Comprehensive phenotypic characterization of an allelic series of zebrafish models of NEB-related nemaline myopathy.

Nemaline myopathy (NM) is a rare congenital neuromuscular disorder characterized by muscle weakness and hypotonia, slow gross motor development, and decreased respiratory function. Mutations in at least twelve genes, all of each encode proteins that are either components of the muscle thin filament or regulate its length and stability, have been associated with NM. Mutations in Nebulin (NEB), a giant filamentous protein localized in the sarcomere, account for more than 50% of NM cases. At present, there remains a lack of understanding of whether NEB genotype influences nebulin function and NM-patient phenotypes. In addition, there is a lack of therapeutically tractable models that can enable drug discovery and address the current unmet treatment needs of patients. To begin to address these gaps, here we have characterized five new zebrafish models of NEB-related NM. These mutants recapitulate most aspects of NEB-based NM, showing drastically reduced survival, defective muscle structure, reduced contraction force, shorter thin filaments, presence of electron-dense structures in myofibers, and thickening of the Z-disks. This study represents the first extensive investigation of an allelic series of nebulin mutants, and thus provides an initial examination in pre-clinical models of potential genotype-phenotype correlations in human NEB patients. It also represents the first utilization of a set of comprehensive outcome measures in zebrafish, including correlation between molecular analyses, structural and biophysical investigations, and phenotypic outcomes. Therefore, it provides a rich source of data for future studies exploring the NM pathomechanisms, and an ideal springboard for therapy identification and development for NEB-related NM.

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

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.

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.

Pathogenic Variants in the Myosin Chaperone UNC-45B Cause Progressive Myopathy with Eccentric Cores.

The myosin-directed chaperone UNC-45B is essential for sarcomeric organization and muscle function from Caenorhabditis elegans to humans. The pathological impact of UNC-45B in muscle disease remained elusive. We report ten individuals with bi-allelic variants in UNC45B who exhibit childhood-onset progressive muscle weakness. We identified a common UNC45B variant that acts as a complex hypomorph splice variant. Purified UNC-45B mutants showed changes in folding and solubility. In situ localization studies further demonstrated reduced expression of mutant UNC-45B in muscle combined with abnormal localization away from the A-band towards the Z-disk of the sarcomere. The physiological relevance of these observations was investigated in C. elegans by transgenic expression of conserved UNC-45 missense variants, which showed impaired myosin binding for one and defective muscle function for three. Together, our results demonstrate that UNC-45B impairment manifests as a chaperonopathy with progressive muscle pathology, which discovers the previously unknown conserved role of UNC-45B in myofibrillar organization.

Molecular Mechanisms of Diaphragm Myopathy in Humans With Severe Heart Failure.

[Figure: see text].

KBTBD13 is a novel cardiomyopathy gene.

KBTBD13 variants cause nemaline myopathy type 6 (NEM6). The majority of NEM6 patients harbors the Dutch founder variant, c.1222C>T, p.Arg408Cys (KBTBD13 p.R408C). Although KBTBD13 is expressed in cardiac muscle, cardiac involvement in NEM6 is unknown. Here, we constructed pedigrees of three families with the KBTBD13 p.R408C variant. In 65 evaluated patients, 12% presented with left ventricle dilatation, 29% with left ventricular ejection fraction< 50%, 8% with atrial fibrillation, 9% with ventricular tachycardia, and 20% with repolarization abnormalities. Five patients received an implantable cardioverter defibrillator, three cases of sudden cardiac death were reported. Linkage analysis confirmed cosegregation of the KBTBD13 p.R408C variant with the cardiac phenotype. Mouse studies revealed that (1) mice harboring the Kbtbd13 p.R408C variant display mild diastolic dysfunction; (2) Kbtbd13-deficient mice have systolic dysfunction. Hence, (1) KBTBD13 is associated with cardiac dysfunction and cardiomyopathy; (2) KBTBD13 should be added to the cardiomyopathy gene panel; (3) NEM6 patients should be referred to the cardiologist.

Lung- and Diaphragm-Protective Ventilation by Titrating Inspiratory Support to Diaphragm Effort: A Randomized Clinical Trial.

OBJECTIVES: Lung- and diaphragm-protective ventilation is a novel concept that aims to limit the detrimental effects of mechanical ventilation on the diaphragm while remaining within limits of lung-protective ventilation. The premise is that low breathing effort under mechanical ventilation causes diaphragm atrophy, whereas excessive breathing effort induces diaphragm and lung injury. In a proof-of-concept study, we aimed to assess whether titration of inspiratory support based on diaphragm effort increases the time that patients have effort in a predefined "diaphragm-protective" range, without compromising lung-protective ventilation. DESIGN: Randomized clinical trial. SETTING: Mixed medical-surgical ICU in a tertiary academic hospital in the Netherlands. PATIENTS: Patients (n = 40) with respiratory failure ventilated in a partially-supported mode. INTERVENTIONS: In the intervention group, inspiratory support was titrated hourly to obtain transdiaphragmatic pressure swings in the predefined "diaphragm-protective" range (3-12 cm H2O). The control group received standard-of-care. MEASUREMENTS AND MAIN RESULTS: Transdiaphragmatic pressure, transpulmonary pressure, and tidal volume were monitored continuously for 24 hours in both groups. In the intervention group, more breaths were within "diaphragm-protective" range compared with the control group (median 81%; interquartile range [64-86%] vs 35% [16-60%], respectively; p < 0.001). Dynamic transpulmonary pressures (20.5 ± 7.1 vs 18.5 ± 7.0 cm H2O; p = 0.321) and tidal volumes (7.56 ± 1.47 vs 7.54 ± 1.22 mL/kg; p = 0.961) were not different in the intervention and control group, respectively. CONCLUSIONS: Titration of inspiratory support based on patient breathing effort greatly increased the time that patients had diaphragm effort in the predefined "diaphragm-protective" range without compromising tidal volumes and transpulmonary pressures. This study provides a strong rationale for further studies powered on patient-centered outcomes.

Assessment of PABPN1 nuclear inclusions on a large cohort of patients and in a human xenograft model of oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a rare muscle disease characterized by an onset of weakness in the pharyngeal and eyelid muscles. The disease is caused by the extension of a polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) protein leading to the formation of intranuclear inclusions or aggregates in the muscle of OPMD patients. Despite numerous studies stressing the deleterious role of nuclear inclusions in cellular and animal OPMD models, their exact contribution to human disease is still unclear. In this study, we used a large and unique collection of human muscle biopsy samples to perform an in-depth analysis of PABPN1 aggregates in relation to age, genotype and muscle status with the final aim to improve our understanding of OPMD physiopathology. Here we demonstrate that age and genotype influence PABPN1 aggregates: the percentage of myonuclei containing PABPN1 aggregates increases with age and the chaperone HSP70 co-localize more frequently with PABPN1 aggregates with a larger polyalanine tract. In addition to the previously described PRMT1 and HSP70 co-factors, we identified new components of PABPN1 aggregates including GRP78/BiP, RPL24 and p62. We also observed that myonuclei containing aggregates are larger than myonuclei without. When comparing two muscles from the same patient, a similar amount of aggregates is observed in different muscles, except for the pharyngeal muscle where fewer aggregates are observed. This could be due to the peculiar nature of this muscle which has a low level of PAPBN1 and contains regenerating fibers. To confirm the fate of PABPN1 aggregates in a regenerating muscle, we generated a xenograft model by transplanting human OPMD muscle biopsy samples into the hindlimb of an immunodeficient mouse. Xenografts from subjects with OPMD displayed regeneration of human myofibers and PABPN1 aggregates were rapidly present-although to a lower extent-after muscle fiber regeneration. Our data obtained on human OPMD samples add support to the dual non-exclusive models in OPMD combining toxic PABPN1 intranuclear inclusions together with PABPN1 loss of function which altogether result in this late-onset and muscle selective disease.

Association of diaphragm thickness and echogenicity with age, sex, and body mass index in healthy subjects.

INTRODUCTION/AIMS: Diaphragm ultrasound is increasingly used in the diagnosis of diaphragm dysfunction and to guide respiratory management in patients with neuromuscular disorders and those who are critically ill. However, the association between diaphragm ultrasound variables and demographic factors like age, sex, and body mass index (BMI) are understudied. Such relationships are important for correct interpretation of normative values and comparison with selected patients groups. The aim of this study was to determine the associations between diaphragm ultrasound variables and subject characteristics. METHODS: B-mode ultrasound was used to image the diaphragm at the zone of apposition in 83 healthy subjects. Diaphragm thickness at resting end-expiration (Tend-exp ), diaphragm thickness at maximal end-inspiration (Tmax-insp ), diaphragm thickening ratio (Tmax-insp /Tend-exp ), and diaphragm echogenicity were measured. Multivariate linear regression was used to explore the associations between diaphragm ultrasound variables and subject characteristics. RESULTS: Tend-exp , Tmax-insp , and thickening ratio do not change with age whereas diaphragm echogenicity increases with age. The thickening ratio had a weak negative association with BMI, while Tend-exp was positively associated with BMI. Men had a larger Tend-exp and Tmax-insp than women (Tend-exp 1.6 ± 0.5 and 1.4 ± 0.3 mm; p = .011, Tmax-insp 3.8 ± 1.0 and 3.2 ± 0.9 mm; p = .004), but similar thickening ratios. DISCUSSION: Diaphragm thickness, thickening, and echogenicity measured with ultrasound are associated with factors such as age, BMI, and sex. Therefore, subject characteristics should be considered when interpreting diaphragm ultrasound measurements. In the absence of normative values, matched control groups are a prerequisite for research and in clinical practice.

Early Restrictive Fluid Strategy Impairs the Diaphragm Force in Lambs with Acute Respiratory Distress Syndrome.

BACKGROUND: The effect of fluid management strategies in critical illness-associated diaphragm weakness are unknown. This study hypothesized that a liberal fluid strategy induces diaphragm muscle fiber edema, leading to reduction in diaphragmatic force generation in the early phase of experimental pediatric acute respiratory distress syndrome in lambs. METHODS: Nineteen mechanically ventilated female lambs (2 to 6 weeks old) with experimental pediatric acute respiratory distress syndrome were randomized to either a strict restrictive fluid strategy with norepinephrine or a liberal fluid strategy. The fluid strategies were maintained throughout a 6-h period of mechanical ventilation. Transdiaphragmatic pressure was measured under different levels of positive end-expiratory pressure (between 5 and 20 cm H2O). Furthermore, diaphragmatic microcirculation, histology, inflammation, and oxidative stress were studied. RESULTS: Transdiaphragmatic pressures decreased more in the restrictive group (-9.6 cm H2O [95% CI, -14.4 to -4.8]) compared to the liberal group (-0.8 cm H2O [95% CI, -5.8 to 4.3]) during the application of 5 cm H2O positive end-expiratory pressure (P = 0.016) and during the application of 10 cm H2O positive end-expiratory pressure (-10.3 cm H2O [95% CI, -15.2 to -5.4] vs. -2.8 cm H2O [95% CI, -8.0 to 2.3]; P = 0.041). In addition, diaphragmatic microvessel density was decreased in the restrictive group compared to the liberal group (34.0 crossings [25th to 75th percentile, 22.0 to 42.0] vs. 46.0 [25th to 75th percentile, 43.5 to 54.0]; P = 0.015). The application of positive end-expiratory pressure itself decreased the diaphragmatic force generation in a dose-related way; increasing positive end-expiratory pressure from 5 to 20 cm H2O reduced transdiaphragmatic pressures with 27.3% (17.3 cm H2O [95% CI, 14.0 to 20.5] at positive end-expiratory pressure 5 cm H2O vs. 12.6 cm H2O [95% CI, 9.2 to 15.9] at positive end-expiratory pressure 20 cm H2O; P < 0.0001). The diaphragmatic histology, markers for inflammation, and oxidative stress were similar between the groups. CONCLUSIONS: Early fluid restriction decreases the force-generating capacity of the diaphragm and diaphragmatic microcirculation in the acute phase of pediatric acute respiratory distress syndrome. In addition, the application of positive end-expiratory pressure decreases the force-generating capacity of the diaphragm in a dose-related way. These observations provide new insights into the mechanisms of critical illness-associated diaphragm weakness.

Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits.

Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function.

Rbm20ΔRRM Mice, Expressing a Titin Isoform with Lower Stiffness, Are Protected from Mechanical Ventilation-Induced Diaphragm Weakness.

Diaphragm weakness frequently develops in mechanically ventilated critically ill patients and is associated with increased morbidity, including ventilator weaning failure, mortality, and health care costs. The mechanisms underlying diaphragm weakness are incompletely understood but may include the elastic properties of titin, a giant protein whose layout in the muscle's sarcomeres makes it an ideal candidate to sense ventilation-induced diaphragm unloading, resulting in downstream signaling through titin-binding proteins. In the current study, we investigated whether modulating titin stiffness affects the development of diaphragm weakness during mechanical ventilation. To this end, we ventilated genetically engineered mice with reduced titin stiffness (Rbm20ΔRRM), and robust (TtnΔIAjxn) or severely (TtnΔ112-158) increased titin stiffness for 8 h, and assessed diaphragm contractility and protein expression of titin-binding proteins. Mechanical ventilation reduced the maximum active tension of the diaphragm in WT, TtnΔIAjxn and TtnΔ112-158 mice. However, in Rbm20ΔRRM mice maximum active tension was preserved after ventilation. Analyses of titin binding proteins suggest that muscle ankyrin repeat proteins (MARPs) 1 and 2 may play a role in the adaptation of the diaphragm to mechanical ventilation, and the preservation of diaphragm contractility in Rbm20ΔRRM mice. Thus, Rbm20ΔRRM mice, expressing titin isoforms with lower stiffness, are protected from mechanical ventilation-induced diaphragm weakness, suggesting that titin elasticity may modulate the diaphragm's response to unloading during mechanical ventilation.

Reduced specific force in patients with mild and severe facioscapulohumeral muscular dystrophy.

BACKGROUND: Specific force, that is the amount of force generated per unit of muscle tissue, is reduced in patients with facioscapulohumeral muscular dystrophy (FSHD). The causes of reduced specific force and its relation with FSHD disease severity are unknown. METHODS: Quantitative muscle magnetic resonance imaging (MRI), measurement of voluntary maximum force generation and quadriceps force-frequency relationship, and vastus lateralis muscle biopsies were performed in 12 genetically confirmed patients with FSHD and 12 controls. RESULTS: Specific force was reduced by ~33% in all FSHD patients independent of disease severity. Quadriceps force-frequency relationship shifted to the right in severe FSHD compared to controls. Fiber type distribution in vastus lateralis muscle biopsies did not differ between groups. CONCLUSIONS: Reduced quadriceps specific force is present in all FSHD patients regardless of disease severity or fatty infiltration. Early myopathic changes, including fibrosis, and non-muscle factors, such as physical fatigue and musculoskeletal pain, may contribute to reduced specific force.

Changes in Respiratory Muscle Thickness during Mechanical Ventilation: Focus on Expiratory Muscles.

BACKGROUND: The lateral abdominal wall muscles are recruited with active expiration, as may occur with high breathing effort, inspiratory muscle weakness, or pulmonary hyperinflation. The effects of critical illness and mechanical ventilation on these muscles are unknown. This study aimed to assess the reproducibility of expiratory muscle (i.e., lateral abdominal wall muscles and rectus abdominis muscle) ultrasound and the impact of tidal volume on expiratory muscle thickness, to evaluate changes in expiratory muscle thickness during mechanical ventilation, and to compare this to changes in diaphragm thickness. METHODS: Two raters assessed the interrater and intrarater reproducibility of expiratory muscle ultrasound (n = 30) and the effect of delivered tidal volume on expiratory muscle thickness (n = 10). Changes in the thickness of the expiratory muscles and the diaphragm were assessed in 77 patients with at least two serial ultrasound measurements in the first week of mechanical ventilation. RESULTS: The reproducibility of the measurements was excellent (interrater intraclass correlation coefficient: 0.994 [95% CI, 0.987 to 0.997]; intrarater intraclass correlation coefficient: 0.992 [95% CI, 0.957 to 0.998]). Expiratory muscle thickness decreased by 3.0 ± 1.7% (mean ± SD) with tidal volumes of 481 ± 64 ml (P < 0.001). The thickness of the expiratory muscles remained stable in 51 of 77 (66%), decreased in 17 of 77 (22%), and increased in 9 of 77 (12%) patients. Reduced thickness resulted from loss of muscular tissue, whereas increased thickness mainly resulted from increased interparietal fasciae thickness. Changes in thickness of the expiratory muscles were not associated with changes in the thickness of the diaphragm (R2 = 0.013; P = 0.332). CONCLUSIONS: Thickness measurement of the expiratory muscles by ultrasound has excellent reproducibility. Changes in the thickness of the expiratory muscles occurred in 34% of patients and were unrelated to changes in diaphragm thickness. Increased expiratory muscle thickness resulted from increased thickness of the fasciae.

Lung- and Diaphragm-Protective Ventilation.

Mechanical ventilation can cause acute diaphragm atrophy and injury, and this is associated with poor clinical outcomes. Although the importance and impact of lung-protective ventilation is widely appreciated and well established, the concept of diaphragm-protective ventilation has recently emerged as a potential complementary therapeutic strategy. This Perspective, developed from discussions at a meeting of international experts convened by PLUG (the Pleural Pressure Working Group) of the European Society of Intensive Care Medicine, outlines a conceptual framework for an integrated lung- and diaphragm-protective approach to mechanical ventilation on the basis of growing evidence about mechanisms of injury. We propose targets for diaphragm protection based on respiratory effort and patient-ventilator synchrony. The potential for conflict between diaphragm protection and lung protection under certain conditions is discussed; we emphasize that when conflicts arise, lung protection must be prioritized over diaphragm protection. Monitoring respiratory effort is essential to concomitantly protect both the diaphragm and the lung during mechanical ventilation. To implement lung- and diaphragm-protective ventilation, new approaches to monitoring, to setting the ventilator, and to titrating sedation will be required. Adjunctive interventions, including extracorporeal life support techniques, phrenic nerve stimulation, and clinical decision-support systems, may also play an important role in selected patients in the future. Evaluating the clinical impact of this new paradigm will be challenging, owing to the complexity of the intervention. The concept of lung- and diaphragm-protective ventilation presents a new opportunity to potentially improve clinical outcomes for critically ill patients.

Troponin Variants in Congenital Myopathies: How They Affect Skeletal Muscle Mechanics.

The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies. TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca2+ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.

Nebulin: big protein with big responsibilities.

Nebulin, encoded by NEB, is a giant skeletal muscle protein of about 6669 amino acids which forms an integral part of the sarcomeric thin filament. In recent years, the nebula around this protein has been largely lifted resulting in the discovery that nebulin is critical for a number of tasks in skeletal muscle. In this review, we firstly discussed nebulin's role as a structural component of the thin filament and the Z-disk, regulating the length and the mechanical properties of the thin filament as well as providing stability to myofibrils by interacting with structural proteins within the Z-disk. Secondly, we reviewed nebulin's involvement in the regulation of muscle contraction, cross-bridge cycling kinetics, Ca2+-homeostasis and excitation contraction (EC) coupling. While its role in Ca2+-homeostasis and EC coupling is still poorly understood, a large number of studies have helped to improve our knowledge on how nebulin affects skeletal muscle contractile mechanics. These studies suggest that nebulin affects the number of force generating actin-myosin cross-bridges and may also affect the force that each cross-bridge produces. It may exert this effect by interacting directly with actin and myosin and/or indirectly by potentially changing the localisation and function of the regulatory complex (troponin and tropomyosin). Besides unravelling the biology of nebulin, these studies are particularly helpful in understanding the patho-mechanism of myopathies caused by NEB mutations, providing knowledge which constitutes the critical first step towards the development of therapeutic interventions. Currently, effective treatments are not available, although a number of therapeutic strategies are being investigated.