Aberrant myonuclear domains and impaired myofiber contractility despite marked hypertrophy in MYMK-related, Carey-Fineman-Ziter Syndrome.

Carey Fineman Ziter Syndrome (CFZS) is a rare autosomal recessive disease caused by mutations in the MYMK locus which encodes the protein, myomaker. Myomaker is essential for fusion and concurrent myonuclei donation of muscle progenitors during growth and development. Strikingly, in humans, MYMK mutations appear to prompt myofiber hypertrophy but paradoxically, induce generalised muscle weakness. As the underlying cellular mechanisms remain unexplored, the present study aimed to gain insights by combining myofiber deep-phenotyping and proteomic profiling. Hence, we isolated individual muscle fibers from CFZS patients and performed mechanical, 3D morphological and proteomic analyses. Myofibers from CFZS patients were ~ 4x larger than controls and possessed ~ 2x more myonuclei than those from healthy subjects, leading to disproportionally larger myonuclear domain volumes. These greater myonuclear domain sizes were accompanied by smaller intrinsic cellular force generating-capacities in myofibers from CFZS patients than in control muscle cells. Our complementary proteomic analyses indicated remodelling in 233 proteins particularly those associated with cellular respiration. Overall, our findings suggest that myomaker is somewhat functional in CFZS patients, but the associated nuclear accretion may ultimately lead to non-functional hypertrophy and altered energy-related mechanisms in CFZS patients. All of these are likely contributors of the muscle weakness experienced by CFZS patients.

Muscle fibre size and myonuclear positioning in trained and aged humans.

Changes in myonuclear architecture and positioning are associated with exercise adaptations and ageing. However, data on the positioning and number of myonuclei following exercise are inconsistent. Additionally, whether myonuclear domains (MNDs; i.e., the theoretical volume of cytoplasm within which a myonucleus is responsible for transcribing DNA) and myonuclear positioning are altered with age remains unclear. The aim of this investigation was to investigate relationships between age and activity status and myonuclear domains and positioning. Vastus lateralis muscle biopsies from younger endurance-trained (YT) and older endurance-trained (OT) individuals were compared with age-matched untrained counterparts (YU and OU; OU samples were acquired during surgical operation). Serial, optical z-slices were acquired throughout isolated muscle fibres and analysed to give three-dimensional coordinates for myonuclei and muscle fibre dimensions. The mean cross-sectional area (CSA) of muscle fibres from OU individuals was 33%-53% smaller compared with the other groups. The number of nuclei relative to fibre CSA was 90% greater in OU compared with YU muscle fibres. Additionally, scaling of MND volume with fibre size was altered in older untrained individuals. The myonuclear arrangement, in contrast, was similar across groups. Fibre CSA and most myonuclear parameters were significantly associated with age in untrained individuals, but not in trained individuals. These data indicate that regular endurance exercise throughout the lifespan might better preserve the size of muscle fibres in older age and maintain the relationship between fibre size and MND volumes. Inactivity, however, might result in reduced muscle fibre size and altered myonuclear parameters.

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.

rAAV-related therapy fully rescues myonuclear and myofilament function in X-linked myotubular myopathy.

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.

Effect of PGC1-beta ablation on myonuclear organisation.

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 1ß (PGC1-ß) 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-ß 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-1ß play different roles in regulating nuclear organisation in skeletal muscle; however, further work is required to pinpoint their exact functions.

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.

Reducing dynamin 2 (DNM2) rescues DNM2-related dominant centronuclear myopathy.

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 (Dnm2RW/+) 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 Dnm2RW/+mice for 5 wk. While muscle mass, histopathology, and muscle ultrastructure were perturbed in Dnm2RW/+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.

Prelamin A causes aberrant myonuclear arrangement and results in muscle fiber weakness.

Physiological and premature aging are frequently associated with an accumulation of prelamin A, a precursor of lamin A, in the nuclear envelope of various cell types. Here, we aimed to underpin the hitherto unknown mechanisms by which prelamin A alters myonuclear organization and muscle fiber function. By experimentally studying membrane-permeabilized myofibers from various transgenic mouse lines, our results indicate that, in the presence of prelamin A, the abundance of nuclei and myosin content is markedly reduced within muscle fibers. This leads to a concept by which the remaining myonuclei are very distant from each other and are pushed to function beyond their maximum cytoplasmic capacity, ultimately inducing muscle fiber weakness.

SIRT1 regulates nuclear number and domain size in skeletal muscle fibers.

Skeletal muscle fibers are giant multinucleated cells wherein individual nuclei govern the protein synthesis in a finite volume of cytoplasm; this is termed the myonuclear domain (MND). The factors that control MND size remain to be defined. In the present study, we studied the contribution of the NAD+ -dependent deacetylase, sirtuin 1 (SIRT1), to the regulation of nuclear number and MND size. For this, we isolated myofibers from mice with tissue-specific inactivation (mKO) or inducible overexpression (imOX) of SIRT1 and analyzed the 3D organisation of myonuclei. In imOX mice, the number of nuclei was increased whilst the average MND size was decreased as compared to littermate controls. Our findings were the opposite in mKO mice. Muscle stem cell (satellite cell) numbers were reduced in mKO muscles, a possible explanation for the lower density of myonuclei in these mice; however, no change was observed in imOX mice, suggesting that other factors might also be involved, such as the functional regulation of stem cells/muscle precursors. Interestingly, however, the changes in the MND volume did not impact the force-generating capacity of muscle fibers. Taken together, our results demonstrate that SIRT1 is a key regulator of MND sizes, although the underlying molecular mechanisms and the cause-effect relationship between MND and muscle function remain to be fully defined.

Exploring the Role of PGC-1α in Defining Nuclear Organisation in Skeletal Muscle Fibres.

Muscle fibres are multinucleated cells, with each nucleus controlling the protein synthesis in a finite volume of cytoplasm termed the myonuclear domain (MND). What determines MND size remains unclear. In the present study, we aimed to test the hypothesis that the level of expression of the transcriptional coactivator PGC-1α and subsequent activation of the mitochondrial biogenesis are major contributors. Hence, we used two transgenic mouse models with varying expression of PGC-1α in skeletal muscles. We isolated myofibres from the fast twitch extensor digitorum longus (EDL) and slow twitch diaphragm muscles. We then membrane-permeabilised them and analysed the 3D spatial arrangements of myonuclei. In EDL muscles, when PGC-1α is over-expressed, MND volume decreases; whereas, when PGC-1α is lacking, no change occurs. In the diaphragm, no clear difference was noted. This indicates that PGC-1α and the related mitochondrial biogenesis programme are determinants of MND size. PGC-1α may facilitate the addition of new myonuclei in order to reach MND volumes that can support an increased mitochondrial density. J. Cell. Physiol. 232: 1270-1274, 2017. © 2016 Wiley Periodicals, Inc.

Modulating myosin restores muscle function in a mouse model of nemaline myopathy.

OBJECTIVE: Nemaline myopathy, one of the most common congenital myopathies, is associated with mutations in various genes including ACTA1. This disease is also characterized by various forms/degrees of muscle weakness, with most cases being severe and resulting in death in infancy. Recent findings have provided valuable insight into the underlying pathophysiological mechanisms. Mutations in ACTA1 directly disrupt binding interactions between actin and myosin, and consequently the intrinsic force-generating capacity of muscle fibers. ACTA1 mutations are also associated with variations in myofiber size, the mechanisms of which have been unclear. In the present study, we sought to test the hypotheses that the compromised functional and morphological attributes of skeletal muscles bearing ACTA1 mutations (1) would be directly due to the inefficient actomyosin complex and (2) could be restored by manipulating myosin expression. METHODS: We used a knockin mouse model expressing the ACTA1 His40Tyr actin mutation found in human patients. We then performed in vivo intramuscular injections of recombinant adeno-associated viral vectors harboring a myosin transgene known to facilitate muscle contraction. RESULTS: We observed that in the presence of the transgene, the intrinsic force-generating capacity was restored and myofiber size was normal. INTERPRETATION: This demonstrates a direct link between disrupted attachment of myosin molecules to actin monomers and muscle fiber atrophy. These data also suggest that further therapeutic interventions should primarily target myosin dysfunction to alleviate the pathology of ACTA1-related nemaline myopathy. Ann Neurol 2016;79:717-725.