Super-relaxed myosins contribute to respiratory muscle hibernation in mechanically ventilated patients.

Patients receiving mechanical ventilation in the intensive care unit (ICU) frequently develop contractile weakness of the diaphragm. Consequently, they may experience difficulty weaning from mechanical ventilation, which increases mortality and poses a high economic burden. Because of a lack of knowledge regarding the molecular changes in the diaphragm, no treatment is currently available to improve diaphragm contractility. We compared diaphragm biopsies from ventilated ICU patients (N = 54) to those of non-ICU patients undergoing thoracic surgery (N = 27). By integrating data from myofiber force measurements, x-ray diffraction experiments, and biochemical assays with clinical data, we found that in myofibers isolated from the diaphragm of ventilated ICU patients, myosin is trapped in an energy-sparing, super-relaxed state, which impairs the binding of myosin to actin during diaphragm contraction. Studies on quadriceps biopsies of ICU patients and on the diaphragm of previously healthy mechanically ventilated rats suggested that the super-relaxed myosins are specific to the diaphragm and not a result of critical illness. Exposing slow- and fast-twitch myofibers isolated from the diaphragm biopsies to small-molecule compounds activating troponin restored contractile force in vitro. These findings support the continued development of drugs that target sarcomere proteins to increase the calcium sensitivity of myofibers for the treatment of ICU-acquired diaphragm weakness.

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.

Characterization of NEB mutations 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. Mutations in the nebulin gene ( NEB ) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations 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 splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a 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 I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, 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 splicing mutations. 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.