Factors Underlying the Early Limb Muscle Weakness in Acute Quadriplegic Myopathy Using an Experimental ICU Porcine Model

The basic mechanisms underlying acquired generalized muscle weakness and paralysis in critically ill patients remain poorly understood and may be related to prolonged mechanical ventilation/immobilization (MV) or to other triggering factors such as sepsis, systemic corticosteroid (CS) treatment and administration of neuromuscular blocking agents (NMBA). The present study aims at exploring the relative importance of these factors by using a unique porcine model. Piglets were all exposed to MV together with different combinations of endotoxin-induced sepsis, CS and NMBA for five days. Peroneal motor nerve conduction velocity and amplitude of the compound muscle action potential (CMAP) as well as biceps femoris muscle biopsy specimens were obtained immediately after anesthesia on the first day and at the end of the 5-day experimental period. Results showed that peroneal nerve motor conduction velocity is unaffected whereas the size of the CMAP decreases independently of the type of intervention, in all groups after 5 days. Otherwise, despite a preserved size, muscle fibre specific force (maximum force normalized to cross-sectional area) decreased dramatically for animals exposed to MV in combination with CS or/and sepsis. These results suggest that the rapid declines in CMAP amplitude and in force generation capacity are triggered by independent mechanisms with significant clinical and therapeutic implications

Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy

Nemaline myopathy (NM) is a skeletal muscle disorder caused by mutations in genes that are generally involved in muscle contraction, in particular those related to the structure and/or regulation of the thin filament. Many pathogenic aspects of this disease remain largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mouse models and patients with NM: irregular spacing and morphology of nuclei; disrupted nuclear envelope; altered chromatin arrangement; and disorganisation of the cortical cytoskeleton. Impairments in contractility are the primary cause of these nuclear defects. We also establish the role of microtubule organisation in determining nuclear morphology, a phenomenon which is likely to contribute to nuclear alterations in this disease. Our results overlap with findings in diseases caused directly by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to explain some of the hallmarks of NM, including contractile filament disarray, altered mechanical properties and broad transcriptional alterations

Molecular Consequences of the Myopathy-Related D286G Mutation on Actin Function

Myopathies are notably associated with mutations in genes encoding proteins known to be essential for the force production of skeletal muscle fibers, such as skeletal alpha-actin. The exact molecular mechanisms by which these specific defects induce myopathic phenotypes remain unclear. Hence, in the present study, to better understand actin dysfunction, we conducted a molecular dynamic simulation together with ex vivo experiments of the specific muscle disease-causing actin mutation, D286G located in the actin-actin interface. Our computational study showed that D286G impairs the flexural rigidity of actin filaments. However, upon activation, D286G did not have any direct consequences on actin filament extension. Hence, D286G may alter the structure of actin filaments but, when expressed together with normal actin molecules, it may only have minor effects on the ex vivo mechanics of actin filaments upon skeletal muscle fiber contraction

Activation of eIF4E-binding-protein-1 rescues mTORC1-induced sarcopenia by expanding lysosomal degradation capacity

Background: Chronic mTORC1 activation in skeletal muscle is linked with age-associated loss of muscle mass and strength, known as sarcopenia. Genetic activation of mTORC1 by conditionally ablating mTORC1 upstream inhibitor TSC1 in skeletal muscle accelerates sarcopenia development in adult mice. Conversely, genetic suppression of mTORC1 downstream effectors of protein synthesis delays sarcopenia in natural aging mice. mTORC1 promotes protein synthesis by activating ribosomal protein S6 kinases (S6Ks) and inhibiting eIF4E-binding proteins (4EBPs). Whole-body knockout of S6K1 or muscle-specific over-expression of a 4EBP1 mutant transgene (4EBP1mt), which is resistant to mTORC1-mediated inhibition, ameliorates muscle loss with age and preserves muscle function by enhancing mitochondria activities, despite both transgenic mice showing retarded muscle growth at a young age. Why repression of mTORC1-mediated protein synthesis can mitigate progressive muscle atrophy and dysfunction with age remains unclear. Methods: Mice with myofiber-specific knockout of TSC1 (TSC1mKO), in which mTORC1 is hyperactivated in fully differentiated myofibers, were used as a mouse model of sarcopenia. To elucidate the role of mTORC1-mediated protein synthesis in regulating muscle mass and physiology, we bred the 4EBP1mt transgene or S6k1 floxed mice into the TSC1mKO mouse background to generate 4EBP1mt-TSC1mKO or S6K1-TSC1mKO mice, respectively. Functional and molecular analyses were performed to assess their role in sarcopenia development. Results: Here, we show that 4EBP1mt-TSC1mKO, but not S6K1-TSC1mKO, preserved muscle mass (36.7% increase compared with TSC1mKO, P < 0.001) and strength (36.8% increase compared with TSC1mKO, P < 0.01) at the level of control mice. Mechanistically, 4EBP1 activation suppressed aberrant protein synthesis (two-fold reduction compared with TSC1mKO, P < 0.05) and restored autophagy flux without relieving mTORC1-mediated inhibition of ULK1, an upstream activator of autophagosome initiation. We discovered a previously unidentified phenotype of lysosomal failure in TSC1mKO mouse muscle, in which the lysosomal defect was also conserved in the naturally aged mouse muscle, whereas 4EBP1 activation enhanced lysosomal protease activities to compensate for impaired autophagy induced by mTORC1 hyperactivity. Consequently, 4EBP1 activation relieved oxidative stress to prevent toxic aggregate accumulation (0.5-fold reduction compared with TSC1mKO, P < 0.05) in muscle and restored mitochondrial homeostasis and function. Conclusions: We identify 4EBP1 as a communication hub coordinating protein synthesis and degradation to protect proteostasis, revealing therapeutic potential for activating lysosomal degradation to mitigate sarcopenia

Human Skeletal myopathy myosin mutations disrupt myosin head sequestration

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies

Diaphragm Muscle Weakness in an Experimental Porcine Intensive Care Unit Model

In critically ill patients, mechanisms underlying diaphragm muscle remodeling and resultant dysfunction contributing to weaning failure remain unclear. Ventilator-induced modifications as well as sepsis and administration of pharmacological agents such as corticosteroids and neuromuscular blocking agents may be involved. Thus, the objective of the present study was to examine how sepsis, systemic corticosteroid treatment (CS) and neuromuscular blocking agent administration (NMBA) aggravate ventilator-related diaphragm cell and molecular dysfunction in the intensive care unit. Piglets were exposed to different combinations of mechanical ventilation and sedation, endotoxin-induced sepsis, CS and NMBA for five days and compared with sham-operated control animals. On day 5, diaphragm muscle fibre structure (myosin heavy chain isoform proportion, cross-sectional area and contractile protein content) did not differ from controls in any of the mechanically ventilated animals. However, a decrease in single fibre maximal force normalized to cross-sectional area (specific force) was observed in all experimental piglets. Therefore, exposure to mechanical ventilation and sedation for five days has a key negative impact on diaphragm contractile function despite a preservation of muscle structure. Post-translational modifications of contractile proteins are forwarded as one probable underlying mechanism. Unexpectedly, sepsis, CS or NMBA have no significant additive effects, suggesting that mechanical ventilation and sedation are the triggering factors leading to diaphragm weakness in the intensive care unit

Nebulin nemaline myopathy recapitulated in a compound heterozygous mouse model with both a missense and a nonsense mutation in Neb

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, Neb(Y2303H, Y935X), has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, Neb(Y2303H,Y935X) mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.Peer reviewe

Nebulin nemaline myopathy recapitulated in a compound heterozygous mouse model with both a missense and a nonsense mutation in Neb

Nemaline myopathy (NM) caused by mutations in the gene encoding nebulin (NEB) accounts for at least 50% of all NM cases worldwide, representing a significant disease burden. Most NEB-NM patients have autosomal recessive disease due to a compound heterozygous genotype. Of the few murine models developed for NEB-NM, most are Neb knockout models rather than harbouring Neb mutations. Additionally, some models have a very severe phenotype that limits their application for evaluating disease progression and potential therapies. No existing murine models possess compound heterozygous Neb mutations that reflect the genotype and resulting phenotype present in most patients. We aimed to develop a murine model that more closely matched the underlying genetics of NEB-NM, which could assist elucidation of the pathogenetic mechanisms underlying the disease. Here, we have characterised a mouse strain with compound heterozygous Neb mutations; one missense (p.Tyr2303His), affecting a conserved actin-binding site and one nonsense mutation (p.Tyr935*), introducing a premature stop codon early in the protein. Our studies reveal that this compound heterozygous model, Neb(Y2303H, Y935X), has striking skeletal muscle pathology including nemaline bodies. In vitro whole muscle and single myofibre physiology studies also demonstrate functional perturbations. However, no reduction in lifespan was noted. Therefore, Neb(Y2303H,Y935X) mice recapitulate human NEB-NM and are a much needed addition to the NEB-NM mouse model collection. The moderate phenotype also makes this an appropriate model for studying NEB-NM pathogenesis, and could potentially be suitable for testing therapeutic applications.Peer reviewe

Rationale and design of EXPLORE: a randomized, prospective, multicenter trial investigating the impact of recanalization of a chronic total occlusion on left ventricular function in patients after primary percutaneous coronary intervention for acute ST-elevation myocardial infarction

<p>Abstract</p> <p>Background</p> <p>In the setting of primary percutaneous coronary intervention, patients with a chronic total occlusion in a non-infarct related artery were recently identified as a high-risk subgroup. It is unclear whether ST-elevation myocardial infarction patients with a chronic total occlusion in a non-infarct related artery should undergo additional percutaneous coronary intervention of the chronic total occlusion on top of optimal medical therapy shortly after primary percutaneous coronary intervention. Possible beneficial effects include reduction in adverse left ventricular remodeling and preservation of global left ventricular function and improved clinical outcome during future coronary events.</p> <p>Methods/Design</p> <p>The Evaluating Xience V and left ventricular function in Percutaneous coronary intervention on occLusiOns afteR ST-Elevation myocardial infarction (EXPLORE) trial is a randomized, prospective, multicenter, two-arm trial with blinded evaluation of endpoints. Three hundred patients after primary percutaneous coronary intervention for ST-elevation myocardial infarction with a chronic total occlusion in a non-infarct related artery are randomized to either elective percutaneous coronary intervention of the chronic total occlusion within seven days or standard medical treatment. When assigned to the invasive arm, an everolimus-eluting coronary stent is used. Primary endpoints are left ventricular ejection fraction and left ventricular end-diastolic volume assessed by cardiac Magnetic Resonance Imaging at four months. Clinical follow-up will continue until five years.</p> <p>Discussion</p> <p>The ongoing EXPLORE trial is the first randomized clinical trial powered to investigate whether recanalization of a chronic total occlusion in a non-infarct related artery after primary percutaneous coronary intervention for ST-elevation myocardial infarction results in a better preserved residual left ventricular ejection fraction, reduced end-diastolic volume and enhanced clinical outcome.</p> <p>Trial registration</p> <p>trialregister.nl NTR1108.</p

Developmental regulation of MURF E3 ubiquitin ligases in skeletal muscle

The striated muscle-specific tripartite motif (TRIM) proteins TRIM63/MURF1, TRIM55/MURF2 and TRIM54/MURF3 can function as E3 ubiquitin ligases in ubiquitin-mediated muscle protein turnover. Despite the well-characterised role of MURF1 in skeletal muscle atrophy, the dynamics of MURF isogene expression in the development and early postnatal adaptation of skeletal muscle is unknown. Here, we show that MURF2 is the isogene most highly expressed in embryonic skeletal muscle at E15.5, with the 50 kDa A isoform predominantly expressed. MURF1 and MURF3 are upregulated only postnatally. Knockdown of MURF2 p50A by isoform-specific siRNA results in delayed myogenic differentiation and myotube formation in vitro, with perturbation of the stable, glutamylated microtubule population. This underscores that MURF2 plays an important role in the earliest stages of skeletal muscle differentiation and myofibrillogenesis. During further development, there is a shift towards the 60 kDa A isoform, which dominates postnatally. Analysis of the fibre-type expression shows that MURF2 A isoforms are predominantly slow-fibre associated, whilst MURF1 is largely excluded from these fibres, and MURF3 is ubiquitously distributed in both type I and II fibres