Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1-related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

Diagnostic interest of whole-body MRI in early- and late-onset LAMA2 muscular dystrophies: a large international cohort.

BACKGROUND: LAMA2-related muscular dystrophy (LAMA2-RD) encompasses a group of recessive muscular dystrophies caused by mutations in the LAMA2 gene, which codes for the alpha-2 chain of laminin-211 (merosin). Diagnosis is straightforward in the classic congenital presentation with no ambulation and complete merosin deficiency in muscle biopsy, but is far more difficult in milder ambulant individuals with partial merosin deficiency. OBJECTIVE: To investigate the diagnostic utility of muscle imaging in LAMA2-RD using whole-body magnetic resonance imaging (WBMRI). RESULTS: 27 patients (2-62 years, 21-80% with acquisition of walking ability and 6 never ambulant) were included in an international collaborative study. All carried two pathogenic mutations, mostly private missense changes. An intronic variant (c.909 + 7A > G) was identified in all the Chilean cases. Three patients (two ambulant) showed intellectual disability, epilepsy, and brain structural abnormalities. WBMRI T1w sequences or T2 fat-saturated images (Dixon) revealed abnormal muscle fat replacement predominantly in subscapularis, lumbar paraspinals, gluteus minimus and medius, posterior thigh (adductor magnus, biceps femoris, hamstrings) and soleus. This involvement pattern was consistent for both ambulant and non-ambulant patients. The degree of replacement was predominantly correlated to the disease duration, rather than to the onset or the clinical severity. A "COL6-like sandwich sign" was observed in several muscles in ambulant adults, but different involvement of subscapularis, gluteus minimus, and medius changes allowed distinguishing LAMA2-RD from collagenopathies. The thigh muscles seem to be the best ones to assess disease progression. CONCLUSION: WBMRI in LAMA2-RD shows a homogeneous pattern of brain and muscle imaging, representing a supportive diagnostic tool.

A muscle growth-promoting treatment based on the attenuation of activin/myostatin signalling results in long-term testicular abnormalities.

Activin/myostatin signalling acts to induce skeletal muscle atrophy in adult mammals by inhibiting protein synthesis as well as promoting protein and organelle turnover. Numerous strategies have been successfully developed to attenuate the signalling properties of these molecules, which result in augmenting muscle growth. However, these molecules, in particular activin, play major roles in tissue homeostasis in numerous organs of the mammalian body. We have recently shown that although the attenuation of activin/myostatin results in robust muscle growth, it also has a detrimental impact on the testis. Here, we aimed to discover the long-term consequences of a brief period of exposure to muscle growth-promoting molecules in the testis. We demonstrate that muscle hypertrophy promoted by a soluble activin type IIB ligand trap (sActRIIB) is a short-lived phenomenon. In stark contrast, short-term treatment with sActRIIB results in immediate impact on the testis, which persists after the sessions of the intervention. Gene array analysis identified an expansion in aberrant gene expression over time in the testis, initiated by a brief exposure to muscle growth-promoting molecules. The impact on the testis results in decreased organ size as well as quantitative and qualitative impact on sperm. Finally, we have used a drug-repurposing strategy to exploit the gene expression data to identify a compound - N6-methyladenosine - that may protect the testis from the impact of the muscle growth-promoting regime. This work indicates the potential long-term harmful effects of strategies aimed at promoting muscle growth by attenuating activin/myostatin signalling. Furthermore, we have identified a molecule that could, in the future, be used to overcome the detrimental impact of sActRIIB treatment on the testis.

Very Low Residual Dystrophin Quantity Is Associated with Milder Dystrophinopathy.

OBJECTIVE: This study was undertaken to determine whether a low residual quantity of dystrophin protein is associated with delayed clinical milestones in patients with DMD mutations. METHODS: We performed a retrospective multicentric cohort study by using molecular and clinical data from patients with DMD mutations registered in the Universal Mutation Database-DMD France database. Patients with intronic, splice site, or nonsense DMD mutations, with available muscle biopsy Western blot data, were included irrespective of whether they presented with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD). Patients were separated into 3 groups based on dystrophin protein levels. Clinical outcomes were ages at appearance of first symptoms; loss of ambulation; fall in vital capacity and left ventricular ejection fraction; interventions such as spinal fusion, tracheostomy, and noninvasive ventilation; and death. RESULTS: Of 3,880 patients with DMD mutations, 90 with mutations of interest were included. Forty-two patients expressed no dystrophin (group A), and 31 of 42 (74%) developed DMD. Thirty-four patients had dystrophin quantities < 5% (group B), and 21 of 34 (61%) developed BMD. Fourteen patients had dystrophin quantities ≥ 5% (group C), and all but 4 who lost ambulation beyond 24 years of age were ambulant. Dystrophin quantities of <5%, as low as <0.5%, were associated with milder phenotype for most of the evaluated clinical outcomes, including age at loss of ambulation (p < 0.001). INTERPRETATION: Very low residual dystrophin protein quantity can cause a shift in disease phenotype from DMD toward BMD. ANN NEUROL 2021;89:280-292.

Asymmetric muscle weakness due to ACTA1 mosaic mutations.

OBJECTIVE: To characterize 2 unrelated patients with either asymmetric or unilateral muscle weakness at the clinical, genetic, histologic, and ultrastructural level. METHODS: The patients underwent thorough clinical examination, whole-body MRI, and exome sequencing. Muscle morphology was assessed by histology and electron microscopy. RESULTS: Both patients presented with early-onset hypotonia, delayed motor milestones, scoliosis, and reduced pulmonary function. Patient P1 manifested unilateral muscle weakness exclusively affecting the left side of the body; the asymmetry was less pronounced in patient P2. Muscle biopsies from both patients showed nemaline rods as the main histopathologic hallmark, and MRI revealed major fatty infiltrations in selective head, proximal, and distal muscles, correlating with the degree of muscle weakness asymmetry. Exome sequencing on blood DNA from both patients identified de novo ACTA1 missense mutations in a small number of reads, suggesting mutation mosaicism. Subsequent Sanger sequencing confirmed the presence of the mutations on muscle DNA, while they were barely detectable on blood DNA. CONCLUSIONS: De novo mutations can occur anytime during embryonic development and may result in a mosaic pattern of affected cells and tissues and lead to the development of an asymmetric clinical picture. The present study points out that mosaic mutations might not be easily detectable on leukocyte DNA and thereby escape routine genetic analysis, and possibly account for a significant number of molecularly undiagnosed patients.

Inhibition of Activin/Myostatin signalling induces skeletal muscle hypertrophy but impairs mouse testicular development.

Numerous approaches are being developed to promote post-natal muscle growth based on attenuating Myostatin/Activin signalling for clinical uses such as the treatment neuromuscular diseases, cancer cachexia and sarcopenia. However there have been concerns about the effects of inhibiting Activin on tissues other than skeletal muscle. We intraperitoneally injected mice with the Activin ligand trap, sActRIIB, in young, adult and a progeric mouse model. Treatment at any stage in the life of the mouse rapidly increased muscle mass. However at all stages of life the treatment decreased the weights of the testis. Not only were the testis smaller, but they contained fewer sperm compared to untreated mice. We found that the hypertrophic muscle phenotype was lost after the cessation of sActRIIB treatment but abnormal testis phenotype persisted. In summary, attenuation of Myostatin/Activin signalling inhibited testis development. Future use of molecules based on a similar mode of action to promote muscle growth should be carefully profiled for adverse side-effects on the testis. However the effectiveness of sActRIIB as a modulator of Activin function provides a possible therapeutic strategy to alleviate testicular seminoma development.

SIX1 and SIX4 homeoproteins regulate PAX7+ progenitor cell properties during fetal epaxial myogenesis.

Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7.

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration.

STAC3 variants cause a congenital myopathy with distinctive dysmorphic features and malignant hyperthermia susceptibility.

SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.

Lack of myostatin impairs mechanical performance and ATP cost of contraction in exercising mouse gastrocnemius muscle in vivo.

Although it is well established that the lack of myostatin (Mstn) promotes skeletal muscle hypertrophy, the corresponding changes regarding force generation have been studied mainly in vitro and remain conflicting. Furthermore, the metabolic underpinnings of these changes are very poorly documented. To clarify this issue, we have investigated strictly noninvasively in vivo the impact of the lack of Mstn on gastrocnemius muscle function and energetics in Mstn-targeted knockout (Mstn-/-) mice using ¹H-magnetic resonance (MR) imaging and ³¹P-MR spectroscopy during maximal repeated isometric contractions induced by transcutaneous electrostimulation. In Mstn-/- animals, although body weight, gastrocnemius muscle volume, and absolute force were larger (+38, +118, and +34%, respectively) compared with wild-type (Mstn+/+) mice, specific force (calculated from MR imaging measurements) was significantly lower (-36%), and resistance to fatigue was decreased. Besides, Mstn deficiency did not affect phosphorylated compound concentrations and intracellular pH at rest but caused a large increase in ATP cost of contraction (up to +206% compared with Mstn+/+) throughout the stimulation period. Further, Mstn deficiency limits the shift toward oxidative metabolism during muscle activity despite the fact that oxidative ATP synthesis capacity was not altered. Our data demonstrate in vivo that the absence of Mstn impairs both mechanical performance and energy cost of contraction in hypertrophic muscle. These findings must be kept in mind when considering Mstn as a potential therapeutic target for increasing muscle mass in patients suffering from muscle-wasting disorders.

Combined effect of AAV-U7-induced dystrophin exon skipping and soluble activin Type IIB receptor in mdx mice.

Adeno-associated virus (AAV)-U7-mediated skipping of dystrophin-exon-23 restores dystrophin expression and muscle function in the mdx mouse model of Duchenne muscular dystrophy. Soluble activin receptor IIB (sActRIIB-Fc) inhibits signaling of myostatin and homologous molecules and increases muscle mass and function of wild-type and mdx mice. We hypothesized that combined treatment with AAV-U7 and sActRIIB-Fc may synergistically improve mdx muscle function. Bioactivity of sActRIIB-Fc on skeletal muscle was first demonstrated in wild-type mice. In mdx mice we show that AAV-U7-mediated dystrophin restoration improved specific muscle force and resistance to eccentric contractions when applied alone. Treatment of mdx mice with sActRIIB-Fc increased body weight, muscle mass and myofiber size, but had little effect on muscle function. Combined treatment stimulated muscle growth comparable to the effect of sActRIIB-Fc alone and dystrophin rescue was similar to AAV-U7 alone. Moreover, combined treatment improved maximal tetanic force and the resistance to eccentric contraction to similar extent as AAV-U7 alone. In conclusion, combination of dystrophin exon skipping with sActRIIB-Fc brings together benefits of each treatment; however, we failed to evidence a clear synergistic effect on mdx muscle function.

Exercise training attenuates the hypermuscular phenotype and restores skeletal muscle function in the myostatin null mouse.

Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN(-/-)) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force-generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN(-/-) mice that can be induced by training. To this end, we subjected MSTN(-/-) mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross-sectional area of hypertrophic myofibres from MSTN(-/-) mice decreased towards wild-type values in response to exercise and, in this context, Bnip3, a key autophagy-related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear-to-cytoplasmic ratio towards wild-type values. Importantly, both training regimes increased muscle force in MSTN(-/-) mice. We conclude that impaired skeletal muscle function in myostatin-deficient mice can be improved through endurance exercise-mediated remodelling of muscle fibre size and metabolic profile.

Myostatin knockout mice increase oxidative muscle phenotype as an adaptive response to exercise.

Myostatin-deficient mice (MSTN (-/-)) display excessive muscle mass and this is associated with a profound loss of oxidative metabolic properties. In this study we analysed the effect of two endurance-based exercise regimes, either a forced high-impact swim training or moderate intensity voluntary wheel running on the adaptive properties of the tibialis anterior and plantaris muscle from MSTN (-/-) mice. MSTN (-/-) and wild type (MSTN (+/+)) animals had comparable performances in the wheel running regime in terms of distance, average speed and time, but MSTN (-/-) mice showed a reduced ability to sustain a high-impact activity via swimming. Swim training elicited muscle specific adaptations on fibre type distribution in MSTN (-/-); the tibialis anterior displaying a partial transformation in contrast to the plantaris which showed no change. Conversely, wheel running induced similar changes in fibre type composition of both muscles, favouring transitions from IIB-to-IIA. Succinate dehydrogenase activity, an indicator of mitochondrial oxidative potential was increased in response to either exercise regime, with wheel running eliciting more robust changes in the MSTN (-/-) muscles. Examination of the cross sectional area of individual fibre types showed genotype-specific responses with MSTN (-/-) mice exhibiting an incapability of fibre enlargement following the wheel running regime, as opposed to MSTN (+/+) mice and a greater susceptibility to muscle fibre area loss following swimming. In conclusion, the muscle fibre hypertrophy, oxidative capacity and glycolytic phenotype of myostatin deficient muscle can be altered with endurance exercise regimes.

Activin A inhibits formation of skeletal muscle during chick development.

In this study we investigated the effect of recombinant activin A on the differentiation of limb muscle precursors of chick embryos. We show that treatment with activin resulted in a downregulation of Pax-3 and MyoD expression within 6 h after treatment, whereas expression of Myf-5 and Pax-7 was largely unaffected. The effect on gene expression was transient because 1 day after activin exposure the development of the premuscle masses had proceeded, and Pax-3 and MyoD expression was reexpressed at normal levels. Unlike other transforming growth factors-beta, activin did not induce programmed cell death in limb mesenchyme, thus myogenic cells were not permanently lost. In high-density cultures of embryonic chick limb mesenchyme (micromass cultures), activin repressed the generation of Pax-7-expressing muscle precursors. Furthermore, in the presence of activin, fewer muscle precursors differentiated, and the population of differentiating cells failed to fuse and form myotubes. Our data suggest that activin reversibly inhibited expression of two transcription factors, Pax-3 and MyoD, and thus transiently inhibited proliferation and differentiation of limb muscle precursors. However, myogenic cells were not lost as they continued to express Pax-7 and Myf-5, and this may have allowed precursors to commence development after the activin effect faded. We suggest that activin acts in conjunction with a closely related signalling molecule, myostatin, to prevent excessive growth of skeletal muscle.