Skeletal Muscle

Manual analysis of skeletal muscle cross-sections is time-consuming and subject to error and user bias. To overcome these limitations, we developed and validated a semi-automated, quantitative, and reproducible image-analysis pipeline specifically tailored to quantify Pax7+ satellite cells, myonuclei, and cross-sectional area by fiber type. The workflow combines FIJI/ImageJ-based image preprocessing with CellProfiler, Cellpose, and a custom Python script to process and analyze immunohistological images of muscle tissue cross-sections. Outcomes include Pax7+ satellite cells and myonuclei quantified per fiber by fiber type, along with cross-sectional area, perimeter, and fiber type classification. This semi-automated approach provides a robust and efficient platform for high-throughput analysis of muscle tissue cross-sections from large datasets. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729866v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a3401dorg.highwire.dtl.DTLVardef@1c63145org.highwire.dtl.DTLVardef@ccbf76org.highwire.dtl.DTLVardef@2e0da0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pompe disease (glycogen storage disease type II) is an autosomal recessive lysosomal storage disorder characterized by progressive glycogen accumulation within lysosomes. It leads to their enlargement, autophagosome build-up and defective autophagic flux. Among the pathophysiological features, mitochondrial abnormalities have long been regarded as secondary consequences of lysosomal dysfunction. Typically, they have been described in electron microscopy, revealing paracrystalline inclusions, cristae lost, swollen mitochondria, and glycogen-filled structures. However, the spatial organization and interplay between mitochondria and lysosomes in skeletal muscle remain poorly understood, as does the progression of these alterations with respect to muscle metabolic profile. Here, we present a novel approach combining super-resolution imaging with a deep learning- based image analysis workflow to quantitatively assess mitochondrial and lysosomal remodeling as well as their interactions in skeletal muscle of the main murine model of the Pompe disease. Organelles were analyzed at two specific stages of the disease, according to muscle type, fiber type and subcellular location of the mitochondria. We show that the overall structure of the mitochondrial network is affected as early as the pre-symptomatic stage (1 month), while changes in mitochondrial density are more restricted at this stage and become more widespread as disease progresses (4 months). Importantly, these pathophysiological modifications are highly dependent on the muscle, fiber type and subcellular location. Alongside a rapid and widespread increase in lysosomal size, and a subsequent shift toward tighter lysosomal clustering at the later stage, we observe a progressive, region-specific increase in mitochondria-lysosome interactions that is most pronounced in the intermyofibrillar region. Our findings establish that this original imaging approach provides a relevant and powerful framework for quantitatively analyzing interactions between organelles within skeletal muscle fibers, thus offering new opportunities to explore the subcellular changes underlying disease progression. As such, it represents an interesting tool for monitoring pathophysiology and evaluating the effectiveness of therapeutic interventions.

This study examined how lower-body lengthened partial (LP) versus full range of motion (FULL) resistance training affects acute post-exercise signaling, chronic hypertrophy, and cellular adaptations of the vastus lateralis (VL) muscle in resistance-trained men. Eight males (22{+/-}1 years old, 5.6{+/-}1.4 years training) completed a crossover study whereby VL biopsies were collected pre-exercise and 0, 3, and 24 hours following LP and FULL leg extension bouts for transcriptomic and anabolic signaling analyses (Experiment 1). Another 16 males (26{+/-}5 years old; 8.0{+/-}4.9 years training) completed an 8-week, twice-weekly lower-body intervention using a within-subject design (Experiment 2). One leg was assigned to FULL and the contralateral leg to LP training across three exercises (leg press, leg extension, and lying leg curl). Pre- and post-intervention outcomes included VL muscle cross-sectional area (mCSA) summed across five equidistant MRI-derived transverse slices and mid-thigh VL biopsy outcomes. As a secondary outcome, other hip and thigh muscles from Experiment 2 MRI scans were assessed. Condition x Time interactions for all outcomes were assessed using linear mixed-effects models. In Experiment 1, both conditions produced similar time-dependent changes in the VL transcriptome and anabolic (mTORC1 and Hippo) signaling, but minimal between-protocol interactions. In Experiment 2, VL summed mCSA significantly increased over time (mean change: 9.3 cm{superscript 2}, 95% CI [6.8, 11.8], P<0.001), but there was no clear evidence of differential change between protocols (LP-FULL change: -1.4 cm{superscript 2}, 95% CI [-6.1, 3.8], P=0.640). Additionally, no significant interactions existed for type I fiber CSA (P=0.476), type II fiber CSA (P=0.350), type I fiber myonuclei (P=0.813), type II fiber myonuclei (P=0.589), type I and II satellite cell number (P=0.102 and P=0.797, respectively), or total RNA content (P=0.537). Despite these null VL-centric findings, secondary Experiment 2 analyses provided some evidence that whole hamstring hypertrophy was greater following LP versus FULL (LP-FULL change: 3.9 cm{superscript 2}, 95% CI [-0.2, 7.9], P=0.058). In conclusion, 8 weeks of LP and FULL resistance training broadly elicit similar acute and chronic VL responses in previously trained men, though secondary hamstring findings suggest that differential responses may depend on exercises included in the resistance training program.

Skeletal muscle contractile units or sarcomeres require constant maintenance as they are subjected to continuous chemical and mechanical stress. When sarcomere maintenance is disrupted, as occurs in progressive neuromuscular diseases, muscle progressively atrophies, reducing muscle strength and motor control. Transient cytoplasmic ribonucleoprotein aggregates comprised of TDP-43 bound to mRNAs encoding sarcomeric structural proteins (myo-granules) are implicated in building muscle. Ablating TDP-43 in differentiated skeletal muscle causes phenotypes remarkably similar to those of progressive neuromuscular diseases, including muscle atrophy, loss of muscle mass, and aberrantly organized sarcomeres. When injured, differentiated muscle lacking TDP-43 is incapable of repair, failing to build sarcomeres, severely disrupting muscle morphology with fibrotic tissue replacing muscle tissue. TDP-43 is thus required to build and maintain sarcomeres, likely protecting and transporting mRNAs encoding sarcomeric structural proteins in myo-granules.

Introduction. Skeletal muscle loss in sarcopenia and neuromuscular disorders remains a major unmet medical need. AAV9-delivered follistatin (FST), a myostatin/activin antagonist, induces muscle hypertrophy; however, fibre growth without adequate vascular adaptation may limit therapeutic efficacy. We evaluated whether co-administration of a VEGF-A165 plasmid enhances the hypertrophic and angiogenic effects of intramuscular AAV-FST gene transfer in C57BL/6 mice. Methods. Thirty-six C57BL/6 mice (18 males, 18 females) were assigned to PBS vehicle (n=10), AAV-FST (1 x 10^11 vg; n=10), VEGF plasmid (100 ug; n=6), or combination treatment (VEGF plus AAV-FST; n=10). The contralateral hindlimb served as an internal control. Endpoints at Day 115 included hindlimb muscle mass ratio (R/L), transgene expression, FST protein levels, muscle fibre morphometry, capillary density, and safety assessments. Results. Combination therapy produced the highest R/L ratio (1.176 +/- 0.091; p=0.004; d=2.04), whereas AAV-FST alone showed a borderline effect (R/L=1.113; p=0.050). Compared with AAV-FST monotherapy, combination treatment increased muscle FST mRNA approximately 2.1-fold, protein levels approximately 2.0-fold, and the muscle-to-liver expression ratio 2.6-fold. It also induced larger muscle fibres and doubled CD31+ vessel counts versus AAV-FST alone, indicating simultaneous hypertrophy and angiogenesis. No adverse haematological, biochemical, or histopathological findings were observed. Discussion. Combined AAV-FST and VEGF therapy enhanced local muscle hypertrophy, increased capillary density, and improved the muscle-to-liver transgene expression profile compared with AAV-FST monotherapy. The regimen was well tolerated and supports further evaluation of angiogenic preconditioning as a strategy to improve muscle-directed gene therapy for muscle-wasting disorders.

Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common dominant muscular dystrophies and remains without an approved disease modifying therapy. Caused by the aberrant expression of the cytotoxic gene DUX4, FSHD is typically diagnosed in adulthood, however clinical onset in children (<18 years of age) is often associated with a more severe and rapid disease. While clinical trials are underway, a lack of human-specific pre-clinical models limit effective testing of potential therapies, particularly in children. To fill this gap, we describe here the development of induced pluripotent stem cell-derived 2-and 3-dimensional skeletal muscle models of children with clinically defined mild, moderate, and severe FSHD. These iPSC-derived muscle models replicate key features of FSHD, including aberrant DUX4 mRNA expression, muscle atrophy, and weakness, which correlate with the individuals specific disease severity. Next, we assessed the efficacy of adenine base editing (ABE) as a potential gene therapy approach to treat FSHD. DUX4-targeted ABE reduced DUX4 mRNA expression, improved muscle area and force generation in the most severe individual. Together this work supports the use of iPSC-derived skeletal muscle models as a less invasive method to study childhood-onset FSHD and establishes targeted DUX4 gene editing therapies as a potential treatment approach.

The mitochondrial phosphatase PPTC7 is required to sustain mammalian metabolism, as its global knockout (KO) triggers hypoketotic hypoglycemia and perinatal lethality in mice. However, the extent to which the loss of Pptc7 manifests pathology beyond the perinatal transition is unknown. Furthermore, PPTC7 was recently identified as dual functional, regulating mitochondrial protein phosphorylation and receptor mediated mitophagy, rendering it unclear which function(s) may influence in vivo physiology. Here, we find that sustained, inducible Pptc7 KO decreased lean mass, compromised whole body oxygen consumption, and altered circulating metabolites in adult male mice. We hypothesized that these phenotypes stemmed from skeletal muscle dysfunction and found lower mass and fiber cross-sectional area with shifts in fiber type distribution in select muscles of the hindlimb in Pptc7 KO animals. Loss of PPTC7 increased BNIP3 protein levels and decreased mitochondrial content in skeletal muscle, suggesting elevated mitophagy may drive pathology. Consistently, KO of Bnip3 rescued the lower body weight and lean mass seen in inducible Pptc7 KO adult animals and partially rescued perinatal lethality in global Pptc7 KO mice. These data demonstrate that loss of PPTC7 incites surprisingly variable dysfunction across physiological contexts that at least partially stems from dysregulated BNIP3.

Although microtubules (MT) are established regulators of striated muscle mechanics, how MT lattice organization and post-translational modifications (PTM) individually shape contractility in healthy skeletal muscle fibers remains incompletely understood. We used an ex vivo single muscle fiber culture under unloading, examining acetylation and detyrosination (deTyr). During long-term 2D culture, the MT lattice was disrupted by transverse MT depletion without changes in MT abundance. In individual fibers, MT structure, but not abundance, positively correlated with sarcomere shortening non-linearly. When fibers were cultured in 3D hydrogels, the MT lattice was similarly disrupted yet shortening was preserved, with increased longitudinal MTs and decreased deTyr-MTs. Pharmacologically, contractility increased with either a decrease (parthenolide) or an increase (Taxol) in deTyr-MTs, and Taxol further rescued the MT lattice. Our findings identify MT organization and detyrosination, rather than MT abundance, as key determinants of muscle fiber contractility, positioning deTyr-MT as a load-responsive, bidirectional marker relevant to disuse atrophy and aging.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete epigenetic silencing of a D4Z4 macrosatellite array, leading to pathogenic misexpression of DUX4 in skeletal muscle. Therapeutic development of small molecule drugs for FSHD has been hampered by screens that yield key myogenic regulators as candidates and a lack of mechanistic knowledge regarding their effects on DUX4. To uncover more specific targets, we performed a candidate-based screen which identified several epigenetic facilitators of DUX4 expression in primary FSHD myocytes, including the chromatin remodeling factor BAZ1A. Here, we used a compound that we recently identified as a BAZ1A inhibitor and potent DUX4 suppressor to interrogate the role of BAZ1A at the FSHD locus. Our data suggest a model in which BAZ1A binds to D4Z4 in FSHD muscle, changing the chromatin landscape of the array. BAZ1A binding leads to reduced occupancy of the HP1 repressor, increased occupancy of the p300 coactivator, and increased H3K27 acetylation, promoting transcription of both DUX4 and the long non-coding RNA DBE-T from the disease locus. DBE-T, in turn, recruits the histone methyltransferase ASH1L, which establishes H3K36 methylation in cis, further promoting DUX4 transcription. BAZ1A inhibition disrupts this powerful feed-forward loop, supporting the development of more metabolically stable inhibitors. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/732407v1_ufig1.gif" ALT="Figure 1000"> View larger version (28K): org.highwire.dtl.DTLVardef@13bba8dorg.highwire.dtl.DTLVardef@5485c4org.highwire.dtl.DTLVardef@11a2e02org.highwire.dtl.DTLVardef@1c2a6b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

Amyotrophic lateral sclerosis is characterized by the progressive dismantling of the motor unit. While dying back hypothesis suggests that peripheral neuromuscular dysfunction precedes motor neuron loss, the molecular mechanisms limiting endogenous compensatory responses remain poorly understood. We longitudinally examined neuromuscular decline and GDF5-SMAD1/5/8 signaling in SOD1G93A mice. Our findings revealed a translational checkpoint linked to the lncRNA Myoparr that suppresses GDF5 production at symptom onset. To overcome this deficit, we delivered AAV9-GDF5 at the symptomatic stage. GDF5 supplementation restored SMAD signaling balance, shifting the motor unit from a pro-atrophic TGF-{beta}-SMAD2/3 toward a pro-myogenic SMAD1/5 profile. Treatment preserved muscle mass, reduced mitochondrial reactive oxygen species, and maintained neuromuscular junction integrity, including peri-synaptic glial support. GDF5 also promoted molecular recovery of spinal MNs by enhancing homeostatic marker expression. Together, these findings identify GDF5 as a multimodal stabilizer of the motor unit and highlight its potential as therapeutic target in combinatorial strategies aimed at coupling motor unit stabilization with central neuroprotective interventions.

The circadian clock regulates stem cell responses during tissue remodeling and repair. In skeletal muscle regeneration, successful regenerative myogenesis requires a temporally coordinated transition from Notch- to Wnt-driven signaling. However, the mechanisms governing this timing event remain poorly understood. Here, we show that circadian clock activity marks the cycling population of regeneration-activated myogenic progenitors that is induced in concert with Notch signaling. We identify key components of the Notch pathway as direct circadian clock targets and demonstrate that the clock coordinates Notch and Wnt signaling to drive myogenic progression. Genetic activation of the clock in satellite cells, as well as pharmacological clock stimulation, enhanced both proliferative expansion and subsequent differentiation of myogenic progenitors during regeneration. These effects were mediated by early activation of Notch signaling followed by increased Wnt pathway activity at later regenerative stages. Notably, both clock-dependent mechanisms remained functional in dystrophin-deficient mouse muscle and human myoblasts. Furthermore, clock-activating compounds enhanced regenerative myogenesis following acute injury and improved regeneration in dystrophic muscle. Collectively, these findings establish the circadian clock as a temporal regulator of regenerative signaling programs that orchestrate muscle repair with potential for targeted interventions.

BackgroundDynamic power declines earlier across the lifespan and shows a more pronounced and complex pattern than isometric strength, particularly in ageing females. However, the functional, skeletal muscle and molecular mechanisms underpinning power loss across the female lifespan remain to be collectively examined. MethodsEighty-six females aged 18-80 years and stratified per decade of age completed a series of maximal voluntary knee extensions to construct torque-velocity and power-velocity relationships of the quadriceps. Data points corresponding to >95% maximal power were selected for the evaluation of rate of torque development (RTD) and quadriceps surface electromyography (EMG). Outcomes were quantified within discrete 50ms time bins from torque onset to +200ms and included absolute RTD, RTD normalised to peak force, and EMG amplitude and rate of rise normalised to the maximal compound action potential. Quadriceps morphology was assessed via computed tomography, and a vastus lateralis muscle biopsy was collected to assess markers of denervation and expression of genes associated with the neuromuscular junction and calcium-handling transcriptome. ResultsAgeing led to linear reductions in maximal power (-1.39 {+/-} 0.01% p/year), torque (-0.98 {+/-} 0.13% p/year) and velocity (-0.38 {+/-} 0.01% p/year) (all p < 0.05). Quadriceps skeletal muscle CSA attenuated power loss by [~]40% (p < 0.001), largely through reduction of the decline in torque ([~]50%), with no effect on the decline in velocity. During early time bins, older females generated higher relative RTD accompanied by higher EMG amplitude, whereas during later time bins, older females generated less absolute and relative RTD accompanied by lower EMG amplitude and rate of rise (all p < 0.05). Ageing increased neural cell adhesion molecule (NCAM) positive fibres and fibrosis (both p < 0.05). The presence of NCAM{square} fibres was associated with attenuation of the age-related decline in maximal power ([~]15%), torque ([~]35%) and velocity ([~]60%), suggesting that NCAM{square} fibre prevalence may partially explain the observed age associations. Within the neuromuscular junction transcriptome, ageing reduced acetylcholinesterase and increased laminin alpha-2 and muscle-specific kinase (all FDR < 0.05), whereas lesser changes were observed within the calcium-handling transcriptome. ConclusionsSkeletal muscle CSA explains [~]40% of the age-related decline in quadriceps dynamic maximal power across the female lifespan, whereas a neurodegenerative profile mainly evidenced by age-related changes in voluntary neural drive, denervation and markers of neuromuscular junction instability further contribute to the decline.

The mitochondrial fatty acid synthesis (mtFAS) pathway is a highly conserved process in mitochondria implicated in metabolic state sensing. Aberrant functioning of this pathway leads to neurodegenerative diseases in humans. Animal experiments indicates that the mtFAS pathway is essential in mammals, and mtFAS inactivation leads to neuronal cell death. Nuclear encoded mitochondrial 3-ketoacyl-acyl carrier protein reductase (KAR) is a heterotetrameric enzyme in this process, consisting of two CBR4 and two HSD17B8 polypeptides. CBR4 works as the catalytic subunit of the enzyme. Here, we provide evidence that CBR4 function is essential in mammals. In contrast, a skeletal muscle-specific Cbr4 KO in mice did not result in any measurable defects in muscle strength and endurance, and the overall structure of the muscle remained unchanged. The Cbr4 KO did not affect the lipoylation process in quadriceps muscle samples, and high-resolution respirometry analysis of soleus muscle samples showed no defects in mitochondrial respiration capacity. The lack of a phenotype of a muscle-specific Cbr4 KO is consistent with previous reports on a lack of effects of mtFAS inactivation in muscle and re-iterates the question about the existence of bypass mechanisms that can alleviate mtFAS deficiencies in non-neuronal cell types.

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder marked by lack of dystrophin expression. Symptoms include loss of ambulation, respiratory problems, and cardiac complications with heart failure being the leading cause of death. Dystrophin transduces force from the actin cytoskeleton to the extracellular matrix to protect cells during muscle contraction. Restoration of dystrophin expression by gene transfer holds promise in addressing the root cause of disease. We compared the changes to transcriptional profiles after gene transfer by adeno-associated virus or lentivirus to examine whether viral treatment alone impacts cell homeostasis. We delivered GFP to cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs) with DMD mutations. Global transcriptional profiling revealed a downregulation of metabolic genes after lentiviral transduction compared to untreated controls. In both AAV and lentivirus-treated DMD iPSC-cardiomyocytes, we observed an activation of the p53 DNA damage response in addition to a downregulation of cell cycle genes, suggesting stress-induced G2/M checkpoint arrest following viral delivery. These findings demonstrate that gene therapy mediated by viral vectors activates cell stress pathways. Interventions to mitigate these stress responses may be necessary for safe and effective gene transfer in diseased cells.

Facioscapulohumeral muscular dystrophy (FSHD) is a rare neuromuscular disease caused by aberrant re-expression of the embryonic transcription factor DUX4 in skeletal muscle, which activates a toxic transcriptional program that drives progressive muscle wasting. No approved disease-modifying therapies currently exist. Prior work in mammalian and zebrafish models has shown that a truncated form of DUX4 retaining only its DNA-binding domain (DBD) lacks transactivation capacity and can suppress DUX4-FL-driven pathology; separately, dCas9/KRAB-based epigenetic repressors have demonstrated efficacy in silencing DUX4 expression, though CRISPR-based strategies face challenges from the repetitive nature of the D4Z4 locus, the immunogenicity associated with bacterial Cas proteins, and the payload limitations of gene delivery vehicles. Building on these findings, we corroborate that the DUX4 DBD, comprising both homeodomains, acts as a non-toxic competitive inhibitor of full-length DUX4 (DUX4-FL) at its genomic target sites, and extend this strategy by fusing the DBD to a human KRAB(ZNF10) domain, converting DUX4 from a transcriptional activator into a fully humanized epigenetic silencer of its own targets. Using a fluorescent DUX4-responsive reporter, we show that DBD alone produces dose-dependent repression of DUX4-FL transcriptional activity in HEK293T cells (200-fold at the highest inducible dose tested), while a constitutively expressed DBD-KRAB fusion produces significantly greater repression than DBD alone (949-fold versus 17-fold at a 25x molar ratio), with a similar trend observed in C2C12 myoblasts (47-fold versus 3.3-fold knockdown). To contextualize these findings and explore dosing considerations, we developed three complementary computational models - a transcription factor competitive binding model, a myotube diffusion model, and an ordinary differential equation (ODE) compartmental model - that illustrate how DBD concentration, intracellular diffusion, and population-level cell state transitions may relate to therapeutic efficacy. Together, these results corroborate and extend existing approaches into a single, fully humanized construct that may help circumvent the immunogenicity and delivery limitations of Cas-based systems.

Regular exercise induces adaptations in skeletal muscle and other organ systems to improve physical performance and overall health. Exercise results in phosphorylation of 5 AMP-activated protein kinase (AMPK) at threonine 172 (T172) of the 2 subunit; however, the role of this activation in cellular and functional adaptations has not been elucidated. To this end, we subjected non-activatable Ampk2(T172A) knock-in (KI) adult mice and wild-type (WT) littermates to 4 weeks of voluntary wheel running (VWR). Exercise training led to significant improvements in endurance capacity, maximal oxygen consumption ([Formula]O2max), and glucose tolerance, as well as skeletal muscle IIb-to-IIa fiber type shift in both WT and KI mice. Contrastingly, VWR resulted in increased mitochondrial OxPhos protein expression, mitochondrial volume density, and capillary density in skeletal muscle of WT but not KI mice. Exercise-induced improvements of mitochondrial respiration and conductance revealed by high-resolution respirometry of isolated mitochondria were blunted in KI mice. Therefore, for the first time, we reveal that AMPK2 T172 activation is required for exercise training-induced mitochondrial biogenesis, improvement of mitochondrial respiratory function, and angiogenesis in skeletal muscle, but that these adaptations are not solely responsible for improved [Formula]O2max and exercise endurance capacity. Significance StatementExercise is the most effective lifestyle intervention for promoting health and preventing chronic diseases through adaptive changes in skeletal muscle and many other tissues/organs. AMPK is an energy sensor and signaling regulator for exercise-induced skeletal muscle adaptation, yet its functional role and the impact on exercise capacity have been studied in mouse genetic models wherein protein stoichiometry is disrupted. Using non-activatable Ampk2(T172A) knock-in mice, we ascertained that AMPK2 activation via T172 phosphorylation is required for endurance training-induced mitochondrial and angiogenic adaptations in skeletal muscle. Importantly, these adaptations are not required for improved exercise capacity, challenging the prevailing concept that increased mitochondrial content and function and microvasculature are the sole driving factors for the performance gains with endurance training.

Skeletal muscle stem cells (MuSCs) give rise to a fusogenic cell population that provide new myonuclei to muscle fibers. Myonuclear functional heterogeneity has recently become appreciated, but the terminal identity of MuSC-Derived myonuclei remains undefined. We performed single-nucleus RNA-sequencing of myonuclei in Adult and Aged muscle to define MuSC-Derived and resident myonuclear responses to mechanical overload (MOV), which induces a hypertrophic stimulus. We found a MuSC-dependent induction of a youthful transcriptional signature in resident myonuclei after MOV in Aged muscle. Age determined terminal transcriptional states of MuSC-Derived myonuclei toward MTJ in Adult, NMJ in Aged, and muscle spindles in both ages. Microtubule-remodeling genes, Macf1, Map1b, and Nav3, along with the transcription factor Runx1, identified this post-fusion specialization with greater expression of these genes in Adult than in Aged MuSC-Derived myonuclei. In-silico transcription factor KO screen identified Runx1 as a regulator of post-fusion specialization and Esrrg as a driver of spindle (intrafusal) MuSC-Derived myonuclear maturation. By defining the age-associated fate of MuSC fusion to muscle fibers, we provide potential targets for modulating muscle plasticity.

IntroductionThe quality of preclinical studies for rare diseases, such as Duchenne muscular dystrophy (DMD), relies on the availability of comprehensive natural disease history data. In addition to the classic BL10-mdx mouse, in recent years, the D2-mdx model has increasingly been used as an alternative model due to its reportedly more severely impaired phenotype. To improve our understanding of disease progression in these two DMD models, we conducted a comprehensive natural history study. Materials and MethodsThis involved a cross-sectional analysis of key in vivo and ex vivo outcome measures performed in two independent laboratories, using the same study setup in compliance with TREAT-NMD Standard Operating Procedures (SOPs), while also taking advantage of site-specific expertise. Globally, largely comparable results were obtained across the two study sites. ResultsBody composition showed pronounced differences between the strains, with BL10-mdx mice displaying a hypertrophic and D2-mdx mice displaying an atrophic phenotype. Dystrophic mice of each strain exhibited significant alterations of disease-relevant indices related to muscle functionality and integrity, mostly worsening with age, in comparison to their wildtypes. Cardiac function was affected earlier and more severely in D2-mdx mice. DiscussionNotably, for some parameters, genetic-background related differences were observed, emphasizing the need to include control groups with matching genetic backgrounds in experimental designs. ConclusionsCollectively, our natural history study provides benchmark data for these two mdx mouse strains to guide model selection for preclinical DMD studies, allowing accurate data interpretation. HighlightsO_LIDistinct body composition phenotypes: BL10-mdx mice exhibit pseudohypertrophy while D2-mdx mice display pronounced atrophy. C_LIO_LIEarlier cardiac dysfunction in D2-mdx: D2-mdx mice develop reduced ejection fraction and stroke volume from 28 weeks, while BL10-mdx only at 52 weeks. C_LIO_LIGenetic background-dependent variations: Intrinsic deficits in wildtype D2 mice demonstrate that genetic background influences outcome measures independent of dystrophic pathology. C_LIO_LIComparable ex vivo muscle physiology: Despite divergent in vivo phenotypes, isolated muscle contractile parameters show similar impairment in both dystrophic models. C_LIO_LIMulti-site standardized validation: Cross-sectional study at two independent laboratories following harmonized TREAT-NMD Standard Operating Procedures. C_LI

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas. An obstacle to treating MPNSTs is a lack of effective systemic therapies. Although over 70% of human MPNSTs have lost or inactivated the epigenome regulator polycomb repressive complex 2 (PRC2), its activity and contribution to canine PNST progression remain unclear. This study compared canine peripheral nerve sheath tumors (PNSTs) and human MPNSTs across biological and clinical features, including PRC2 activity. Immunohistochemical analysis was performed for a human tissue microarray of 54 neurofibromas and 139 MPNSTs, and 63 canine PNSTs for H3K27me3, a repressive histone mark deposited by intact PRC2, and H3K27ac, which increases globally upon H3K27me3 loss. To understand the genomic alterations present in canine PNSTs, we analyzed tumor mutation burden, copy number alteration, and transcriptomes of eight canine PNST/normal pairs. The results suggested that H3K27me3 loss and associated gain of H3K27ac epigenetically drive human and canine tumors. These findings warrant further studies to evaluate whether these epigenetic deregulations alter similar gene signatures across species.

Skeletal muscle homeostasis depends on the coordinated regulation of protein turnover and mitochondrial quality control; however, the molecular mechanisms linking these processes remain unclear. In this study, we examined the physiological role of leucine aminopeptidase 3 (LAP3), a post-proteolytic aminopeptidase, using constitutive LAP3-deficient mice. LAP3 deficiency preferentially affected skeletal muscle, causing reduced muscle mass and mitochondrial enlargement in both sexes. Female LAP3-deficient mice also showed reduced myofiber size, impaired endurance capacity, increased energy expenditure, elevated lipid oxidation, and lipid droplet accumulation adjacent to the mitochondria. Proteomic analyses revealed remodeling of pathways related to lipid metabolism and protein homeostasis. Consistent with these findings, LAP3 deficiency increased the expression of Pink1 and Tax1bp1 and promoted the accumulation of ubiquitinated proteins, suggesting alterations in mitochondrial quality control and proteostatic regulation. In cultured myogenic cells, LAP3 localized to mitochondrial fractions, and both LAP3 knockdown and overexpression altered mitochondrial morphology. Taken together, these results identify LAP3 as a regulator of skeletal muscle homeostasis and support a role for LAP3 in linking intracellular peptide turnover to mitochondrial homeostasis, with female skeletal muscle showing greater susceptibility to LAP3 deficiency.