisms of NEB pathogenic variants, including the activation of cryptic splice sites leading to aberrant splicing and its impact on nebulin and thin filament length. More importantly, the study suggests that OM could become an effective drug for improving muscle function in NEM2 patients, especially those with low nebulin levels. These findings enrich the understanding of the molecular mechanisms underlying nemaline myopathy and provide valuable information for developing targeted treatments.

Research Highlights

1. Reveals New Pathogenic Mechanisms: First discovery of aberrant RNA splicing due to activation of cryptic splice sites and its impact on protein function.
2. Potential of OM Therapy: Finds that OM can significantly enhance muscle force in NEM2 patients, particularly those with low nebulin levels.
3. Relationship between Protein Levels and Function: Establishes a negative correlation between nebulin protein levels, thin filament length, and muscle force through detailed studies, providing important evidence for future research.

This study offers new ideas and potential solutions for the treatment of nemaline myopathy, possessing high scientific and clinical application value.