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.
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.
What is the central question of the study?
How do endurance exercise and the ageing process affect the positioning and number of myonuclei within muscle fibres?
What are the main findings and their importance?
There was reduced muscle fibre size, with smaller myonuclear domains and altered myonuclear domain scaling in older, untrained individuals. However, older trained individuals maintained similar muscle and myonuclear characteristics to younger trained individuals. This suggests that inactivity, rather than ageing, influences muscle fibre and myonuclear characteristics. These observations suggest that consistent endurance exercise over one's lifetime might help to preserve muscle fibre size and quality and myonuclear arrangement into old age.
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.
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 × 10
vg kg
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.
Abstract only
Skeletal myopathies are a broad spectrum of genetic disorders that affect muscle, and are caused by mutations in a variety of genes required for muscle function and cellular ...homeostasis. Actin is an essential component of the contraction machinery. Mutations in actin‐associated proteins cause nemaline myopathy, which is characterized by the presence of protein aggregates, as well as muscle fiber atrophy, contractile dysfunction and weakness. At present, there are no effective therapies for nemaline myopathy.
Myostatin is a secreted factor responsible for negatively regulating muscle growth. Inhibition of myostatin (MI) results in muscle fiber hypertrophy, and has received much interest as a possible therapeutic strategy for a variety of muscle diseases. Previous work has been carried out in two mouse models of nemaline myopathy with mutations in
ACTA1
(skeletal muscle actin): an H40Y mutant, in which MI confers only modest therapeutic benefits; and a D286G mutant, in which MI is able to induce marked hypertrophy and functional improvements. We aim to investigate the reasons for this differing response.
Muscle fibers are giant cells which require hundreds of internal nuclei to generate sufficient gene products. It has been hypothesized that insufficient myonuclear density is a pathological factor in a variety of muscle diseases, leading to reduced expression of proteins required for e.g. contraction and energy metabolism. We hypothesized that nuclear density might also be a pathological factor in
ACTA1
mutants, and that differences in this parameter might explain the variable responses to MI. Thus, using confocal microscopy and single muscle fibers, we assessed nuclear number and organization in 3 dimensions, in mutants with and without MI treatment.
We find that nuclei are positioned irregularly in the severe H40Y mutant. MI treatment is able to rescue this defect, and cause insertion of extra nuclei; yet these effects are not accompanied by significant hypertrophy. No underlying nuclear defects were observed in the milder D286G mutant; MI treatment did not alter nuclear arrangement but caused marked hypertrophy. Nuclear organisation defects will also be discussed in light of our findings in patients with
ACTA1
mutations. These findings suggest that aberrant nuclear arrangement may be a pathological factor in diseases of muscle contraction. MI treatment may not be an effective treatment when force production and/or pathology is too severe. Unravelling the mechanisms of myostatin action at the subcellular level is of importance for the design, modulation and improvement of therapies.
Support or Funding Information
Medical Research Council, UK
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
Sarcopenia, a condition characterised by progressive loss of skeletal muscle mass and strength, has been associated with an intrinsic deterioration of the force-generating capacity of individual ...myofibres. As the reduction in force-generation is greater than the loss of muscle fibre volume (specific force loss), a decrease in cellular quality may contribute to the aforementioned weakness. The work presented in this thesis aimed to further elucidate the mechanisms by which myofibre force is depressed during the ageing process, using a plethora of animal and human models, with specific reference to myonuclear proteins and organisation. Existent ex vivo methods to study myonuclear organisation in single muscle fibres did not fulfil the methodological necessities of this project. Thus, methods of varying degrees of automation were developed to study the (i) three-dimensional nuclear organisation, and (ii) nuclear pixel intensities of a range of antibodies, in confocal image stacks of isolated skinned single muscle fibres. Analyses were performed using custom-made MATLAB scripts and Fiji macros. Both methods were used in the present thesis and various related publications. Physiological and premature ageing are frequently associated with an accumulation of prelamin A, a precursor of lamin A, in the nuclear envelope of various cell types. The current work aimed to underpin the hitherto unknown mechanisms by which accumulation of prelamin A in the nucleus alters myonuclear organisation and muscle fibre function. By applying the methods developed in this work on single muscle fibres from various mouse models of lamin A misprocessing, results indicated that postnatal accumulation of farnesylated prelamin A and absence of Zmpste24 in myonuclei can lead to a low number of nuclei and enlarged myonuclear domains in single muscle fibres, concomitant with decreased transcriptional activity, reduction in contractile protein content and low specific force. ABSTRACT 3 Scientific studies on human ageing are limited by inclusion of sedentary cohorts. Hence, the role of myonuclear organisation is unknown in biological ageing, i.e. ageing that is not contaminated by inactivity-related pathologies. The final objective in this thesis was to verify whether levels of activity and age can lead to changes in nuclear organisation in single muscle fibres. Results showed an elevated number of nuclei in single muscle fibres from physically inactive patients and nuclear mispositioning in all the elderly cohorts. Transcriptional activity in the active cohort was >2x that of all other groups, revealing a possible association between level of physical activity, but not age. Consequently, biological ageing and muscle disuse do not share the same myonuclear alterations, demonstrating the importance of differentiating between the two conditions in ageing research. Future work should deepen the understanding of the mechanisms by which the persistence of farnesylated prelamin A and absence of Zmpste24 in myonuclei lead to a smaller nuclear population in single muscle fibres. Moreover, research should continue to characterise the specific contribution of muscle disuse to human ageing. Importantly, future studies should identify whether any correlation exists between the existence of prelamin A and progerin in single muscle fibres, and age and physical activity.
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.
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.
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