Characterization of NEB mutations in Nemaline myopathy patients and effect of Omecamtiv mecarbil on force.

Nebulin, a critical protein of the skeletal muscle thin filaments, is integral to vital physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Mutations in the nebulin gene (NEB) underlie 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 distinct mutations, aiming to understand their impact on mRNA and protein levels, along with the mechanical properties of skeletal muscle. Obtained results support that truncation mutations affect NEB mRNA stability and lead to NMD (nonsense mediated decay) of the mutated transcript. Moreover, our results show cryptic donor splice site activation and consequent aberrant splicing in all patients with splicing mutations. Protein findings revealed two patient groups based on nebulin expression: those with relatively normal levels (NEM2-N) and those with reduced nebulin levels (NEM2-R). We observed a positive correlation between nebulin levels and TFL, as well between nebulin levels and tension (both maximal and submaximal), however, a direct correlation between nebulin levels and mRNA was not evident. Interestingly, our study unveiled that a duplication mutation in nebulin resulted in an elongated nebulin protein and subsequently longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM) on force production of skeletal muscle of NEM2 patients. OM treatment substantially increased submaximal tension of slow skeletal fibers across all NEM2 patients ranging from 87-318% and tension increase due to OM was inversely proportional to nebulin content. In summary, our study indicates the engagement of post-transcriptional or post-translational mechanisms in nebulin expression regulation. Furthermore, we propose that not only shortened but also elongated thin filaments are implicated in the pathomechanism of NEM2. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with diminished nebulin levels.