Journal of Cachexia, Sarcopenia and Muscle

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.

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.

BackgroundAging and disuse are two of the most clinically relevant conditions associated with the loss of skeletal muscle mass, yet the ultrastructural adaptations that drive these losses remain poorly defined. In particular, it is unclear whether radial atrophy of muscle fibers is driven by a reduction in the size of the existing myofibrils, and/or the loss of myofibrils. Accordingly, the objective of this study was to define the macro-to-ultrastructural adaptations that mediate aging- and disuse-induced loss of muscle mass. MethodsSkeletal muscle structure was assessed at the macroscopic, microscopic, and ultrastructural levels in humans and mice. In humans, magnetic resonance imaging was used to quantify knee extensor muscle volume and cross-sectional area (CSA) in young (19 - 40 years) and old (65 - 84 years) adults, and vastus lateralis biopsies were analyzed for microscopic and ultrastructural adaptations using immunohistochemistry and fluorescence imaging of myofibrils with image deconvolution (FIM-ID). Parallel studies were performed in young (4 months) and aged (24 months) mice, along with the use of unilateral hindlimb immobilization to model disuse. ResultsAging led to a robust loss of skeletal muscle mass that was mediated by coordinated macro-to-ultrastructural adaptations. In humans, aging reduced knee extensor muscle volume (34%, P < 0.005) and CSA (32%, P < 0.001) in a sex-independent manner, and these effects were associated with radial atrophy of SERCA1-positive fibers (23%, P < 0.05). Ultrastructural analyses revealed that the radial atrophy was driven by a reduction in the number of myofibrils per fiber (23%, P < 0.05) without changes in myofibril CSA. In mice, aging produced similar macro-to-ultrastructural adaptations in various flexor muscles; however, radial atrophy of the highly glycolytic/Type IIb fibers, which are not present in human limb muscles, was also associated with a decrease in the CSA of the myofibrils (9%, P < 0.005). We also determined that disuse led to radial atrophy of SERCA1-positive fibers (24%, P < 0.001), and this was mediated by a decrease in both the number (22%, P < 0.005) and size of the myofibrils (4%, P < 0.05). Notably, the results also revealed that the magnitude of the disuse-induced adaptations was significantly blunted with aging. ConclusionThis study identifies the loss of myofibrils as a central and conserved mediator of the radial atrophy of muscle fibers that occurs in response to disuse and aging, while also highlighting smaller context-dependent contributions that can arise from changes in myofibril size.

BackgroundDiabetes mellitus leads to skeletal muscle dysfunction associated with loss of strength, impaired blood perfusion, lipid accumulation, and inflammation. The opening of large-pore channels has been linked to increased membrane permeability and inflammatory signaling in several pathologies. Boldine, an alkaloid from Peumus boldus, blocks large-pore channel activity and exhibits antioxidant and anti-inflammatory properties. This study evaluated whether boldine prevents skeletal muscle alterations induced by diabetes and explored potential underlying mechanisms. MethodsDiabetes was induced in male C57BL/6J mice using streptozotocin (STZ, 40 mg/kg/day for 5 days). Diabetic mice were treated with boldine (50 mg/kg/day) for four weeks. Muscle strength and resting membrane potential were analyzed in vivo. Also, right gastrocnemius muscle blood perfusion at basal and after acetylcholine (10 M) stimulation were analyzed in vivo. Lipid accumulation was assessed using Oil Red O staining, and CD31 immunodetection was used to evaluate capillary density. mRNA levels of NLRP3 were evaluated in muscle by qPCR. In human myoblasts (AB1167) cultured under low (8 mM) or high glucose (25 mM) conditions, with or without boldine, membrane permeability (ethidium uptake), intracellular Ca{superscript 2} (Fura-2), nitric oxide (DAF-FM), and levels of NLRP3 and Casp1 (qPCR) and reactivity PPAR{gamma} (Immunofluorescence) were determined. ResultsSTZ mice showed reduced muscle strength and depolarized resting membrane potential, both prevented by boldine. Basal muscle perfusion was [~]20% lower in diabetic mice (160.1 {+/-} 17.2 vs. 199.1 {+/-} 13.8 units), whereas boldine preserved perfusion (184.6 {+/-} 14.3 units). Oil Red O-positive fibers increased to 52.4 {+/-} 3.6% in diabetic mice and decreased to 15.2 {+/-} 4.1% with boldine (control: 3.1 {+/-} 1.3%; p<0.05). NLRP3 mRNA increased 17.7 {+/-} 2.8-fold in diabetic muscle and was reduced by [~]50% with boldine. In myoblasts, high glucose increased ethidium uptake, nitric oxide production, NLRP3 and caspase-1 expression, and nuclear PPAR{gamma} ([~]45% positive nuclei); all effects were prevented by boldine. ConclusionsBoldine preserves skeletal muscle function and vascular reactivity in diabetes and prevents lipid accumulation and inflammasome activation both in vivo and in vitro. These effects are associated with inhibition of large-pore channel activity and attenuation of downstream calcium-dependent, inflammatory, and adipogenic pathways, supporting boldine as a promising therapeutic candidate for diabetes-associated skeletal muscle dysfunction. Graphical abstractIn myoblasts, high glucose activates large-pore channels, elevating cytoplasmic Ca{superscript 2} concentration and nitric oxide generation, which increases the activity of Cx-formed hemichannels, raises the levels of inflammasome components, and promotes lipid accumulation. In STZ-diabetic mice, de novo expression of large-pore channels in skeletal muscles contributes to reduced blood perfusion, accumulation of intramuscular fat, muscle weakness, and reduced resting membrane potential of myofibers. Boldine inhibits large-pore channel activity, preventing these alterations and preserving muscle physiology in vivo. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/707704v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@19179b4org.highwire.dtl.DTLVardef@1cd3d21org.highwire.dtl.DTLVardef@16851d6org.highwire.dtl.DTLVardef@1d4e77c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Age-related reductions in whole-muscle function are attributed, in part, to pronounced atrophy of muscle fibers expressing the fast myosin heavy chain (MyHC) II isoforms. Senescence, a state of irreversible cell cycle arrest that can be characterized by DNA damage ({gamma}H2AX) and chromatin remodeling (loss of nuclear HMGB1), may contribute to skeletal muscle aging. Muscle nuclei (myonuclei) maintain fiber size and function and could exhibit senescence-associated features; however, the prevalence of senescent myonuclei and whether they contribute to fast fiber atrophy in older adults remains unknown. Vastus lateralis biopsies from 20 young (19-34yr; 10 females) and 20 older (65-84yr; 10 females) adults were analyzed via immunohistochemistry for myonuclei positive for {gamma}H2AX ({gamma}H2AX+) and negative for HMGB1 (HMGB1-). MyHC II cross-sectional area (CSA) was [~]70% larger in young compared with old, whereas MyHC I CSA did not differ with age. The relative abundance of {gamma}H2AX+/HMGB1- myonuclei did not differ with age and was not associated with CSA in either fiber type. Single-nucleus RNA-sequencing corroborated no age-related difference in the prevalence of myonuclei with senescence-associated features. Myonuclear content of MyHC II fibers was [~]30% higher in young compared with old and was closely associated with CSA in both fiber types. Size-cluster analysis revealed a pronounced age-related leftward shift in MyHC II CSA that paralleled the reductions in myonuclear number, consistent with myonuclear loss. These data suggest that age-related fast fiber atrophy is not attributed to an increased prevalence of senescent myonuclei but instead occurs concomitantly with fiber type-specific myonuclear loss across the lifespan.

Advances in our understanding of the biology of skeletal muscle ageing are being made at pace, with great potential for these findings to inform the identification of novel treatments for sarcopenia. However, translation of findings from animal models to humans has been hampered by limitations of existing human muscle biopsy studies. Devised to directly address this challenge, the Muscle Ageing and Sarcopenia Study (MASS) Lifecourse is a unique resource for the study of human muscle ageing across adulthood. This deep-phenotyped observational study of 260 community-dwelling men and women aged 18 to 85 years living in North East England includes muscle biopsy samples and detailed characterisation of physical function, health status and sociodemographic and behavioural risk factors. Underpinned by broad interdisciplinary research and clinical expertise this study is catalysing cutting-edge translational research on human muscle ageing across the adult life course.

Abnormal iron metabolism is closely linked to sarcopenia; however, the specific iron metabolism features of fat-infiltrating sarcopenia remain poorly understood. Proteomic sequencing revealed that in skeletal muscle with severe fat infiltration, the expression of iron utilization-related proteins was significantly downregulated, whereas that of iron uptake and storage proteins was markedly upregulated, thereby presenting an abnormal iron deficiency (ID) phenotype. Nevertheless, the mechanism by which ID drives intramuscular fat infiltration has not been fully elucidated. Using clinical samples, aged murine ID models, and in vitro cell assays, this study is the first to demonstrate that ID stabilizes hypoxia-inducible factor-1 (HIF-1), promoting aberrant adipogenic differentiation of fibro-adipogenic progenitors (FAPs), disrupting the homeostatic balance between satellite cells (SCs) and FAPs, exacerbating skeletal muscle fat infiltration, and impairing muscle repair capacity. Notably, treatment with the HIF-1 inhibitor PX478 reversed these pathological alterations and improved muscle function. Collectively, our findings identify the ID-HIF-1-FAPs axis as a key driver of intramuscular fat infiltration, offering a novel therapeutic target for the clinical management of fat-infiltrating sarcopenia.

BackgroundSkeletal muscle in wasting conditions often exhibits a common set of phenotypes that include atrophy, mitochondrial respiratory dysfunction, and fragmentation of the acetylcholine receptor (AChR) cluster at the endplate. Mitochondria are frequently implicated in driving muscle pathology in these conditions, although which aspects of mitochondrial function are most relevant is poorly understood. MethodsTo address this gap, we focused on mitochondrial permeability transition (mPT), a well-established pathological mechanism in ischemia-reperfusion injury and neurodegeneration but poorly studied in skeletal muscle. We performed a broad assessment of the consequences of mPT in skeletal muscle, focusing on features that are common in wasting conditions. We then tested whether tumor-host factors could promote mPT and compared differentially expressed genes (DEGs) with mPT and a mouse model of pancreatic cancer cachexia. ResultsInducing mPT in mouse skeletal muscle bundles in a Ca2+ retention capacity assay progressively altered mitochondrial morphology, beginning with cristae swirling and condensation, progressing to mitochondrial cristae displacement, and culminating in breach of the outer mitochondrial membrane; features that are common in wasting conditions. Inducing mPT with Bz423 in single mouse muscle fibers increased mROS and Caspase 3 (Casp3) activity and was prevented by inhibitors of mPT, mROS or Casp3. Incubating single muscle fibers with Bz423 for 24 h reduced fiber diameter by [~]20% which was prevented by inhibiting mPT, mROS, or Casp3. Inducing mPT caused a complex I-specific mitochondrial respiratory impairment and increased co-localization of lysosomes with mitochondria. Inducing mPT also fragmented the AChR cluster at the muscle endplate and was prevented by inhibiting mPT or Casp3. The Ca2+ threshold for mPT and mitochondrial calcein colocalization were reduced by pancreatic tumor-conditioned media in skeletal muscle or C2C12 myoblasts, respectively, and these effects were counteracted by mPT inhibition or cyclophilin D knockout. Finally, there was significant overlap between the transcriptome of mPT and that seen in diaphragm muscle in a mouse model of pancreatic cancer cachexia, particularly during the muscle wasting phase. ConclusionsWe conclude that inducing mPT in skeletal muscle recapitulates muscle phenotypes common with muscle wasting conditions like cachexia. Furthermore, mPT is engaged by tumor-host factors and had significant overlap with DEGs seen during the muscle wasting phase in a mouse model of pancreatic cancer cachexia, warranting further investigation of mPT as a therapeutic target.

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by skeletal muscle degeneration and cardiomyopathy without a cure. This study examined the therapeutic potential of the sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin (EMPA) on cardiac function in the dystrophin-deficient mdx mouse model of DMD. Male mice were fed control chow or EMPA-containing chow ([~]25 mg/kg/day), and cardiac function was evaluated longitudinally by four-dimensional ultrasound imaging. EMPA did not alter left ventricular mass or chamber volume but preserved ejection fraction (EF) for 12 weeks, maintained significantly higher EF through 24 weeks, and attenuated global impairment of systolic and diastolic myocardial deformation. These functional improvements were accompanied by reduced cardiomyocyte hypertrophy and decreased expression of cardiac stress genes. EMPA reduced mitochondrial DNA damage, increased mitochondrial DNA copy number, and induced transcriptional signatures consistent with enhanced fatty acid and ketone metabolism, contributing to increased myocardial ATP content. Systemically, EMPA improved body mass trajectory, preserved relative lean mass, enhanced skeletal muscle torque, and did not adversely affect renal function. Together, these findings demonstrate that EMPA improves cardiac performance and mitochondrial integrity while enhancing myocardial energy availability in mdx mice, supporting SGLT2 inhibitors as a promising therapeutic strategy for individuals with DMD.

BackgroundPeripheral nerve trauma denervates skeletal muscle resulting in paralysis and atrophy that is reversible if timely reinnervation occurs, due to its regenerative capacity. If reinnervation is delayed muscles regenerative ability is exhausted and resident fibroadipogenic progenitors (FAPs) differentiate into adipocytes and fibroblasts that replace muscle with non-contractile fibrotic tissue and fat, resulting in physical disability. Prostaglandin E2 (PGE2) inhibits adipogenesis and fibrosis in other tissues. We determined whether PGE2 could inhibit fibro-fatty degradation of long-term denervated muscle. MethodsWe utilized the rat tibial nerve transection model, denervating the gastrocnemius and selected a 5 week post-denervation time point to represent short-term muscle denervation injury (reversible with reinnervation), and 12 weeks to represent sustained, irreversible injury. Gastrocnemius FAPs were isolated via FACS and grown in culture to assess endogenous PGE2 production and the proliferative and differentiation response to exogenous PGE2. We evaluated transcript and protein expression of PGE2 synthesizing enzyme PTGS2, PGE2 degrading enzyme 15-PGDH and markers of proliferation, adipogenesis and fibrogenesis using RT-qPCR, immunofluorescence and SDS-PAGE/Western blotting. Paracrine impact of FAPs produced PGE2 was assessed by treating C2C12 myoblasts with FAPs conditioned media. ResultsTranscript expression of PTGS2 was increased and 15-PGDH decreased (4.37{+/-}2.63 and -3.06{+/-}0.85 fold change respectively, p<0.05) in 5 week, but not 12 week denervated gastrocnemius, consistent with increased PGE2 production in 5 week denervated muscle. Similarly, PTGS2 transcript levels were significantly increased (2.58{+/-}0.33 fold change, p<0.05) and 15-PGDH decreased (-5.24{+/-}3.19 fold change, p<0.05) in FAPs isolated from 5 week, but not 12 week denervated muscle, demonstrating that FAPs are a source of PGE2 in short-term denervated muscle. 16,16-dimethyl PGE2 did not impact naive FAPs in vitro proliferation, but significantly inhibited their differentiation as demonstrated by 88.9%, 82.3% and 94.2% decreases in FAPs expression of adipogenic marker perilipin-1, fibrogenic marker -smooth muscle actin (-SMA) and lipid content respectively, mediated via PGE2 binding to the FAPs EP4 receptor. FAPs isolated from 12 week denervated muscle demonstrated increased adipogenesis and fibrogenesis vs. naive FAPs (perilipin-1 and -SMA 7.93{+/-}2.96 and 2.00{+/-}0.33 fold increase respectively, p<0.05) and remained fully susceptible to PGE2 inhibition of fibro-adipogenic differentiation. Conditioned media from FAPs derived from 5 week, but not 12 week, denervated gastrocnemius stimulated C2C12 myoblast proliferation which was prevented by EP4 blockade. ConclusionsPGE2 is identified as a novel negative regulator of FAPs differentiation in traumatically denervated muscle, suggesting the therapeutic potential of PGE2 to prevent fibro-fatty degradation of long-term denervated muscle awaiting reinnervation.

Unlike mammals and birds, where new muscle fiber formation (hyperplasia) ceases around birth, large and fast-growing fish such as rainbow trout undergo a spectacular post-hatching surge of hyperplasia, followed by a considerably delayed hyperplasia decline. This study investigated the roles of the satellite cells (SCs) and their niche in this decline by determining the number and the myogenic capacity of the muscle progenitors as well as the functionality of their direct tissue environment. Histological analysis revealed a significant decrease in hyperplasia (fibers <25 {micro}m) and SC numbers (Pax7+) between 10 g and 500 g trout. Transplantation experiments using muscle-derived cells (MDCs) from mlc2-GFP transgenic trout (10 g to 2 kg donors into 10 g to 2 kg recipients) demonstrated a marked decline in both intrinsic myogenic capacity and niche functionality as trout grow from 10 g to 500 g. Detailed analyses of GFP+ fibers produced after transplantation showed an enrichment of small-diameter GFP+ fibers in 10 g but not 100 g trout recipient muscles, showing a rapid impairment in niche ability to support hyperplasia. In addition, transplantation of MDCs from trout of different ages but the same weight, showed that increasing trout weight, but not aging, is associated with an impairment of the myogenic capacity of progenitors and their niche. Overall, these findings show that the muscle hyperplasia decline in trout is primarily driven by early impairment of the SC niche, followed by a reduction in their myogenic capacity and number, with weight gain playing a more critical role than aging.

Skeletal muscle is the most abundant tissue in the human body and is essential for locomotion and the regulation of whole-body metabolism. The maintenance of skeletal muscle mass is essential for health, yet the molecular and signaling mechanisms that control skeletal muscle mass remain poorly understood. Transforming growth factor-{beta}-activated kinase 1 (TAK1) is a key signaling protein that regulates multiple intracellular pathways. Recent studies have demonstrated that TAK1 is a critical regulator of skeletal muscle mass. However, the mechanisms by which TAK1 regulates muscle mass and whether its role is sex dependent remain incompletely understood. In this study, we show that targeted inactivation of TAK1 induces muscle atrophy more rapidly in male than in female mice. Loss of TAK1 activity also abolished mechanical overload-induced phosphorylation of p70S6K and rpS6, and the induction of myofiber hypertrophy in both sexes. RNA-Seq analysis further revealed that TAK1 inactivation in skeletal muscle disrupts the gene expression of various molecules involved in catabolic processes, calcium signaling, muscle structure development, and aerobic respiration. Moreover, TAK1 inactivation impairs fatty acid oxidation and promotes lipid accumulation in skeletal muscle of adult mice in a sex-independent manner. Collectively, our findings demonstrate that TAK1 regulates skeletal muscle mass and growth by coordinating distinct intracellular pathways in both male and female mice.

Aging is associated with a progressive loss of skeletal muscle function, known as sarcopenia; however, the molecular mechanisms coordinating cellular stress responses and structural adaptations permissive of sarcopenia remain incompletely understood. In our previous studies, we found aging differentially impacted mitochondrial networks by muscle, suggesting unique stress thresholds and response activation. Here, we investigate the role of activating transcription factor 4 (ATF4), a master regulator of the integrated stress response (ISR), in aged quadriceps muscle using complementary patient and aging mouse models. Older adults exhibited a marked decrease in aerobic capacity, muscle strength, and endurance when compared with young participants. These results paralleled findings in aged mice, with significant loss of muscle mass across multiple hindlimb muscles. Ultrastructural analysis revealed substantial age-related changes in mitochondrial morphology, including increased volume, surface area, and branching index, as well as a shift toward larger, more complex mitochondria. Our data indicate that ATF4 binds directly to the promoter region of the gene encoding TFAM, suggesting a transcriptional regulatory relationship to support DNA stability. These structural and transcriptional changes likely impair oxidative capacity and drive a feed-forward cycle of mitochondrial dysfunction and ISR activation. Our findings indicate that ATF4 coordinates transcriptomic and structural adaptations in aging muscle, identifying the ISR pathway as a potential therapeutic target for preserving muscle function in older adults.

BackgroundIn vertebrates, skeletal muscle grows postnatally through different strategies. While mammals predominantly rely on fiber hypertrophy after birth, many teleost fish retain the unique ability to generate new fibers via hyperplasia well into juvenile stages. The molecular mechanisms governing the transition between hyperplastic-hypertrophic and hypertrophic growth modes in fish muscle remain poorly understood. ResultsWe generated a single-cell transcriptomic atlas of muscle-derived cells from juvenile Oncorhynchus mykiss (rainbow trout) at five growth stages. Fifteen tissue resident cell populations were identified, including eight myogenic subpopulations spanning from quiescent stem cells to terminally differentiating myocytes. Two distinct transcriptional trajectories were uncovered thanks to RNA velocity analysis: one present only during hyperplastic growth and another maintained throughout growth, indicating specialization of satellite cells toward hyperplasia or hypertrophy. Comparative analyses with human single-cell atlases indicate that subpopulations specifically related to hyperplasia and hypertrophy are conserved, depending on stage (fetal or adult). Strikingly, we identified a population of pax7+/pdgfr+ cells, indicating plasticity toward fibroblastic lineage and associating these cells with hypertrophic growth. Furthermore, both intrinsic changes in muscle stem cells and extrinsic remodeling of the extracellular matrix accompanied the decline of hyperplasia, highlighting dynamic crosstalk between myogenic and mesenchymal compartments. ConclusionsOur findings reveal the existence of two transcriptionally distinct muscle stem cell fates that underlie hyperplastic versus hypertrophic growth in fish. The identification of a tissue-resident pax7+/pdgfr+ subpopulation provides new insights into muscle stem cell plasticity and niche remodeling. This work establishes a comparative framework to explore the regulation of postnatal muscle growth across vertebrates.

Progressive cardiomyopathy is the leading cause of death in Duchenne muscular dystrophy (DMD). Dysregulation of calcium handling has been implicated in cardiomyopathy progression in DMD. Here we describe a therapeutic approach to improve calcium homeostasis in a mouse model of DMD using the novel therapeutic NDC-1171, which is a positive allosteric modulator of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump. We synthesized NDC-1171 and treated 4-week-old D2.mdx mice (n=9) via oral gavage. A group of D2.mdx mice (n=9) and a group of DBA/2J mice (n=9; background strain) received a vehicle on the same schedule. We used ultrasound to assess left ventricular function, followed by a treadmill exhaustion test and a 4-paw grip strength test to assess skeletal muscle function. NDC-1171 attenuated cardiac functional decline in D2.mdx mice. At 16 weeks of age, left ventricular ejection fraction (LVEF) was significantly preserved in mice treated with NDC-1171 (57.7{square}{+/-}{square}0.5%) compared to mice treated with a vehicle (50.7{square}{+/-}{square}0.9%, p{square}<{square}0.05), though remained lower than background strain controls (62.4{square}{+/-}{square}0.6%). In contrast, functional behavior testing revealed no significant improvement in skeletal muscle function with treatment. These data suggest that treatment with the SERCA pump modulator NDC-1171 helps preserve cardiac function in a murine model of DMD, even as skeletal muscle function was impaired. Future work will be needed to determine if the benefits of this novel SERCA activator translate to large animal and clinical studies, but these initial results are promising and could help guide development of future treatments for pediatric patients with muscular dystrophy.

Rhabdomyolysis is the acute breakdown of skeletal muscle resulting from failure of cellular homeostasis in response to metabolic stress. Recurrent forms are frequently linked to inherited defects affecting energy metabolism or calcium handling. Ryanodine receptor type 3 (RyR3) is an intracellular calcium release channel, expressed in skeletal muscle, that contributes to the fine-tuning of calcium signaling. Although variants in other calcium-handling proteins have been implicated in rhabdomyolysis, the role of RyR3 has not been established. In this study, we report rare compound heterozygous missense variants in RYR3 identified in two unrelated individuals with severe, fever-triggered recurrent rhabdomyolysis. Muscle biopsies revealed mild structural changes with triadic disorganization, mitochondrial alterations, lipid accumulation, and autophagic material, while overall muscle architecture was largely preserved. Structural modeling supports the pathogenicity of the variants, and calcium flux analysis demonstrated significantly reduced ryanodine receptor-mediated calcium release in patient-derived myoblasts. Functional analyses showed that RyR3 deficiency impaired starvation-induced autophagy, characterized by defective autophagosome formation and reduced autophagic flux, and increased susceptibility to metabolic stress. Mitochondrial bioenergetic profiling revealed reduced oxidative phosphorylation capacity and decreased membrane potential under stress conditions, consistent with compromised mitochondrial adaptation. In zebrafish, ryr3 knockdown resulted in structural and functional muscle abnormalities, including reduced myotome area and decreased locomotor activity, associated with impaired autophagic flux. This study establishes a novel association between recessive RYR3 variants and recurrent rhabdomyolysis and identifies RyR3 as a critical regulator of skeletal muscle stress adaptation through calcium-dependent control of autophagy and mitochondrial homeostasis. More broadly, our findings further highlight autophagy as a central determinant of muscle resilience in the context of rhabdomyolysis.

Skeletal muscle atrophy and deconditioning contribute to functional limitation and disability in COPD. While transcriptome and DNA methylation changes accompany exercise in healthy muscle, their interaction with COPD status and ageing, and integrative analyses of methylome-transcriptome responses have not been explored. We performed gene expression and DNA methylation profiling in skeletal muscle of sedentary volunteers with COPD, age-matched older adults, and younger healthy individuals, before and during (1,4 and 8 weeks) supervised aerobic exercise training and after four weeks of detraining. Exercise induced transcriptomic and DNA methylation changes, but these responses were unaffected by COPD status or age. Subsequent analysis focusing on temporal exercise effects independent of disease or age revealed differential transcriptomic changes across time points, a subset of which significantly associated with DNA methylome alterations. Transient transcriptomic changes not linked to DNA methylation were enriched for inflammatory and oxidative stress pathways, whereas persistent methylation-associated adaptations were related to immunomodulation and tissue remodelling. Together, this study provides insight into molecular mechanisms contributing to skeletal muscle adaptation to aerobic exercise training in sedentary individuals.

Spinal muscular atrophy (SMA) is characterized by motor neuron degeneration caused by deficiency of the survival motor neuron (SMN) protein. However, evidence increasingly supports broader systemic involvement. This study aimed to examine cardiac pathology in SMA patients and to investigate how reduced SMN levels impact cardiomyocyte homeostasis. We analyzed postmortem data from 14 SMA type I patients from the pre-treatment era, integrating gross anatomical, histopathological, and clinical findings. To investigate cardiomyocyte-intrinsic effects of SMN deficiency, healthy human cardiomyocytes were subjected to SMN knockdown and assessed using metabolic assays and transcriptomic profiling. Key findings were further investigated in vivo using the Smn2B/- mouse model of SMA. We found heterogeneous cardiac involvement in SMA patients, including cardiomegaly, variable fat deposition and interstitial fibrosis. SMN knockdown in human cardiomyocytes induced a metabolic shift and widespread transcriptional dysregulation, with pathway analyses identifying selective upregulation of PTEN signaling. Elevated PTEN protein levels were observed in a subset of human SMA hearts and in early postnatal hearts of Smn2B/- mice. Our results demonstrate that the heart remains a biologically relevant target of SMN deficiency and highlights cardiomyocyte-specific metabolic and PTEN signaling alterations as potential contributors to cardiac involvement in SMA.

The knock-out mutation of the unique M13 family member, the Drosophila melanogaster (fruit fly) Neprilysin-like 15 (Nepl15), resulted in marked reductions of glycogen and glycerolipid storage in adult male flies, but a significant increase of glycogen storage in adult female flies, although the mutant flies consumed the same amount of food as the isogenic w1118 controls. The findings prompted us to characterize sex and age-specific effects of Nepl15 knock-out (Nepl15KO) mutation on lifespan, fertility and fecundity, physiology, cytophysiology, and overall health. The current study shows Nepl15 transcripts are expressed in all embryonic stages of the control flies. The mutant embryos show more glycogen storage, likely due to more maternal glycogen deposition in the eggs. Moreover, there are slight increases in the number of eggs laid, the percentage of pupariation, and the percentage of adult fly eclosion from pupae in the Nepl15KO mutant flies. Interestingly, Nepl15KO female, but not male flies, outlive the respective control flies when cultured on a standard diet. The mutant adult females show significantly less Target of Rapamycin (TOR) and more Sirtuin 6 (Sirt6) expression, changes that may synergistically contribute to their lifespan extension. In contrast, mutant males exhibit significant reductions in both TOR and Sirt6 expression, potentially offsetting their effects on longevity. Cellular health is further improved in mutant females, as evidenced by a marked reduction in reactive oxygen species (ROS), associated with a 1.5-fold increase in the Superoxide dismutase 2 (Sod2) expression at 7 days of age. Both sexes demonstrate improved gut barrier integrity at 40 days, with reduced "Smurf" leakage compared to age-matched controls. Optical cardiography reveals that heart rate in 40-day-old mutants is better preserved, resembling that of 7-day-old flies, whereas control flies show a pronounced age-associated decline. Functionally, Nepl15KO males and females outperform controls in a 6-cm climbing assay at 10, 20, 30, and 40 days of age, with the greatest difference observed at day 40. Following a 45-minute exercise bout at 10 rpm, mutant females continue to outperform controls at both 7 and 40 days, indicating preserved neuromuscular performance. Consistently, ATP levels are significantly elevated in 7- and 40-day-old mutant females, but not in mutant males. Interestingly, only 7-day-old mutant males exhibit increased mitochondrial inner membrane potential, which may enable more rapid ATP turnover when energy demand arises. No detectable differences are observed in thoracic muscle or mitochondrial ultrastructure, nor in overall mitochondrial number. However, no observable changes are noticed in the ultrastructure of the thoracic muscle and mitochondria, and the overall number of mitochondria in the mutant flies. Collectively, our findings demonstrate that Nepl15 loss-of-function confers health benefits at cellular, organ, and organismal levels, with pronounced sex-specific differences. However, the mechanisms by which aging mutant males sustain enhanced functional performance remain elusive.

Spinal muscular atrophy (SMA) is caused by loss of SMN protein and is increasingly recognized as a multisystem disorder involving molecular pathology beyond motor neurons. Recently, we identified NRF2-KEAP1 signaling as dysregulated in SMA mice. Because NRF2 coordinates transcriptional programs that maintain cellular redox homeostasis and adaptive stress responses, we investigated whether NRF2 signaling is similarly altered in SMA type I patient-derived fibroblasts and whether it can be pharmacologically engaged. Compared with control fibroblasts, SMA fibroblasts displayed reduced basal expression of NRF2 target proteins, including NQO1 and xCT (SLC7A11), along with decreased levels of PGC1. Omaveloxolone (OMAV), a pharmacological NRF2 activator approved for the treatment of Friedreichs ataxia, increased cell viability and upregulated NRF2 target proteins in both control and SMA fibroblasts. Notably, OMAV produced a modest increase in SMN protein abundance and PGC1 levels selectively in SMA cells. Together, these findings support diminished NRF2 pathway output as a feature of SMA fibroblasts and demonstrate that OMAV induces NRF2 target proteins in this human SMA cellular model, consistent with enhanced cytoprotective signaling. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/712434v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1904bfeorg.highwire.dtl.DTLVardef@6d20e2org.highwire.dtl.DTLVardef@89f365org.highwire.dtl.DTLVardef@ca9638_HPS_FORMAT_FIGEXP M_FIG C_FIG