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Mammalian cells exhibit remarkable diversity in cell size, but the factors that regulate establishment and maintenance of these sizes remain poorly understood. This is especially true for skeletal muscle, comprised of syncytial myofibers that each accrue hundreds of nuclei during development. Here, we directly explore the assumed causal relationship between multinucleation and establishment of normal size through titration of myonuclear numbers during mouse neonatal development. Three independent mouse models, where myonuclear numbers were reduced by 75, 55, or 25%, led to the discovery that myonuclei possess a reserve capacity to support larger functional cytoplasmic volumes in developing myofibers. Surprisingly, the results revealed an inverse relationship between nuclei numbers and reserve capacity. We propose that as myonuclear numbers increase, the range of transcriptional return on a per nuclear basis in myofibers diminishes, which accounts for both the absolute reliance developing myofibers have on nuclear accrual to establish size, and the limits of adaptability in adult skeletal muscle., Skeletal muscle is composed of syncytial myofibres, each containing hundreds of nuclei. Through genetic reduction of the number of nuclei per myofibre, the authors confirm that more nuclei produce larger cells but myofibres with fewer nuclei adaptively compensate leading to larger and functional myonuclear domains.

Skeletal muscle weakness is an important determinant of age-related declines in independence and quality of life but its causes remain unclear. Accelerated ageing syndromes such as Hutchinson–Gilford Progerin Syndrome, caused by mutations in genes encoding nuclear envelope proteins, have been extensively studied to aid our understanding of the normal biological ageing process. Like several other pathologies associated with genetic defects to nuclear envelope proteins including Emery–Dreifuss muscular dystrophy, Limb–Girdle muscular dystrophy and congenital muscular dystrophy, these disorders can lead to severe muscle dysfunction. Here, we first describe the structure and function of nuclear envelope proteins, and then review the mechanisms by which mutations in genes encoding nuclear envelope proteins induce premature ageing diseases and muscle pathologies. In doing so, we highlight the potential importance of such genes in processes leading to skeletal muscle weakness in old age.

Aims: This study was designed to evaluate the effects of Efavirenz-based highly active antiretroviral therapy (EFVb-HAART, Efavirenz/Lamivudine/Tenofovir) with emphasis on survival, longevity, climbing ability, and reproductive capacity in D. melanogaster. Methods: The experiments were carried out at the Africa Center of Excellence in Phytomedicine Research and Development (ACEPRD), University of Jos, Nigeria between January 2017 and August 2018. D. melanogaster (both sexes) 1-4 days old were exposed to different concentrations of EFVb-HAART (range 10-1200 mg) in the fly food for initial 7 days to determine the LD50, then 5 day fly exposure to 93.11 mg, 46.56 mg, 23.28 mg or 11.64 mg for negative geotaxis assay, and acetylcholinesterase (AChE) activity. Furthermore, 28-day fly survival and longevity were determined. Statistical significance was presumed at P< 0.05. Results: The LD50 of EFVb-HAART in D. melanogaster was 93.11 mg. The HAART exposed flies showed significantly (P

Objective: To screen the toxic effect of Mangifera indica aqueous leaf extract in Drosophila melanogaster. Materials and Methods: Phytochemical screening was carried out. 20 Adult flies were exposed to 7.5 mg, 15 mg, 30 mg, 45 mg and 100 mg /10 g diet for acute toxicity (168hrs) while 50 flies were exposed to 2.5 mg, 5 mg and 10 mg/10 g diet for sub chronic (28 days). All concentrations were prepared in 1000µl of distilled water and replicated three (3) times. Diet+1000µl of Distilled water served as control. Fecundity/developmental toxicity, Climbing and AChE activities were carried out by exposing flies to the sub-chronic concentrations for 5 days. Results: Phytochemical screening revealed the presence of alkaloids, phenols, flavonoids, tannins, saponins and terpenoids. The acute toxicity test showed 100% mortality at 100 mg/ 10 g diet and 168hrs LC50 was 72.4 mg/10 g diet. The sub chronic toxicity test showed decrease in flies survival along concentration with a least survival at 10 mg/10 g diet. There was a slight reduction and elevation in the Climbing and AChE activities respectively but not statistically significant (p>0.05) compared to control. At 5 mg and 10 mg/10 g diet there was a delay in the development with few emerged flies. Conclusion: From the Results, it can be concluded that Mangiferaindica aqueous leaf extract may be toxic at high dose from 72.4 mg/10 g diet and might have an adverse effect on the development and survival of flies at sub chronic concentration as low as 2.5 mg/10g diet.

Skeletal muscle fibres are large, elongated multinucleated cells. Each nucleus within a myofibre is responsible for generating gene products for a finite volume of cytoplasm-the myonuclear domain (MND). Variation in MND sizes during atrophy, hypertrophy and disease states, are common. The factors that contribute to definitive MND sizes are not yet fully understood. Previous work has shown that peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1-α) modulates MND volume, presumably to support increased biogenesis of mitochondria. The transcriptional co-regulator peroxisome proliferator-activated receptor gamma coactivator 1β (PGC1-β) is a homologue of PGC1-α with overlapping functions. To investigate the role of this protein in MND size regulation, we studied a mouse skeletal muscle specific knockout (cKO). Myofibres were isolated from the fast twitch extensor digitorum longus (EDL) muscle, membrane-permeabilised and analysed in 3 dimensions using confocal microscopy. PGC1-β ablation resulted in no significant difference in MND size between cKO and wild type (WT) mice, however, subtle differences in nuclear morphology were observed. To determine whether these nuclear shape changes were associated with alterations in global transcriptional activity, acetyl histone H3 immunostaining was carried out. We found there was no significant difference in nuclear fluorescence intensity between the two genotypes. Overall, the results suggest that PGC-1α and PGC-1β play different roles in regulating nuclear organisation in skeletal muscle; however, further work is required to pinpoint their exact functions.

Objective: To evaluate in vivo antioxidant activity of Mangifera indica cold aqueous leaf extract. Methods: A number of 50 adult flies were exposed to graded concentrations of Mangifera indca cold aqueous leaf extract, 2.5 mg/10 g diet, 5 mg/10 g diet and 10 mg/10 g diet for 5 days. Each concentration was prepared in 200 µl of distilled water and replicated five times. 10 g diet with 200 µl distilled water served as control. Mortality reading was taken at 24 hours interval. The flies were homogenized, centrifuged and the supernatant was used to assay for Glutathione-S-transferase (GST), Catalase (CAT) and Total thiol content. Results: The % mortality of flies after 5 days showed 32.5%, 0%, 15.5% and 37% in the control (10 g diet with 200 µl of distilled water), 2.5 mg/10 g diet, 5 mg/10 g diet and 10 mg/ 10 g diet respectively. There was elevation in total thiol content and high GST and CAT activity in 2.5 mg/10 g diet and 5 mg/10 g diet treated flies. Conclusion: The 100% and 85% survival of 2.5 mg/10 g and 5 mg/10 g diet-treated flies respectively and increase of fly antioxidant system after 5 days exposure at these concentrations may suggest protective activity of Mangifera indica in D. melanogaster.

BackgroundMyofibrillar myopathy (MFM) of unknown aetiology has recently been identified in Warmblood (WB) horses. In humans, 16 genes have been implicated in various MFM-like disorders.ObjectivesTo identify variants in 16 MFM candidate genes and compare allele frequencies of all variants between MFM WB and non-MFM WB and coding variants with moderate or severe predicted effects in MFM WB with publicly available data of other breeds. To compare differential gene expression and muscle fibre contractile force between MFM and non-MFM WB.Study designCase-control.Animals8 MFM WB, 8 non-MFM WB, 33 other WB, 32 Thoroughbreds, 80 Quarter Horses and 77 horses of other breeds in public databases.MethodsVariants were called within transcripts of 16 candidate genes using gluteal muscle mRNA sequences aligned to EquCab3.0 and allele frequencies compared by Fisher's exact test among MFM WB, non-MFM WB and public sequences across breeds. Candidate gene differential expression was determined between MFM and non-MFM WB by fitting a negative binomial generalised log-linear model per gene (false discovery rate

The transcriptional demands of skeletal muscle fibres are high and require hundreds of nuclei (myonuclei) to produce specialised contractile machinery and multiple mitochondria along their length. Each myonucleus spatially regulates gene expression in a finite volume of cytoplasm, termed the myonuclear domain (MND), which positively correlates with fibre cross-sectional area (CSA). Endurance training triggers adaptive responses in skeletal muscle, including myonuclear accretion, decreased MND sizes and increased expression of the transcription co-activator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). Previous work has shown that overexpression of PGC-1α in skeletal muscle regulates mitochondrial biogenesis, myonuclear accretion and MND volume. However, whether PGC-1α is critical for these processes in adaptation to endurance training remained unclear. To test this, we evaluated myonuclear distribution and organisation in endurance-trained wild-type mice and mice lacking PGC-1α in skeletal muscle (PGC-1α mKO). Here, we show a differential myonuclear accretion response to endurance training that is governed by PGC-1α and is dependent on muscle fibre size. The positive relationship of MND size and muscle fibre CSA trended towards a stronger correlation in PGC-1a mKO versus control after endurance training, suggesting that myonuclear accretion was slightly affected with increasing fibre CSA in PGC-1α mKO. However, in larger fibres, the relationship between MND and CSA was significantly altered in trained versus sedentary PGC-1α mKO, suggesting that PGC-1α is critical for myonuclear accretion in these fibres. Accordingly, there was a negative correlation between the nuclear number and CSA, suggesting that in larger fibres myonuclear numbers fail to scale with CSA. Our findings suggest that PGC-1α is an important contributor to myonuclear accretion following moderate-intensity endurance training. This may contribute to the adaptive response to endurance training by enabling a sufficient rate of transcription of genes required for mitochondrial biogenesis.

X-linked myotubular myopathy (XLMTM) is a life-threatening skeletal muscle disease caused by mutations in the MTM1 gene. XLMTM fibres display a population of nuclei mispositioned in the centre. In the present study, we aimed to explore whether positioning and overall distribution of nuclei affects cellular organization and contractile function, thereby contributing to muscle weakness in this disease. We also assessed whether gene therapy alters nuclear arrangement and function. We used tissue from human patients and animal models, including XLMTM dogs that had received increasing doses of recombinant AAV8 vector restoring MTM1 expression (rAAV8-cMTM1). We then used single isolated muscle fibres to analyze nuclear organization and contractile function. In addition to the expected mislocalization of nuclei in the centre of muscle fibres, a novel form of nuclear mispositioning was observed: irregular spacing between those located at the fibre periphery, and an overall increased number of nuclei, leading to dramatically smaller and inconsistent myonuclear domains. Nuclear mislocalization was associated with decreases in global nuclear synthetic activity, contractile protein content and intrinsic myofilament force production. A contractile deficit originating at the myofilaments, rather than mechanical interference by centrally positioned nuclei, was supported by experiments in regenerated mouse muscle. Systemic administration of rAAV8-cMTM1 at doses higher than 2.5 × 1013 vg kg−1 allowed a full rescue of all these cellular defects in XLMTM dogs. Altogether, these findings identify previously unrecognized pathological mechanisms in human and animal XLMTM, associated with myonuclear defects and contractile filament function. These defects can be reversed by gene therapy restoring MTM1 expression in dogs with XLMTM.

Classical dynamins are large GTPases regulating membrane and cytoskeleton dynamics, and they are linked to different pathological conditions ranging from neuromuscular diseases to encephalopathy and cancer. Dominant dynamin 2 (DNM2) mutations lead to either mild adult onset or severe autosomal dominant centronuclear myopathy (ADCNM). Our objectives were to better understand the pathomechanism of severe ADCNM and test a potential therapy. Here, we created the Dnm2SL/+ mouse line harboring the common S619L mutation found in patients with severe ADCNM and impairing the conformational switch regulating dynamin self-assembly and membrane remodeling. The Dnm2SL/+ mouse faithfully reproduces severe ADCNM hallmarks with early impaired muscle function and force, together with myofiber hypotrophy. It revealed swollen mitochondria lacking cristae as the main ultrastructural defect and potential cause of the disease. Patient analysis confirmed this structural hallmark. In addition, DNM2 reduction with antisense oligonucleotides after disease onset efficiently reverted locomotor and force defects after only 3 weeks of treatment. Most histological defects including mitochondria alteration were partially or fully rescued. Overall, this study highlights an efficient approach to revert the severe form of dynamin-related centronuclear myopathy. These data also reveal that the dynamin conformational switch is key for muscle function and should be targeted for future therapeutic developments., The dynamin 2 S619L mouse model displays defects in skeletal muscle that are rescued by reducing dynamin 2 protein levels with antisense oligonucleotide treatment.

Centronuclear myopathies (CNM) are a group of severe muscle diseases for which no effective therapy is currently available. We have previously shown that reduction of the large GTPase DNM2 in a mouse model of the X-linked form, due to loss of myotubularin phosphatase MTM1, prevents the development of the skeletal muscle pathophysiology. As DNM2 is mutated in autosomal dominant forms, here we tested whether DNM2 reduction can rescue DNM2-related CNM in a knock-in mouse harboring the p.R465W mutation (Dnm2(RW/+)) and displaying a mild CNM phenotype similar to patients with the same mutation. A single intramuscular injection of adeno-associated virus-shRNA targeting Dnm2 resulted in reduction in protein levels 5 wk post injection, with a corresponding improvement in muscle mass and fiber size distribution, as well as an improvement in histopathological CNM features. To establish a systemic treatment, weekly i.p. injections of antisense oligonucleotides targeting Dnm2 were administered to Dnm2(RW/+)mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2(RW/+)mice compared with wild-type mice, these features were indistinguishable from wild-type mice after reducing DNM2. Therefore, DNM2 knockdown via two different strategies can efficiently correct the myopathy due to DNM2 mutations, and it provides a common therapeutic strategy for several forms of centronuclear myopathy. Furthermore, we provide an example of treating a dominant disease by targeting both alleles, suggesting that this strategy may be applied to other dominant diseases.

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, NebY2303H, 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, NebY2303H,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.

Aims The Acute Cardiac Care Association (ACCA)–European Association of Percutaneous Coronary Intervention (EAPCI) Registry on ST-elevation myocardial infarction (STEMI) of the EurObservational programme (EORP) of the European Society of Cardiology (ESC) registry aimed to determine the current state of the use of reperfusion therapy in ESC member and ESC affiliated countries and the adherence to ESC STEMI guidelines in patients with STEMI. Methods and results Between 1 January 2015 and 31 March 2018, a total of 11 462 patients admitted with an initial diagnosis of STEMI according to the 2012 ESC STEMI guidelines were enrolled. Individual patient data were collected across 196 centres and 29 countries. Among the centres, there were 136 percutaneous coronary intervention centres and 91 with cardiac surgery on-site. The majority of centres (129/196) were part of a STEMI network. The main objective of this study was to describe the demographic, clinical, and angiographic characteristics of patients with STEMI. Other objectives include to assess management patterns and in particular the current use of reperfusion therapies and to evaluate how recommendations of most recent STEMI European guidelines regarding reperfusion therapies and adjunctive pharmacological and non-pharmacological treatments are adopted in clinical practice and how their application can impact on patients’ outcomes. Patients will be followed for 1 year after admission. Conclusion The ESC ACCA-EAPCI EORP ACS STEMI registry is an international registry of care and outcomes of patients hospitalized with STEMI. It will provide insights into the contemporary patient profile, management patterns, and 1-year outcome of patients with STEMI.

Acta neuropathologica 138(3), 477-495 (2019). doi:10.1007/s00401-019-02034-8, Published by Springer, Heidelberg

Nemaline myopathy (NM) is a heterogeneous congenital skeletal muscle disease with cytoplasmic rod-like structures (nemaline bodies) in muscle tissue. While weakness in NM is related to contractile abnormalities, myofiber smallness is an additional abnormality in NM that may be treatable. We evaluated the effects of mRK35 (a myostatin inhibitor developed by Pfizer) treatment in the TgACTA1D286G mouse model of NM. mRK35 induced skeletal muscle growth that led to significant increases in animal bodyweight, forelimb grip strength and muscle fiber force, although it should be noted that animal weight and forelimb grip strength in untreated TgACTA1D286G mice was not different from controls. Treatment was also associated with an increase in the number of tubular aggregates found in skeletal muscle. These findings suggest that myostatin inhibition may be useful in promoting muscle growth and strength in Acta1-mutant muscle, while also further establishing the relationship between low levels of myostatin and tubular aggregate formation

In humans, mutant skeletal muscle α-actin proteins are associated with contractile dysfunction, skeletal muscle weakness and a wide range of primarily skeletal muscle diseases. Despite this knowledge, the exact molecular mechanisms triggering the contractile dysfunction remain unknown. Here, we aimed to unravel these. Hence, we used a transgenic mouse model expressing a well-described D286G mutant skeletal muscle α-actin protein and recapitulating the human condition of contractile deregulation and severe skeletal muscle weakness. We then recorded and analyzed the small-angle X-ray diffraction patterns of isolated membrane-permeabilized myofibers. Results showed that upon addition of Ca(2+), the intensity changes of the second (1/19 nm(-1)) and sixth (1/5.9 nm(-1)) actin layer lines and of the first myosin meridional reflection (1/14.3 nm(-1)) were disrupted when the thin-thick filament overlap was optimal (sarcomere length of 2.5-2.6 μm). However these reflections were normal when the thin and thick filaments were not interacting (sarcomere length>3.6 μm). These findings demonstrate, for the first time, that the replacement of just one amino acid in the skeletal muscle α-actin protein partly prevents actin conformational changes during activation, disrupting the strong binding of myosin molecules. This leads to a limited myosin-related tropomyosin movement over the thin filaments, further affecting the amount of cross-bridges, explaining the contractile dysfunction.

Novel experimental methods, including a modified single fiber in vitro motility assay, X-ray diffraction experiments, and mass spectrometry analyses, have been performed to unravel the molecular events underlying the aging-related impairment in human skeletal muscle function at the motor protein level. The effects of old age on the function of specific myosin isoforms extracted from single human muscle fiber segments, demonstrated a significant slowing of motility speed (P < 0.001) in old age in both type I and IIa myosin heavy chain (MyHC) isoforms. The force-generating capacity of the type I and IIa MyHC isoforms was, on the other hand, not affected by old age. Similar effects were also observed when the myosin molecules extracted from muscle fibers were exposed to oxidative stress. X-ray diffraction experiments did not show any myofilament lattice spacing changes, but unraveled a more disordered filament organization in old age as shown by the greater widths of the 1, 0 equatorial reflections. Mass spectrometry (MS) analyses revealed eight age-specific myosin post-translational modifications (PTMs), in which two were located in the motor domain (carbonylation of Pro79 and Asn81) and six in the tail region (carbonylation of Asp900, Asp904, and Arg908; methylation of Glu1166; deamidation of Gln1164 and Asn1168). However, PTMs in the motor domain were only observed in the IIx MyHC isoform, suggesting PTMs in the rod region contributed to the observed disordering of myosin filaments and the slowing of motility speed. Hence, interventions that would specifically target these PTMs are warranted to reverse myosin dysfunction in old age.

The congenital myopathies − including Central Core Disease (CCD), Multi-minicore Disease (MmD), Centronuclear Myopathy (CNM), Nemaline Myopathy (NM) and Congenital Fibre Type Disproportion (CFTD) − are a genetically heterogeneous group of early-onset neuromuscular conditions characterized by distinct histopathological features, and associated with a substantial individual and societal disease burden. Appropriate supportive management has substantially improved patient morbidity and mortality but there is currently no cure. Recent years have seen an exponential increase in the genetic and molecular understanding of these conditions, leading to the identification of underlying defects in proteins involved in calcium homeostasis and excitation-contraction coupling, thick/thin filament assembly and function, redox regulation, membrane trafficking and/or autophagic pathways. Based on these findings, specific therapies are currently being developed, or are already approaching the clinical trial stage. Despite undeniable progress, therapy development faces considerable challenges, considering the rarity and diversity of specific conditions, and the size and complexity of some of the genes and proteins involved. The present review will summarize the key genetic, histopathological and clinical features of specific congenital myopathies, and outline therapies already available or currently being developed in the context of known pathogenic mechanisms. The relevance of newly discovered molecular mechanisms and novel gene editing strategies for future therapy development will be discussed.

Nemaline myopathy, the most common congenital myopathy, is characterized by mutations in genes encoding myofilament proteins such as skeletal α-actin. These mutations are thought to ultimately lead to skeletal muscle weakness. Interestingly, some of the mutations appear to be more potent in males than in females. The underlying mechanisms remain obscure but may be related to sex-specific differences in the myofilament function of both limb and respiratory muscles. To verify this, in the present study, we used skeletal muscles (tibialis anterior and diaphragm) from a transgenic mouse model harbouring the His40Tyr amino acid substitution in skeletal α-actin. In this animal model, 60% of males die by 13weeks of age (the underlying causes of death are obscure but probably due to respiratory insufficiency) whereas females have a normal lifespan. By recording and analysing the mechanics of membrane-permeabilized myofibres, we only observed sex-related differences in the tibialis anterior muscles. Indeed, the concomitant deficits in maximal steady-state isometric force and stiffness of myofibres were less exacerbated in transgenic females than in males, potentially explaining the lower potency in limb muscles. However, the absence of sex-difference in the diaphragm muscles was rather unexpected and suggests that myofilament dysfunction does not solely underlie the sexually dimorphic phenotypes.

Human tropomyosin mutations deregulate skeletal muscle contraction at the cellular level. One key feature is the slowing of the kinetics of force development. The aim of the present study was to characterize the potential underlying molecular mechanisms by recording and analyzing the X-ray diffraction patterns of human membrane-permeabilized muscle cells expressing a particular β-tropomyosin mutation (E41K). During resting conditions, the d1,0 lattice spacing, Δ1,0 and I1,1 to I1,0 ratio were not different from control values. These results suggest that, in presence of the E41K β-tropomyosin mutation, the myofilament lattice geometry is well maintained and therefore may not have any detrimental influence on the contraction mechanisms and thus, on the rate of force generation.

Summary Myosinopathies have emerged as a new group of diseases and are caused by mutations in genes encoding myosin heavy chain (MyHC) isoforms. One major hallmark of these diseases is skeletal muscle weakness or paralysis, but the underlying molecular mechanisms remain unclear. Here, we have undertaken a detailed functional study of muscle fibers from Myh4arl mice, which carry a mutation that provokes an L342Q change within the catalytic domain of the type IIb skeletal muscle myosin protein MYH4. Because homozygous animals develop rapid muscle-structure disruption and lower-limb paralysis, they must be killed by postnatal day 13, so all experiments were performed using skeletal muscles from adult heterozygous animals (Myh4arl/+). Myh4arl/+ mice contain MYH4L342Q expressed at 7% of the levels of the wild-type (WT) protein and are overtly and histologically normal. However, mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized fibers revealed, upon maximal Ca2+ activation, higher stiffness as well as altered meridional and equatorial reflections in Myh4arl/+ mice when compared with age-matched WT animals. Under rigor conditions, by contrast, no difference was observed between Myh4arl/+ and WT mice. Altogether, these findings prove that, in adult MYH4L342Q heterozygous mice, the transition from weak to strong myosin cross-bridge binding is facilitated, increasing the number of strongly attached myosin heads, thus enhancing force production. These changes are predictably exacerbated in the type IIb fibers of homozygous mice, in which the embryonic myosin isoform is fully replaced by MYH4L342Q, leading to a hypercontraction, muscle-structure disruption and lower-limb paralysis. Overall, these findings provide important insights into the molecular pathogenesis of skeletal myosinopathies.

BackgroundThe greater susceptibility to contraction-induced skeletal muscle injury (fragility) is an important dystrophic feature and tool for testing preclinic dystrophin-based therapies for Duchenne muscular dystrophy. However, how these therapies reduce the muscle fragility is not clear.MethodsTo address this question, we first determined the event(s) of the excitation-contraction cycle which is/are altered following lengthening (eccentric) contractions in the mdx muscle.ResultsWe found that the immediate force drop following lengthening contractions, a widely used measure of muscle fragility, was associated with reduced muscle excitability. Moreover, the force drop can be mimicked by an experimental reduction in muscle excitation of uninjured muscle. Furthermore, the force drop was not related to major neuromuscular transmission failure, excitation-contraction uncoupling, and myofibrillar impairment. Secondly, and importantly, the re-expression of functional truncated dystrophin in the muscle of mdx mice using an exon skipping strategy partially prevented the reductions in both force drop and muscle excitability following lengthening contractions.ConclusionWe demonstrated for the first time that (i) the increased susceptibility to contraction-induced muscle injury in mdx mice is mainly attributable to reduced muscle excitability; (ii) dystrophin-based therapy improves fragility of the dystrophic skeletal muscle by preventing reduction in muscle excitability.

Nemaline myopathy (NM) is the most common congenital myopathy and is caused by mutations in various genes including NEB (nebulin), TPM2 (beta-tropomyosin), TPM3 (gamma-tropomyosin), and ACTA1 (skeletal alpha-actin). 20-25% of NM cases carry ACTA1 defects and these particular mutations usually induce substitutions of single residues in the actin protein. Despite increasing clinical and scientific interest, the contractile consequences of these subtle amino acid substitutions remain obscure. To decipher them, in the present study, we originally recorded and analysed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibres with a particular peptide substitution in actin, i.e. p.Phe352Ser. Results unravelled an unexpected cascade of molecular and cellular events. During contraction, p.Phe352Ser greatly enhances the strain of individual cross-bridges. Paradoxically, p.Phe352Ser also slightly lowers the number of cross-bridges by altering the rate of myosin head attachment to actin monomers. Overall, at the cell level, these divergent mechanisms conduct to an improved steady-state force production. Such results provide new surprising scientific insights and crucial information for future therapeutic strategies.

Ariel is a mouse mutant that suffers from skeletal muscle myofibrillar degeneration due to the rapid accumulation of large intracellular protein aggregates. This fulminant disease is caused by an ENU-induced recessive mutation resulting in an L342Q change within the motor domain of the skeletal muscle myosin protein MYH4 (MyHC IIb). Although normal at birth, homozygous mice develop hindlimb paralysis from Day 13, consistent with the timing of the switch from developmental to adult myosin isoforms in mice. The mutated myosin (MYH4(L342Q)) is an aggregate-prone protein. Notwithstanding the speed of the process, biochemical analysis of purified aggregates showed the presence of proteins typically found in human myofibrillar myopathies, suggesting that the genesis of ariel aggregates follows a pathogenic pathway shared with other conformational protein diseases of skeletal muscle. In contrast, heterozygous mice are overtly and histologically indistinguishable from control mice. MYH4(L342Q) is present in muscles from heterozygous mice at only 7% of the levels of the wild-type protein, resulting in a small but significant increase in force production in isolated single fibres and indicating that elimination of the mutant protein in heterozygotes prevents the pathological changes observed in homozygotes. Recapitulation of the L342Q change in the functional equivalent of mouse MYH4 in human muscles, MYH1, results in a more aggregate-prone protein.

Acute quadriplegic myopathy (AQM) is a common debilitating acquired disorder in critically ill intensive care unit (ICU) patients that is characterized by tetraplegia/generalized weakness of limb and trunk muscles. Masticatory muscles, on the other hand, are typically spared or less affected, yet the mechanisms underlying this striking muscle-specific difference remain unknown. This study aims to evaluate physiological parameters and the gene expression profiles of masticatory and limb muscles exposed to factors suggested to trigger AQM, such as mechanical ventilation, immobilization, neuromuscular blocking agents, corticosteroids (CS), and sepsis for 5 days by using a unique porcine model mimicking the ICU conditions. Single muscle fiber cross-sectional area and force-generating capacity, i.e., maximum force normalized to fiber cross-sectional area (specific force), revealed maintained masseter single muscle fiber cross-sectional area and specific-force after 5 days' exposure to all triggering factors. This is in sharp contrast to observations in limb and trunk muscles, showing a dramatic decline in specific force in response to 5 days' exposure to the triggering factors. Significant differences in gene expression were observed between craniofacial and limb muscles, indicating a highly complex and muscle-specific response involving transcription and growth factors, heat shock proteins, matrix metalloproteinase inhibitor, oxidative stress responsive elements, and sarcomeric proteins underlying the relative sparing of cranial vs. spinal nerve innervated muscles during exposure to the ICU intervention.

Nebulin is a giant protein expressed at high levels in skeletal muscle. Mutations in the nebulin gene (NEB) lead to muscle weakness and various congenital myopathies. Despite increasing clinical and scientific interest, the pathogenesis of weakness remains unknown. The present study, therefore, aims at unraveling the underlying molecular mechanisms. Hence, we recorded and analyzed the mechanics as well as the X-ray diffraction patterns of human membrane-permeabilized single muscle fibers expressing nebulin mutations. Results demonstrated that, during contraction, the cycling rate of myosin heads attaching to actin is dramatically perturbed, causing a reduction in the fraction of myosin-actin interactions in the strong binding state. This phenomenon prevents complete thin-filament activation, more especially proper and full tropomyosin movement, further limiting additional binding of myosin cross-bridges. At the cell level, this reduces the force-generating capacity and, overall, provokes muscle weakness. To reverse such a negative cascade of events, future potential therapeutic interventions should, therefore, focus on the triggering component, the altered myosin cross-bridge cycling kinetics.—Ochala, J., Lehtokari, V.-L., Iwamoto, H., Li, M., Feng, H.-Z., Jin, J. P., Yagi, N., Wallgren-Pettersson, C. Pénisson-Besnier, I., Larsson, L. Disrupted myosin cross-bridge cycling kinetics triggers muscle weakness in nebulin-related myopathy.

Human point mutations in β- and γ-tropomyosin induce contractile deregulation, skeletal muscle weakness, and congenital myopathies. The aim of the present study was to elucidate the hitherto unknown underlying molecular mechanisms. Hence, we recorded and analyzed the X-ray diffraction patterns of human membrane-permeabilized muscle cells expressing a particular β-tropomyosin mutation (R133W) associated with a loss in cell force production, in vivo muscle weakness, and distal arthrogryposis. Upon addition of calcium, we notably observed less intensified changes, compared with controls, ( i ) in the second (1/19 nm −1 ), sixth (1/5.9 nm −1 ), and seventh (1/5.1 nm −1 ) actin layer lines of cells set at a sarcomere length, allowing an optimal thin-thick filament overlap; and ( ii ) in the second actin layer line of overstretched cells. Collectively, these results directly prove that during activation, switching of a positive to a neutral charge at position 133 in the protein partially hinders both calcium- and myosin-induced tropomyosin movement over the thin filament, blocking actin conformational changes and consequently decreasing the number of cross-bridges and subsequent force production.

Ca(2+) ions are key regulators of skeletal muscle contraction. By binding to contractile proteins, they initiate a cascade of molecular events leading to cross-bridge formation and ultimately, muscle shortening and force production. The ability of contractile proteins to respond to Ca(2+) attachment, also known as Ca(2+) sensitivity, is often compromised in acquired and congenital skeletal muscle disorders. It constitutes, undoubtedly, a major physiological cause of weakness for patients. In this review, we discuss recent studies giving strong molecular and cellular evidence that pharmacological modulators of some of the contractile proteins, also termed Ca(2+) sensitizers, are efficient agents to improve Ca(2+) sensitivity and function in diseased skeletal muscle cells. In fact, they compensate for the impaired contractile proteins response to Ca(2+) binding. Currently, such Ca(2+) sensitizing compounds are successfully used for reducing problems in cardiac disorders. Therefore, in the future, under certain conditions, these agents may represent an emerging class of agents to enhance the quality of life of patients suffering from skeletal muscle weakness.

BACKGROUND: Myofibrillar myopathies constitute a rare group of congenital neuromuscular disorders, frequently associated with mutations in Z-disc proteins such as myotilin. Myotilin location and interactions with other Z-disc proteins are clearly defined, but its role in the regulation of muscle structure and function remains unknown. The present study aims at investigating this specific role of myotilin. METHODS: Skeletal and cardiac muscles were collected from adult mice with a targeted deletion of myotilin (myo(-/-)) and wild-type animals (myo(+/+)). RESULTS AND CONCLUSION: Similar skeletal and cardiac muscle weights were observed in myo(-/-) and myo(+/+) mice. At the muscle cell level, the size and force production of single membrane permeabilized fibers were identical between myo(-/-) and myo(+/+) rodents. Thus, myotilin does not have a significant influence on muscle mass, muscle fiber size, or regulation of muscle contraction. Alternatively, compensatory over-expressions of other elements including proteins from the same subfamily, or Z-disc proteins such as telethonin, or intermediate filaments may compensate for the lack of myotilin.

Skeletal muscle wasting and impaired muscle function in response to mechanical ventilation and immobilization in intensive care unit (ICU) patients are clinically challenging partly due to 1) the poorly understood intricate cellular and molecular networks and 2) the unavailability of an animal model mimicking this condition. By employing a unique porcine model mimicking the conditions in the ICU with long-term mechanical ventilation and immobilization, we have analyzed the expression profile of skeletal muscle biopsies taken at three time points during a 5-day period. Among the differentially regulated transcripts, extracellular matrix, energy metabolism, sarcomeric and LIM protein mRNA levels were downregulated, while ubiquitin proteasome system, cathepsins, oxidative stress responsive genes and heat shock proteins (HSP) mRNAs were upregulated. Despite 5 days of immobilization and mechanical ventilation single muscle fiber cross-sectional areas as well as the maximum force generating capacity at the single muscle fiber level were preserved. It is proposed that HSP induction in skeletal muscle is an inherent, primary, but temporary protective mechanism against protein degradation. To our knowledge, this is the first study that isolates the effect of immobilization and mechanical ventilation in an ICU condition from various other cofactors.

A novel E41K β-tropomyosin (β-Tm) mutation, associated with congenital myopathy and muscle weakness, was recently identified in a woman and her daughter. In both patients, muscle weakness was coupled with muscle fibre atrophy. It remains unknown, however, whether the E41K β-Tm mutation directly affects regulation of muscle contraction, contributing to the muscle weakness. To address this question, we studied a broad range of contractile characteristics in skinned muscle fibres from the two patients and eight healthy controls. Results showed decreases (i) in speed of contraction at saturated Ca2+ concentration (apparent rate constant of force redevelopment (ktr) and unloaded shortening speed (V0)); and (ii) in contraction sensitivity to Ca2+ concentration, in fibres from patients compared with controls, suggesting that the mutation has a negative effect on contractile function, contributing to the muscle weakness. To investigate whether these negative impacts are reversible, we exposed skinned muscle fibres to the Ca2+ sensitizer EMD 57033. In fibres from patients, 30 μm of EMD 57033 (i) had no effect on speed of contraction (ktr and V0) at saturated Ca2+ concentration but (ii) increased Ca2+ sensitivity of contraction, suggesting a potential therapeutic approach in patients carrying the E41K β-Tm mutation.

A novel R133W β-tropomyosin (β-Tm) mutation, associated with muscle weakness and distal limb deformities, has recently been identified in a woman and her daughter. The muscle weakness was not accompanied by progressive muscle wasting or histopathological abnormalities in tibialis anterior muscle biopsy specimens. The aim of the present study was to explore the mechanisms underlying the impaired muscle function in patients with the β-Tm mutation. Maximum force normalized to fibre cross-sectional area (specific force, SF), maximum velocity of unloaded shortening (V0), apparent rate constant of force redevelopment (ktr) and force–pCa relationship were evaluated in single chemically skinned muscle fibres from the two patients carrying the β-Tm mutation and from healthy control subjects. Significant differences in regulation of muscle contraction were observed in the type I fibres: a lower SF (P < 0.05) and ktr (P < 0.01), and a faster V0 (P < 0.05). The force–pCa relationship did not differ between patient and control fibres, indicating an unaltered Ca2+ activation of contractile proteins. Collectively, these results indicate a slower cross-bridge attachment rate and a faster detachment rate caused by the R133W β-Tm mutation. It is suggested that the R133W β-Tm mutation induces alteration in myosin–actin kinetics causing a reduced number of myosin molecules in the strong actin-binding state, resulting in overall muscle weakness in the absence of muscle wasting.

Nemaline myopathy is a congenital disorder characterized by muscle weakness. Dominant-negative disease-causing mutations have been identified in various genes that encode sarcomeric thin filament proteins. of these, mutations in skeletal muscle α-actin (ACTA1) account for ∼25% of all reported cases. To investigate the myopathic phenotypes from the level of whole muscle down through the level of single molecules, we generated multiple transgenic Drosophila fly lines expressing three different mutations - V37L, D288G and F352S - in Act88F, the indirect flight muscle (IFM) actin gene. A gradation in flight behavior was observed (V37L>D288G>F352S), which correlated with the observed differences in muscle fiber morphology. While most of these mutations depress muscle function in patients, the F352S lesion elevates myosin cross-bridge strain and steady-state isometric force production and, thereby, apparently increases contractile function. Confocal and electron microscopy revealed breaks in mutant F352S IFM fibers and extensive myofibrillar and myofilamentous disarray. Z-lines showed streaming and formed “zebra bodies”. The microscopic alterations are consistent with excessive acto-myosin interactions, unevenly distributed force, and destructive hypercontraction. Interestingly, in vitro sliding velocities of purified IFM F-actin from wild-type (3.17±0.12 μm/s) vs. F352S mutant heterozygous (2.88±0.09 μm/s, mean±SEM) flies did not significantly differ. Similarly, no differences in maximum calcium-activated velocity (3.88±0.11 vs. 3.77±0.10 μm/s) or in cooperativity (nH = 2.13±0.27 vs. 2.62±0.46) were observed for F-actin in the presence of vertebrate tropomyosin and troponin. However, relative to control, F352S heterozygous filaments showed a significant (p

Skeletal muscle is one of the most dynamic and plastic tissues of the human body. In humans, skeletal muscle comprises approximately 40 % of total body weight and contains 50-75 % of all body proteins. In general, muscle mass depends on the balance between protein syn- thesis and degradation and both processes are sensitive to factors such as nutritional status, hormonal balance, physi- cal activity/exercise, and injury or disease, among others. In this review, we discuss the various domains of muscle structure and function including its cytoskeletal architec- ture, excitation-contraction coupling, energy metabolism, and force and power generation. We will limit the discussion to human skeletal muscle and emphasize recent scientific literature on single muscle fibers.

In skeletal muscle, thick and thin filaments are arranged in a myofibrillar lattice. Tropomodulin 1 (Tmod1) is a pointed-end capping and tropomyosin-binding protein that controls thin-filament assembly, stability, and lengths. It remains unknown whether Tmods have other functional roles, such as regulating muscle contractility. To investigate this, we recorded and analyzed the mechanical properties and X-ray diffraction patterns of single membrane-permeabilized skeletal muscle fibers from mice lacking Tmod1. Results show that absence of Tmod1 and its replacement by Tmod3 and Tmod4 may impair initial tropomyosin movement over actin subunits during thin-filament activation, thus reducing both the fraction of actomyosin crossbridges in the strongly bound state (−29%) and fiber force-generating capacity (−31%). Therefore, Tmods are novel regulators of actomyosin crossbridge formation and muscle contractility, and future investigations and models of skeletal muscle force production must incorporate Tmods.—Ochala, J., Gokhin, D. S., Iwamoto, H., Fowler, V. M. Pointed-end capping by tropomodulin modulates actomyosin crossbridge formation in skeletal muscle fibers.

Multiple congenital myopathies, including nemaline myopathy, can arise due to mutations in the ACTA1 gene encoding skeletal muscle α-actin. The main characteristics of ACTA1 null mutations (absence of skeletal muscle α-actin) are generalized skeletal muscle weakness and premature death. A mouse model (ACTC(Co)/KO) mimicking these conditions has successfully been rescued by transgenic over-expression of cardiac α-actin in skeletal muscles using the ACTC gene. Nevertheless, myofibres from ACTC(Co)/KO animals generate less force than normal myofibres (-20 to 25%). To understand the underlying mechanisms, here we have undertaken a detailed functional study of myofibres from ACTC(Co)/KO rodents. Mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized myofibres showed, upon maximal Ca(2+) activation and under rigor conditions, lower stiffness and disrupted actin-layer line reflections in ACTC(Co)/KO when compared with age-matched wild-types. These results demonstrate that in ACTC(Co)/KO myofibres, the presence of cardiac α-actin instead of skeletal muscle α-actin alters actin conformational changes upon activation. This later finely modulates the strain of individual actomyosin interactions and overall lowers myofibre force production. Taken together, the present findings provide novel primordial information about actin isoforms, their functional differences and have to be considered when designing gene therapies for ACTA1-based congenital myopathies.

Nemaline myopathy is the most common congenital myopathy and is caused by mutations in various genes such as ACTA1 (encoding skeletal α-actin). It is associated with limb and respiratory muscle weakness. Despite increasing clinical and scientific interest, the molecular and cellular events leading to such weakness remain unknown, which prevents the development of specific therapeutic interventions. To unravel the potential mechanisms involved, we dissected lower limb and diaphragm muscles from a knock-in mouse model of severe nemaline myopathy expressing the ACTA1 His40Tyr actin mutation found in human patients. We then studied a broad range of structural and functional characteristics assessing single-myofiber contraction, protein expression, and electron microscopy. One of the major findings in the diaphragm was the presence of numerous noncontractile areas (including disrupted sarcomeric structures and nemaline bodies). This greatly reduced the number of functional sarcomeres, decreased the force generation capacity at the muscle fiber level, and likely would contribute to respiratory weakness. In limb muscle, by contrast, there were fewer noncontractile areas and they did not seem to have a major role in the pathogenesis of weakness. These divergent muscle-specific results provide new important insights into the pathophysiology of severe nemaline myopathy and crucial information for future development of therapeutic strategies.

The sarcomeric tropomodulin (Tmod) isoforms Tmod1 and Tmod4 cap thin filament pointed ends and functionally interact with the leiomodin (Lmod) isoforms Lmod2 and Lmod3 to control myofibril organization, thin filament lengths, and actomyosin crossbridge formation in skeletal muscle fibers. Here, we show that Tmod4 is more abundant than Tmod1 at both the transcript and protein level in a variety of muscle types, but the relative abundances of sarcomeric Tmods are muscle specific.We then generate Tmod4−/− mice, which exhibit normal thin filament lengths, myofibril organization, and skeletal muscle contractile function owing to compensatory upregulation of Tmod1, together with an Lmod isoform switch wherein Lmod3 is downregulated and Lmod2 is upregulated. However, RNAi depletion of Tmod1 from either wild-type or Tmod4−/− muscle fibers leads to thin filament elongation by ∼15%. Thus, Tmod1 per se, rather than total sarcomeric Tmod levels, controls thin filament lengths in mouse skeletal muscle, whereas Tmod4 appears to be dispensable for thin filament length regulation. These findings identify Tmod1 as the key direct regulator of thin filament length in skeletal muscle, in both adult muscle homeostasis and in developmentally compensated contexts.

In humans, congenital myopathy-linked tropomyosin mutations lead to skeletal muscle dysfunction, but the cellular and molecular mechanisms underlying such dysfunction remain obscure. Recent studies have suggested a unifying mechanism by which tropomyosin mutations partially inhibit thin filament activation and prevent proper formation and cycling of myosin cross-bridges, inducing force deficits at the fiber and whole-muscle levels. Here, we aimed to verify this mechanism using single membrane-permeabilized fibers from patients with three tropomyosin mutations (TPM2-null, TPM3-R167H and TPM2-E181K) and measuring a broad range of parameters. Interestingly, we identified two divergent, mutation-specific pathophysiological mechanisms. (i) The TPM2-null and TPM3-R167H mutations both decreased cooperative thin filament activation in combination with reductions in the myosin cross-bridge number and force production. The TPM3-R167H mutation also induced a concomitant reduction in thin filament length. (ii) In contrast, the TPM2-E181K mutation increased thin filament activation, cross-bridge binding and force generation. In the former mechanism, modulating thin filament activation by administering troponin activators (CK-1909178 and EMD 57033) to single membrane-permeabilized fibers carrying tropomyosin mutations rescued the thin filament activation defect associated with the pathophysiology. Therefore, administration of troponin activators may constitute a promising therapeutic approach in the future.

The final functional unit in skeletal muscle, i.e., the sarcomere, has been extensively investigated for many years. However, muscle disorders have not until recently been identified where sarcomeric proteins are specifically or preferentially affected. In this short review, we have focused our attention on muscle wasting conditions with a selective (preferential) loss of sarcomeric proteins or with inherited mutations in the genes encoding these particular molecules. Specifically, we have discussed studies focusing on how these pathophysiological changes affect regulation of muscle contraction.

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.

Hereditary myosinopathies have emerged as a new group of diseases and are characterized by skeletal muscle weakness. These diseases are notably caused by mutations in genes encoding myosin heavy chain (MyHC) isoforms. A new mouse model with a L342Q MyHC IIb mutation has been developed to mimic such myosinopathies. Homozygous mice experience severe skeletal muscle paralysis and, because of ethical reasons, are killed thirteen days after birth. Heterozygous animals have a normal lifespan and no apparent weakness. The underlying mechanisms for such absence of deteriorated phenotype in heterozygous rodents remain unknown. In the current study, we aimed at investigating this particular point by evaluating whether and how the presence of 50% of L342Q MyHC IIb mutants affects the contractile function. We compared isolated membrane-permeabilized muscle fibers from heterozygote (L342Q+/-) and wild-type young adult mice. Results showed, during full activation, at pCa 4.5, an increase in force production in L342Q+/- when compared with wild-type animals (p

In humans, more than 140 different mutations within seven genes (ACTA1, TPM2, TPM3, TNNI2, TNNT1, TNNT3, and NEB) that encode thin filament proteins (skeletal alpha-actin, beta-tropomyosin, gamma-tropomyosin, fast skeletal muscle troponin I, slow skeletal muscle troponin T, fast skeletal muscle troponin T, and nebulin, respectively) have been identified. These mutations have been linked to muscle weakness and various congenital skeletal myopathies including nemaline myopathy, distal arthrogryposis, cap disease, actin myopathy, congenital fiber type disproportion, rod-core myopathy, intranuclear rod myopathy, and distal myopathy, with a dramatic negative impact on the quality of life. In this review, we discuss studies that use various approaches such as patient biopsy specimen samples, tissue culture systems or transgenic animal models, and that demonstrate how thin filament proteins mutations alter muscle structure and contractile function. With an enhanced understanding of the cellular and molecular mechanisms underlying muscle weakness in patients carrying such mutations, better therapy strategies can be developed to improve the quality of life.