Journal of Cachexia, Sarcopenia and Muscle

BackgroundAgeing is accompanied by progressive declines in skeletal muscle mass and strength, culminating in sarcopenia, a condition that contributes to frailty, multimorbidity, and mortality. Age-related changes to mitochondria lead to oxidative damage and dysfunction and are proposed to occur early in the trajectory of sarcopenia, supporting the candidacy of mitochondrial-protective therapies. Here, we test the efficacy of mitochondrial uncoupler BAM15 in age-dependent sarcopenic mouse models. MethodsMale and female MitoQC mice aged 24 months received either standard chow or chow supplemented with BAM15 (0.033% mg/g) ad libitum for eight weeks (n=13-14/group). Young (3-month-old) mice served as reference controls (n=8/group). Muscle mitochondrial respiration was assessed in permeabilized fib res, and contractile function was measured in isolated extensor digitorum longus and soleus muscles. Mitophagy was quantified by immunofluorescence confocal microscopy. Data were analyzed using one-or two-way ANOVA followed by Dunnetts or Bonferronis multiple comparison tests. ResultsAged male and female mice exhibited reduced gastrocnemius muscle mass relative to body mass compared with young controls (p<0.05; [~]18% and [~]32% loss, respectively). BAM15 did not alter muscle size but reversed the age-related loss of contractile function in EDL muscles, to that of the young reference controls in both sexes (p<0.05; [~]33% in males, [~]16% in females). In male mice, BAM15 improved mitochondrial efficiency, evidenced by restoration of Complex I-linked respiration and decreased proton leak ([~]52% improvement; p<0.05), and normalized protein levels of oxidative stress marker 4 -HNE, without changes in mitophagy or mitochondrial content. In females, BAM15 did not improve mitochondrial parameters, which may be, in part, due to aged female muscle exhibiting unchanged Complex I leak and 4-HNE protein abundance, alongside lower complex I subunit (NDUFB8) protein abundance. ConclusionsBAM15 improved skeletal muscle mitochondrial efficiency and contractile function in aged male mice, supporting the potential of mitochondrial uncoupling as a therapeutic strategy for sarcopenia.

BackgroundOxidative phosphorylation (OXPHOS) is a central function and a key indicator of mitochondrial fitness, yet studies in human tissue remain limited. Inclusion body myositis (IBM) is a progressive myopathy that lies at the intersection of aging, inflammation and mitochondrial dysfunction. We aimed to perform a comprehensive profiling of mitochondrial respiration in muscle tissue from patients with IBM. MethodsA wide battery of complementary tests from RNA level to high-resolution respirometry on permeabilized muscle fibers was performed. The relationship between respiration, mitochondrial content, mitochondrial DNA (mtDNA) abnormalities and mitophagy was examined, along with the correlation with various clinical parameters to determine the clinical significance of the findings. ResultsThe study included a total of 67 patients with IBM and 45 controls. IBM muscle tissue exhibited reduced maximal respiration per tissue weight in State 3 (high substrates, high ADP) and uncoupled state with decreased coupling efficiency and higher leak control ratios. When adjusting for citrate synthase reflecting mitochondrial content, males had decreased State 3 intrinsic respiration, whereas females had greater intrinsic respiration in leak states. Complex II control ratio strongly correlated with disease duration and severity only in females. IBM was associated with decreased RNA and protein expression of OXPHOS complexes. Complex I activity was decreased mainly in females. IBM samples exhibited lower maximal H2O2 emission, accompanied by a higher total antioxidant capacity that correlated with disease duration in females. In IBM, there was decreased mtDNA content, and impaired mitophagy, both of which strongly correlated with respirometry measures and markers of disease severity, indicating these pathways are likely interconnected and of clinical significance. ConclusionIBM is characterized by multilevel impairments in mitochondrial coupling efficiency, revealing several potential therapeutic targets to improve mitochondrial fitness, while accounting for sex-specific differences.

Whether and how ovarian hormone fluctuations mediate the skeletal muscle response to ageing in females remains to be elucidated. We examined a tightly controlled, cross-sectional cohort of 96 females between 18-80 years of age to map the functional and molecular trajectory of muscle ageing and determine its relationship with female sex hormones. Across every decade, we quantified body composition (using dual-energy x-ray absorptiometry), muscle morphology (using peripheral quantitative computed tomography), and voluntary and evoked muscle function. Circulating sex hormone concentrations were measured with gas chromatography mass spectrometry and immunoassays. Morphology and gene expression of vastus lateralis muscle samples were assessed with immunohistochemical staining and RNA sequencing, respectively. Age was negatively associated with muscle mass, strength, and muscle fibre size, and positively associated with hybrid type I/II fibre prevalence and fibrosis. We found 37 unique patterns of gene expression across individual decades of age. Immune signalling, cellular adhesion, and extracellular matrix organisation pathways were the most upregulated with age, while mitochondrial function pathways were the most downregulated. Independently of age, circulating oestradiol and progesterone, but not testosterone, concentrations were positively associated with lean mass and negatively associated with hybrid muscle fibres across the lifespan. Oestrogen receptor binding sites were significantly enriched in upregulated genes in pre- versus post-menopausal muscle, suggesting a reduction in the translation of oestrogen target genes after menopause. Altogether, sex hormone fluctuations across the female lifespan may contribute to age-related muscle wasting, although longitudinal and interventional studies are needed to determine the causal nature of the relationship. Abstract figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/25331955v2_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@15610c9org.highwire.dtl.DTLVardef@168104corg.highwire.dtl.DTLVardef@105ef53org.highwire.dtl.DTLVardef@a34522_HPS_FORMAT_FIGEXP M_FIG C_FIG This study mapped the trajectory of muscle ageing at the whole-body, whole-muscle, and cellular level in 96 healthy females aged between 18 and 80 years old, while controlling for confounding lifestyle factors. Muscle mass and function declined with age, concomitant to a reduction in type I fibre size and increase in hybrid type I/IIa fibres. Patterns of muscle gene expression were mapped across ageing, showing an increase in immune cell signalling and a decline in mitochondrial respiration pathways. Circulating sex hormones were significantly associated with muscle mass, morphology, and gene expression across the lifespan. Key points summary O_LIFemales live longer than males but experience worse disability in the later decades of life, highlighting the need to study female-specific patterns of ageing. C_LIO_LIThis study mapped female body composition, muscle morphology, function, and gene expression across every decade from 18 to 80 years of age in tightly controlled conditions and examined the relationships with circulating sex hormones. C_LIO_LIUnique patterns of muscle gene expression across ageing showed an overall increase in immune signalling and a decrease in mitochondrial respiration pathways, but limited associations with circulating sex hormones. C_LIO_LIIndependently of age, circulating oestradiol and progesterone, but not testosterone, were associated with muscle mass and morphology across the lifespan, after adjusting for influential lifestyle factors (protein intake and physical activity). C_LIO_LIFluctuations in female sex hormones across the lifespan should be considered when developing therapies to mitigate age-related muscle wasting and improve the female health span. C_LI

BackgroundLimb-girdle muscular dystrophies (LGMDs) are inherited myopathies characterized mainly by progressive weakness of the proximal muscles of the shoulder and pelvic girdle areas, leading to functional decline and eventual loss of independent ambulation. Dysferlinopathy (LGMD2B) is an autosomal recessive LGMD subtype, is caused by mutations in the DYSF gene that lead to lack of dysferlin which results in muscle death and chronic muscle fiber degeneration. Preservation of ambulation is a key clinical milestone, as loss of independent gait markedly reduces quality of life and complicates care management. Although patients often perceive functional decline before their initial clinical presentation, current clinical assessments typically detect disease progression after substantial muscle damage has occurred. MethodsIn this multigroup case-control study, we profiled plasma miRNAs from 49 genetically confirmed dysferlinopathy patients (24 ambulatory, 25 non-ambulatory) and 25 age- and sex-matched healthy controls. Total RNA was extracted from blood samples and hybridized to Affymetrix GeneChip miRNA 3.1 arrays. After quality control and filtering, differential expression analysis was performed using linear models for microarrays, adjusting for age and sex, with a false discovery rate cutoff of 10%. Results14 miRNAs were significantly altered between dysferlinopathy patients and controls. Notably, miR-4532 was upregulated in ambulatory patients relative to controls, whereas it was downregulated in non-ambulatory patients compared with ambulatory patients, although expression levels remained higher than in controls. Levels of miR-4532 were positively associated with circulating monocyte levels in ambulatory patients only. ConclusionThese results suggest that miR-4532 may be a circulating marker associated with ambulatory status in dysferlinopathy. Its known involvement in inflammatory signaling and muscle regeneration pathways underscores its potential as an early indicator for disease activity.

Skeletal muscle regeneration is a cardinal feature of muscle pathologies and is crucial for post-exercise recovery and traumatic sports injuries. Regeneration of damaged muscle in humans is a prolonged process and is accompanied by pain and physical dysfunction, highlighting the unmet need for effective interventions to accelerate the regenerative process. Through cellular and preclinical models, we have previously identified nicotinamide (NAM) and pyridoxine (PN) as potent stimulators of Muscle Stem Cells (MuSCs). Herein we investigated if a combination of NAM and PN could enhance MuSC activity and improve muscle regeneration in healthy volunteers during recovery from eccentric contractions. MethodsThis randomized, double-blind, placebo-controlled trial enrolled male participants aged 18-50 years supplemented daily with 714mg NAM and 19mg PN (NAM/PN) or placebo for 8 days following unilateral eccentric muscle contractions using Neuromuscular Electrical Stimulation (NMES). MuSC was quantified by immunohistofluorescence on vastus lateralis muscle biopsies. Results39 out of 43 enrolled participants completed the study. Supplementation of NAM/PN was well tolerated and increased blood concentrations of NAM and PN vitamers. The NMES protocol caused myofiber necrosis and triggered a strong MuSC response. After 8 days, the number of Pax7, MyoD, and myogenin positive cells per damaged fiber was significantly higher in NAM/PN vs placebo groups (+29-67%). NAM/PN also increased the proportion of regenerating fibers re-expressing embryonic myosin (+37%). ConclusionDaily oral NAM/PN supplementation following eccentric muscle damaging contractions enhances MuSC activity and accelerates muscle regeneration. These findings provide new possibilities for targeted therapeutic interventions in muscle repair. Trial registrationNCT04874662 One Sentence SummaryMuscle regeneration is enhanced by nicotinamide and pyridoxine supplementation, accelerating recovery and offering therapeutic potential.

IntroductionChronological ageing is associated with mitochondrial dysfunction and increased reactive oxygen species (ROS) production in skeletal muscle. However, the effects of replicative ageing on skeletal muscle cellular metabolism are not well known. Using an established myoblast model of cellular (replicative) ageing, we investigated the impact of ageing on energy metabolism in murine C2C12 myotubes. MethodsControl (P7-11) and replicatively aged (P48-51) C2C12 myoblasts were differentiated over 72-120 h. Mitochondrial bioenergetics were investigated by respirometry and mitochondrial superoxide and cellular ROS were measured in the absence and presence of antimycin A (AA). Genes related to mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Intracellular metabolites were quantified using an untargeted 1H-NMR metabolomics pipeline. ResultsMitochondrial coupling efficiency (Control: 79.5 vs. Aged: 70.3%, P=0.006) and relative oxidative ATP synthesis (Control: 48.6 vs. Aged: 31.7%, P=0.022) were higher in control vs. aged myotubes, but rates of mitochondrial superoxide production were lower (Control: 2.4x10-5 {+/-} 0.4 x 10-5 vs. Aged: 9.7x10-5 {+/-} 1.6x10-5 RFU/sec/cell; P=0.035). Replicatively aged myotubes had greater mRNA expression of mfn2 and Tfam compared to control. Yet, Nrf2 and PGC-1 expression were 2.8-fold and 3.0-fold higher in control versus aged myotubes over 24 h and 48 h (P<0.05), respectively. Branched chain amino acids L-leucine, L-isoleucine and L-valine, and L-carnitine were less abundant in aged versus control myotubes. Conclusion(s)Replicative ageing is associated with bioenergetic uncoupling, increased ROS production and impaired amino acid metabolism. Our findings suggest that cellular mitochondrial dysfunction and altered energy metabolism may exacerbate the age-related decline in skeletal muscle mass and function.

AimFibrosis is the most common complication from chronic diseases, and yet no therapy capable of mitigating its effects is available. Our goal is to unveil specific signallings regulating the fibrogenic process and to identify potential small molecule candidates that block fibrogenic differentiation of fibro/adipogenic progenitors. MethodWe performed a large-scale drug screen using muscle-resident fibro/adipogenic progenitors from a mouse model expressing EGFP under the Collagen1a1 promotor. We first confirmed that the EGFP was expressed in response to TGF{beta}1 stimulation in vitro. Then we treated cells with TGF{beta}1 alone or with drugs from two libraries of known compounds. The drugs ability to block the fibrogenic differentiation was quantified by imaging and flow cytometry. From a two-rounds screening, positive hits were tested in vivo in the mice model for the Duchenne muscular dystrophy (mdx mice). The histopathology of the muscles was assessed with picrosirius red (fibrosis) and laminin staining (myofiber size). Key findingsFrom the in vitro drug screening, we identified 21 drugs and tested 3 in vivo on the mdx mice. None of the three drugs significantly improved muscle histopathology. SignificanceThe in vitro drug screen identified various efficient compounds, none of them strongly inhibited fibrosis in skeletal muscle of mdx mice. To explain these observations, we hypothesize that in Duchenne Muscular Dystrophy, in which fibrosis is a secondary event due to chronic degeneration and inflammation, the drugs tested could have adverse effect on regeneration or inflammation, balancing off any positive effects and leading to the absence of significant results.

BackgroundSarcopenic obesity, where excess body fat coexists with reduced muscle mass and function, is becoming increasingly common in ageing populations and contributes to poor physical and metabolic health. Although adipose tissue-secreted factors are implicated in muscle decline, the specific mechanisms remain unclear. Extracellular vesicles (EVs), which carry regulatory cargo such as microRNAs (miRNAs) between cells, may play a key role in this adipose-muscle communication. MethodsEVs were isolated from adipose-conditioned media (ACM) collected from lean and non-lean human donors using ultracentrifugation. Donors were grouped by BMI (lean: 20.7-24.4; non-lean: 25.3-39.3) and age (younger: 31-56 years; older: 60-84 years). EVs were characterised using nanoparticle tracking analysis (NTA), ExoView, nanoscale flow cytometry (CytoFLEX Nano), and transmission electron microscopy (TEM). Primary human myoblasts were differentiated into myotubes and treated for 24 hours with lean or non-lean EVs (1.3x10 particles/ml) or left untreated. Myotube thickness was measured by immunofluorescence microscopy. Transcriptomic changes were assessed by bulk RNA sequencing. EV miRNA cargo was profiled by small RNA-seq and validated by qPCR. The role of miR-150-5p was tested using antagomir inhibition. ResultsNon-lean EVs significantly reduced myotube thickness compared to both untreated controls (8.7 {+/-} 1.66 {micro}m vs. 12.4 {+/-} 1.72 {micro}m, p < 0.01) and lean EV-treated myotubes (8.7 {+/-} 1.66 {micro}m vs. 13.2 {+/-} 3.84 {micro}m, p < 0.05), indicating a donor BMI-specific effect. This atrophy was restricted to myotubes derived from older donors. MAFbx expression was significantly increased in response to non-lean EVs (p < 0.05). RNA-seq revealed 471 differentially expressed genes (DEGs) in EV-treated vs. untreated cells and 293 DEGs between lean and non-lean EV conditions, with enrichment in inflammatory (TNF, IL1B), oxidative stress, mitochondrial, and chromatin pathways. Small RNA-seq identified 7 differentially expressed miRNAs, including miR-150-5p and miR-193b-5p, both significantly upregulated in non-lean EVs and validated by qPCR. Inhibiting miR-150-5p partially rescued myotube thickness (10.5 {+/-} 1.37 {micro}m vs. 8.7 {+/-} 1.66 {micro}m, p < 0.05) and reduced MAFbx expression. ConclusionsEVs from non-lean adipose tissue drive muscle atrophy and transcriptional changes in an age-dependent manner. These effects are partially mediated by miR-150-5p, highlighting a mechanistic role for EV cargo in adipose-muscle signalling. Targeting EV-derived miRNAs may offer a novel strategy to combat muscle loss in obesity and ageing.

A detailed analysis of how muscle fiber nuclei (myonuclei) respond to a hypertrophic stimulus would provide a critical step toward understanding compromised skeletal muscle plasticity with age. We used recombination-independent doxycycline-inducible myonucleus-specific fluorescent labelling, tissue RNA-sequencing, myonuclear DNA methylation analysis, multi-omic integration, and single myonucleus RNA-sequencing to define the molecular characteristics of adult (6-8 month) and aged (24 month) murine skeletal muscle after acute mechanical overload (MOV). In adult and aged MOV muscles, we found that: 1) similarities in the transcriptional response to loading - specifically in metabolism genes - were partly explained by a post-transcriptional microRNA-mediated mechanism, which we corroborated using an inducible muscle fiber-specific miR-1 knockout model, 2) differences in age-dependent transcriptional responses were linked to the magnitude and location of differential DNA methylation in resident myonuclei, specifically around hypertrophy-associated genes such as Myc, Runx1, Mybph, Ankrd1, collagen genes, and minichromosome maintenance genes, 3) adult and aged resident myonuclear transcriptomes had differing enrichment for innervation-related transcripts as well as unique transcriptional profiles in an Atf3+ "sarcomere assembly" population after MOV, and 4) cellular deconvolution analysis supports a role for neuromuscular junction regulation in age-specific hypertrophic adaptation. These data are a roadmap for uncovering molecular targets to enhance aged muscle adaptability.

During muscle contraction, increased influx of mitochondrial calcium (mtCa{superscript 2}) from the myocyte cytosol through the mitochondrial calcium uniporter (MCU) couples calcium homeostasis with high ATP provision. The mitochondrial calcium uniporter regulator 1 (MCUR1) is an integral membrane protein that promotes MCU activity. Although its function has been studied in cell models, mutations in MCUR1 have not yet been associated with human disease. Here, we present a case study of a patient exhibiting proximal muscle weakness and atrophy, who carries a novel homozygous loss-of-function mutation in MCUR1. To investigate the underlying mechanisms of muscle pathology, we examined patient fibroblasts and quadriceps muscle specimens. MCUR1 deficiency compromised mitochondrial Ca{superscript 2} uptake upon histamine exposure, but did not alter resting mitochondrial membrane potential or MCU protein complex assembly or subcellular location. Consequently, ATP production and oxygen consumption were reduced, and mitochondrial biogenesis was disturbed in muscle, with histological features of autophagic vacuoles with sarcolemmal features. Our study associates MCUR1 deficiency with mitochondrial dysfunction and autophagic vacuolar myopathy, thereby highlighting the crucial role of mitochondrial Ca{superscript 2} uptake in regulating mitochondrial function and expanding the spectrum of mitochondrial disorders in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=144 HEIGHT=200 SRC="FIGDIR/small/25338070v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@9fa55org.highwire.dtl.DTLVardef@111ebb3org.highwire.dtl.DTLVardef@1895c4corg.highwire.dtl.DTLVardef@10aac6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Mitochondria are composed of phospholipid bilayers rich in phosphatidylcholine (PC). StAR-related lipid transfer domain-containing protein 7 (STARD7) functions as a lipid transfer protein that plays a crucial role in maintaining mitochondrial PC homeostasis. In this study, we investigated the physiological role of STARD7 in skeletal muscle using muscle-specific knockout (mKO) mice. STARD7 expression was markedly higher in the soleus, a mitochondria-dense slow-twitch muscle, compared with fast-twitch fibers. Although muscle fibers from mKO mice exhibited no apparent structural abnormalities, their endurance exercise capacity was markedly reduced. RNA-seq analysis revealed suppressed expression of fast-twitch-related genes accompanied by a reduction in fast-twitch fibers. At the mitochondrial level, respiratory chain complexes remained intact, but oxygen consumption was consistently decreased. Targeted lipidomic analysis showed decreased levels of PC, cardiolipin (CL), and coenzyme Q in mKO mitochondria, particularly in the soleus. Conversely, expression of CL biosynthetic enzymes was unchanged, and an in vitro binding assay indicated that STARD7 preferentially transfers linoleic acid-containing PC required for CL remodeling. Furthermore, electron microscopy revealed disorganized cristae structures, whereas 4-HNE-modified proteins, mtDNA content, and OPA1 processing remained unaffected. Together, these findings demonstrate that STARD7 plays an essential role for maintaining mitochondrial integrity and function in skeletal muscle, and its loss likely contributes to the pathogenesis of mitochondrial myopathy.

Sarcopenia is a degenerative condition characterized by the atrophy and functional decline of myofibers, resulting in disability. While the clinical risk factors are known, there is no validated in vitro model to understand the molecular mechanisms and identify therapeutics. To tackle this challenge, we generated an in vitro post-mitotic muscular system by differentiating mouse myoblast cells, namely C2C12. After 12 days of differentiation, cells were expressing physiological markers of myotubes and became self-contracting. Importantly, transcriptomic analyses demonstrated high similarity (r=0.70) when compared to primary human myotubes (HSkMC) providing evidence of resemblance to human cells. Next, we starved and incubated cells with dexamethasone and observed myotube shrinkage, oxidative stress, modification of anabolic, inflammatory, and catabolic markers recapitulating sarcopenia. Conversely, cell refeeding resulted in a recovery in the model with nutrient deprivation but not when incubated also with dexamethasone. In conclusion, we present a model of sarcopenia due to nutrient deprivation and corticosteroids. This model may allow more efficient and effective future research to identify therapeutics against sarcopenia in humans.

The Master Athletic Laboratory Study of Intramuscular Connective Tissue (MALICoT, DRKS00015764) set out to analyze the endomysium content of the human soleus muscle in response to athletic exercise and aging. Forty-three healthy male study participants were grouped into young (20-35 years) non-physically active controls (n=12), young power-trained athletes (n=10), older (60-75 years) non-physically active controls (n=11), and older power-trained athletes (n=10). A single biopsy was taken from the left soleus muscle of each participant, and cryo-sections were used for i) routine histological staining and myopathological evaluation, ii) deep learning-based image analysis of the H&E- and MHC-stained sections, iii) laminin-{gamma}-1/collagen IV double- and collagen I and III single-immunofluorescence staining, and iv) quantitative proteomic analysis. Examiner-based myopathological evaluation revealed normal skeletal muscle in 26 participants, while 11, 4, 1 and 1 biopsies showed unspecific myopathological changes, chronic neurogenic atrophy, type II fiber atrophy, and unspecific myositic changes, respectively. Analysis of the H&E- and MHC-stained sections as well as the laminin-{gamma}-1/collagen I-, collagen III- and collagen IV-immunostained sections revealed an approximately 1.3-fold increase in the mean fiber area in response to power-training in young participants and aging in unathletic participants. No significant change was detected in endomysium thickness or area. Furthermore, proteomic analysis did not reveal any group-specific change except for plasma membrane calcium-transporting ATPase 2 being less abundant in the soleus muscles of aged power-trained athletes. Overall, the data show that neither athletic exercise nor age significantly affected the content or composition of the endomysium in human soleus muscle tissue. New & NoteworthyMALICoT (DRKS00015764) set out to analyze the endomysium of soleus muscle in response to athletic exercise and aging. Myopathological evaluation of biopsies from 43 asymptomatic male study participants revealed normal skeletal muscle in only 26 of them. Neither exercise nor age significantly affected the endomysium content or proteomic profiles of human soleus muscle tissue. However, power-training in young participants and aging in unathletic participants were associated with significant increases in soleus muscle fiber cross-sectional area.

BackgroundHuman primary muscle cell (HPMC) lines derived from skeletal muscle biopsies are potentially powerful tools to interrogate the molecular pathways underlying fundamental muscle mechanisms. HPMCs retain their genome in culture, but many endogenous circulating factors are not present in the in vitro environment, or at concentrations that do not mirror physiological levels. To address the assumption that HPMCs are valid models of age and sex-specificity in human muscle research, we examined to what extent HPMC lines retain their source phenotype in culture. MethodsBiopsies from the vastus lateralis muscle were collected from ten males aged 18-30, ten females aged 18-30 and ten males aged 60-75 recruited from a general, healthy population. A portion of the muscle was used for the establishment of 30 individual HMPC lines. The remaining sample was immediately snap frozen and stored for further analysis. RNA was extracted from muscle tissue samples and their corresponding, fully differentiated HMPCs and analysed using RNA Sequencing. To compare their transcriptomic signature, principal component analysis (PCA), differential expression analysis, single-cell deconvolution and pathway enrichment analysis were conducted in R. ResultsA comparison of the transcriptomic signature of 30 human muscle biopsies and their corresponding HPMCs indicated a near-complete lack of retention of the genes and pathways differentially regulated in vivo when compared to their in vitro equivalent, with the exception of several genes encoded on the Y-chromosome. ConclusionsThe diversity of resident cell populations in muscle tissue and the lack of sex- and age-dependent circulating factors in the cellular milieu likely contribute to these observations, which call for caution when using HPMCs as an experimental model of human muscle sex or age.

BackgroundThe balance between protein synthesis and degradation (proteostasis) is a determining factor for muscle size and function. Signaling via the mammalian target of rapamycin complex 1 (mTORC1) regulates proteostasis in skeletal muscle by affecting protein synthesis and autophagosomal protein degradation. Indeed, genetic inactivation of mTORC1 in developing and growing muscle causes atrophy resulting in a lethal myopathy. However, systemic dampening of mTORC1 signaling by its allosteric inhibitor rapamycin is beneficial at the organismal level and increases lifespan. Whether the beneficial effect of rapamycin comes at the expense of muscle mass and function is yet to be established.\n\nMethodsWe conditionally ablated the gene coding for the mTORC1-essential component raptor in muscle fibers of adult mice (iRAmKO). We performed detailed phenotypic and biochemical analyses of iRAmKO mice and compared them with RAmKO mice, which lack raptor in developing muscle fibers. We also used polysome profiling and proteomics to assess protein translation and associated signaling in skeletal muscle of iRAmKO mice.\n\nResultsAnalysis at different time points reveal that, as in RAmKO mice, the proportion of oxidative fibers decreases, but slow-type fibers increase in iRAmKO mice. Nevertheless, no significant decrease in body and muscle mass, or muscle fiber area was detected up to 5 months post-raptor depletion. Similarly, ex vivo muscle force was not significantly reduced in iRAmKO mice. Despite stable muscle size and function, inducible raptor depletion significantly reduced the expression of key components of the translation machinery and overall translation rates.\n\nConclusionsRaptor depletion and hence complete inhibition of mTORC1 signaling in fully-grown muscle leads to metabolic and morphological changes without inducing muscle atrophy even after 5 months. Together, our data indicate that maintenance of muscle size does not require mTORC1 signaling, suggesting that rapamycin treatment is unlikely to negatively affect muscle mass and function.

BackgroundSkeletal muscle atrophy is prevalent worldwide and is a major detractor from length and quality of life. It is often diagnosed and treated as a single disorder, but the causal stimuli and progression of atrophy vary widely. Malnutrition and disuse are two common causes of muscle atrophy, and despite their prevalence and extensive characterization, there have been no direct comparisons of how these two types of atrophy progress and whether they differentially affect skeletal muscle fiber types. The purpose of this study is to directly compare atrophy from fasting and disuse and provide a transcriptomic resource for future research on both conditions. MethodsWe fasted or hindlimb suspended (HS) two cohorts of 12-week-old female C57/bl6 mice. Mice were fasted for up to 72 hours to induce malnutrition atrophy or were hindlimb suspended for 0, 3, 7, 14, or 28 days to induce disuse atrophy. At each timepoint, mice were euthanized and three muscles (tibialis anterior (TA), extensor digitorum longus (EDL), and soleus) were weighed and collected for RNA sequencing. Atrophy progression and gene expression changes were compared across muscle fiber types and atrophy stimuli. ResultsWe found differences in atrophy progression between muscle fiber types based on fiber twitch speed and atrophy stimulus. Fasted mice lost 25% of their body weight and 23% of fast-twitch TA mass with little change in soleus. In contrast, HS mice lost 40% of the slower-twitch soleus but the effect on the TA was negligible. Gene expression varied in response to both atrophy stimuli, but a greater number of genes changed with fasting compared to HS in the EDL and soleus. By muscle type, a greater transcriptional shift occurred in the EDL with fasting while the soleus showed more gene changes during HS. Enrichment analysis of transcriptional changes showed similarities (downregulation in muscle growth pathways) and differences (increased fatty acid metabolism in fasting and increased neuronal activity in HS) between atrophy stimuli. ConclusionsAtrophy progression varies based on stimuli and muscle fiber type. This study provides a large, matched data set where the effects of different atrophic stimuli can be easily and directly compared in multiple fiber types. To our knowledge, this is the first study to closely compare these two atrophy stimuli in a muscle type-specific context. This work demonstrates that atrophy is not a single disorder and that the development of therapies may need to be tailored to the atrophic stimulus.

Duchenne Muscular Dystrophy (DMD) is the most common childhood muscular disorder. Mitochondrial dysfunctions are key disease features of the disease, and strategies that improve mitochondrial health have emerged as promising to slow disease progression. Emerging evidence indicates that impaired/insufficient mitophagy may contribute to the accumulation of mitochondrial dysfunction seen in patients and animal models of DMD. We therefore hypothesized that overexpressing Parkin, a key mitophagy regulator, may improve mitochondrial and muscle health in a mouse model of DMD. To this end, Parkin was overexpressed using intramuscular injections of adeno-associated viruses performed in 5-week-old and 18-week-old D2.B10-Dmdmdx/J mice (D2.mdx), a widely used mouse model of DMD. Four and 16 weeks of Parkin overexpression initiated in 5-week-old and 18-week-old D2.mdx, respectively, resulted in muscle hypertrophy, as indicated by an increase in muscle mass and fiber cross-sectional area. While Parkin overexpression did not impact maximal mitochondrial respiration or mitochondrial content, it increased the Acceptor Control Ratio, an index of mitochondrial bioenergetic efficiency. Parkin overexpression also decreased mitochondrial H2O2 emission, a surrogate for mitochondrial ROS production. However, Parkin overexpression failed to reduce the proportion of fibers with central nuclei and markers of muscle damage and/or necrosis. Taken all together, our results indicate that Parkin overexpression can attenuate muscle atrophy, improve mitochondrial bioenergetics and lower mitochondrial ROS production in a mouse model of DMD. These findings showcase the partial beneficial effects of overexpressing Parkin in ameliorating some, but not all, pathological features observed in a mouse model of DMD. Graphical abstractImpact of AAV-mediated Parkin overexpression on Duchenne Muscular Dystrophy (DMD) progression in skeletal muscle of D2.mdx (a mouse model of DMD). Parkin overexpression attenuated muscle atrophy, reduced mitochondrial H2O2 emissions and improved an index of mitochondrial coupling efficiency. Created with BioRender.com. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/659533v3_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@143f08borg.highwire.dtl.DTLVardef@16543a9org.highwire.dtl.DTLVardef@13d1110org.highwire.dtl.DTLVardef@2b5e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Altered mitochondrial structure and function are implicated in the functional decline of skeletal muscle. Numerous cytoskeletal proteins have been reported to affect mitochondrial homeostasis, but this complex network is still being unraveled. Here, we investigated alterations to mitochondrial structure and function in mice lacking the cytoskeletal adapter protein, Xin. Xin deficient (Xin-/-) and wild-type (WT) littermate mice were fed a chow or high-fat diet (HFD; 60% kcal fat) for 8 weeks before high-resolution respirometry, histology, electron microscopy and Western blot analyses of their skeletal muscles were conducted. Immuno-electron microscopy and immunofluorescence staining indicates that Xin is present in the mitochondria and peri-mitochondrial areas, as well as the myoplasm. Intermyofibrillar mitochondria in chow-fed Xin-/- mice were notably different from WT; frequently spanning a whole sarcomere and/or swollen in appearance with abnormal cristae. Succinate Dehydrogenase and Cytochrome Oxidase IV (COX) activity staining indicated greater evidence of mitochondrial enzyme activity in Xin-/- mice. HFD did not result in a difference between cohorts with respect to body mass gains or glucose handling. However, electron microscopy revealed significantly greater mitochondrial density ([~]2.1-fold) with evident structural abnormalities (swelling, reduced cristae density) in Xin-/- mice. Complex I and II-supported respiration were not different between groups per mg muscle, but when made relative to mitochondrial density, were significantly lower in Xin-/- muscles. Western blotting of fusion, fission, and autophagy proteins revealed no differences between groups. These results provide the first evidence for a role of Xin in maintaining mitochondrial morphology and function but not in regulating mitochondrial dynamics.

The cellular and molecular consequences of lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression Duchenne and Becker muscular dystrophies. We analyzed muscle biopsies of DMD patients and controls using single nuclei RNA sequencing (snRNAseq) and correlated the results with clinical data. DMD samples displayed an increase in regenerative fibers, satellite cells and fibro-adipogenic progenitor cells (FAPs) and a decrease in slow fibers and smooth muscle cells. Samples from patients with stable mild weakness were characterized by an increase in regenerative fibers, while those from patients with progressive weakness had fewer muscle fibers and increased FAPs. DMD muscle fibers displayed a strong regenerative signature, while DMD FAPs upregulated genes producing extracellular matrix and molecules involved in several signaling pathways. An analysis of intercellular communication profile identified FAPs as a key regulator of cell signaling in DMD samples. We show significant differences in the gene expression profiled of the different cell populations present in DMD muscle samples compared to controls.

Maintenance of skeletal muscle function is essential for functional independence, quality of life and healthspan. Muscle RING-finger protein-1 (MuRF1) negatively regulates muscle function and mass through ubiquitination and degradation of muscle proteins. Accordingly, genetic and pharmacological inhibition of MuRF1 attenuates muscle wasting and weakness under catabolic stress. To explore the potential of MuRF1 inhibitors (e.g., MyoMed-205) to improve muscle health, we investigated here the long-term effects of MyoMed-205 on functional capacity and muscle physiology in rats under basal conditions. Wistar rats were randomized to control or MyoMed-205 groups and were followed for 4 or 8 weeks. Body weight, food and water intake, and exercise capacity were monitored weekly. At each endpoint, the soleus muscle was collected for histological analyses. MyoMed-205-treated rats showed normal basic survival-related behaviors and body growth. After 8 weeks, MyoMed-205-treated animals exhibited enhanced exercise capacity (speed (m/min): +45%, p = 0.01; endurance (min): +47%, p = 0.03; and distance covered (m): +87%, p = 0.04) compared with baseline performance. Conversely, no differences were found in soleus fiber type distribution, cross-sectional area, or lipid and collagen content. Our findings indicate that MyoMed-205 enhances functional exercise capacity independently of changes in soleus muscle structure in rats under basal conditions.