Journal of Cachexia, Sarcopenia and Muscle

BackgroundThe age-related progressive decline in skeletal muscle function is characterised by declining mitochondrial quality control and perturbed fatty acid metabolism, contributing to frailty and increased mortality. The actin cytoskeleton, a key structural component of skeletal muscle, has recently been implicated in mitochondrial anchoring and dynamics. However, the role of actin-regulating proteins, including the Rho GTPase Rac1, in mitochondrial function and age-associated metabolic and functional muscle deterioration remains undefined. MethodsSkeletal muscle from mice with inducible muscle-specific deletion of Rac1 (Rac1 imKO) underwent unbiased mass spectrometry-based proteomic profiling. Mitochondrial morphology was assessed by transmission electron microscopy, and physiological parameters, including muscle mass and contraction-stimulated palmitate oxidation in isolated soleus muscle, were evaluated. Mitochondrial respiratory function was determined by high-resolution respirometry in permeabilised gastrocnemius skeletal muscle fibre bundles. Biochemically, muscular triacylglycerol (TG) content, mRNA (qPCR) and protein (immunoblotting) content were determined. In vastus lateralis muscle biopsies from healthy, untrained young (20-30 years) and old, sarcopenic (83-94 years) men, Rac1 and mitochondrial respiratory protein abundances were measured. A complementary human genetic association analysis was performed using the FinnGen dataset. ResultsRac1 deficiency triggered muscle wasting in middle-aged mice (Gastrocnemius: -10%; Quadriceps: -7%). Preceding muscle wasting, gene set enrichment analysis identified enrichment in fatty acid metabolism and oxidative phosphorylation pathways, consistent with increased mitochondrial volume density in Rac1 imKO muscle (subsarcolemmal: +467%; intermyofibrillar: +166%). Despite mitochondrial expansion at this stage, Rac1 deficiency attenuated the increase in palmitate oxidation in response to muscle contraction (-62%). At the muscle-wasting stage, Rac1 imKO muscle exhibited reduced mitochondrial respiratory capacity (-25-32%). Additionally, the mitochondrial dysfunction was associated with an accumulation of muscle TG (+78%, p = 0.096) and upregulation of fatty acid transporter, CD36 protein content (+25%), indicative of altered fatty acid handling. In humans, Rac1 muscle protein content was increased in old, sarcopenic subjects compared to young (+41%), and negatively correlated with quadriceps cross-sectional area (CSA) (r = -0.475) and type II fibre CSA (r = -0.466). In old, sarcopenic muscle, Rac1 protein content correlated negatively with protein content of multiple mitochondrial respiratory complexes (CI: r = -0.690, CIV: r = -0.938, CV: r = -0.704). GWAS further identified associations between Rac1 SNP variants and lipid metabolic and muscle-wasting diseases. ConclusionsMuscle Rac1 deficiency reduces mitochondrial respiratory capacity and metabolic flexibility through impaired fatty acid metabolism, leading to muscle wasting and highlighting a potential therapeutic target in age-related functional decline.

BackgroundCancer-associated cachexia is characterized by progressive loss of skeletal muscle and adipose tissue driven by systemic inflammation and metabolic dysregulation. AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis, but its role in cachexia and its therapeutic potential remains incompletely defined. We investigated AMPK signaling during cachexia and whether pharmacological AMPK activation alone or combined with ghrelin could ameliorate disease manifestations. MethodsCachexia was induced in male C57BL/6 mice by Lewis lung carcinoma (LLC) implantation. Additional models included fibrosarcoma (CHX and MN/MCA1) and chronic lymphocytic choriomeningitis virus (LCMV) infection. AMPK was activated using AICAR and BC1618 (AB), alone or combined with ghrelin (AB+G). Metabolic, inflammatory, and functional outcomes were assessed in hypothalamus, skeletal muscle, adipose tissue, and serum. ResultsLLC-bearing mice developed cachexia characterized by reduced body weight, lean and fat mass, hypophagia, and elevated circulating IL-6 and corticosterone. Cachectic LLC mice displayed increased Il6 and Il1{beta} expression in hypothalamus, skeletal muscle, and white adipose tissue (WAT). Furthermore, AMPK activation failed to increase in hypothalamus or peripheral tissues despite profound energy deficit. A similar defect in AMPK responsiveness was observed in CHX and LCMV models, indicating a conserved feature of cachexia. AB treatment in LLC mice reduced circulating IL-6 and corticosterone levels and decreased skeletal muscle atrogene expression and IL-6/STAT3 signaling, partially preserving muscle mass, fiber size, and grip strength. However, food intake remained low, and WAT was largely unresponsive, maintaining elevated Il6 expression and tissue loss. Ghrelin alone increased food intake in LLC mice but did not ameliorate the cachectic phenotype. In contrast, AB+G restored food intake and prevented loss of lean and fat mass. LLC AB+G mice exhibited reduced hypothalamic Il6 and serotonin transporter (Slc6a4) expression, normalized adipocyte morphology and serum leptin levels, decreased adipose Il6 and Atgl expression and reduced WAT sympathetic innervation. AB+G further lowered circulating corticosterone levels, and provided greater protection against muscle wasting, with increased Pgc1 expression and improved muscle function. Neither intervention affected tumor growth or tumor inflammatory gene expression. ConclusionsCancer cachexia is associated with a central and peripheral failure to appropriately activate AMPK signaling in response to the energetic stress imposed by cachexia. Combined AMPK activation and ghrelin administration exerted complementary effects on energy homeostasis, inflammation, and tissue wasting, resulting in greater protection against cachexia than either intervention alone. These findings support combined AMPK-ghrelin targeting as a promising therapeutic strategy for cancer cachexia.

BackgroundAberrant NAD+ metabolism has been implicated in the pathogenesis of cancer cachexia, highlighting this pathway as a potential therapeutic target to mitigate skeletal muscle wasting. However, it remains unclear whether chemotherapeutic agents contribute to the onset of cachexia by disrupting NAD+ metabolism. Here, we investigated the effects of commonly used chemotherapy regimens on NAD+ metabolism in skeletal muscle and liver of healthy mice. MethodsHealthy mice were treated with either 2-week regimens of folfiri or cisplatin, or 5-week regimens of folfiri or folfox, with vehicle-treated mice serving as controls. Cachexia-related outcomes were assessed, while skeletal muscle and liver tissues were analyzed for NAD metabolites and markers of NAD+ metabolism. Given the consistent downregulation of the NAD+ biosynthetic enzyme Nrk2 in cachectic chemotherapy-treated mice, we examined skeletal muscle Nrk2/NRK2 expression across published murine and human cachexia datasets, and in additional models of muscle wasting and hypertrophy. ResultsNAD+ loss was observed in atrophic muscle following administration of cisplatin (2-week treatment; -14% vs controls, p=0.047) and folfiri (5-week treatment; -18%, p=0.069). In contrast, muscle NAD+ levels were preserved in non-atrophic groups (2-week folfiri and 5-week folfox). Muscle Nrk2 was the most responsive NAD+ biosynthetic enzyme, showing consistent downregulation across chemotherapy models with ongoing or developing muscle loss: cisplatin (-93%, p<0.001), folfiri (-84%, p<0.001) and folfox (-92%, p<0.001). In the liver, NAD+ levels declined after prolonged 5-week folfiri (-20%, p=0.013) and folfox (-15%, p=0.043) treatments. These changes were accompanied by distinct alterations in NAD+ biosynthesis pathways, indicating treatment-specific reorganization of hepatic NAD+ metabolism. Cross-study analyses revealed early and consistent skeletal muscle Nrk2 downregulation across multiple murine cachexia models and human inactivity studies, whereas cachexia-targeted interventions in rodents and resistance training in humans increased its expression. ConclusionsThese findings demonstrate that chemotherapy distrupts tissue NAD+ metabolism, with skeletal muscle NAD+ loss accompanying muscle atrophy and hepatic NAD+ levels declining after prolonged treatment. The early and robust responsiveness of muscle Nrk2 expression to changes in muscle mass underscores its potential as a dynamic indicator for predicting treatment-induced changes in muscle mass. Together, these results provide new molecular insight into the metabolic basis of chemotherapy-induced muscle wasting and support further investigation of NAD+-targeted strategies in this context.

BackgroundMYORG (myogenesis-regulating glycosidase) and STRADB (STE20-related kinase adapter protein beta) were previously identified as activity-mediated skeletal muscle genes with potential roles in frailty and sarcopenia. We hypothesized that, if these genes are sustained by neuromuscular contractile activity, their expression should be consistently downregulated in muscular dystrophies, conditions defined by progressive muscle degeneration and secondary functional disuse. MethodsWe performed a systematic cross-dataset transcriptomic analysis of five publicly available GEO microarray datasets of human skeletal muscle. Discovery analysis was conducted in GSE3307 (Affymetrix HG-U133A/B; samples spanning DMD, LGMD2A/B/I, BMD, FSHD, JDM, ALS, AQM versus healthy controls). Independent external validation was performed in GSE38417 (HG-U133 Plus 2.0, DMD; n=16/6), GSE11681 (HG-U133A/B, LGMD2A; n=8-10/9-10), GSE465 (HG-U95Av2/B/C, multi-disease), and GSE1007 (HG-U95B/C/E, DMD; n=10-11/11). Raw CEL files underwent array-level quality assessment using NUSE and RLE diagnostics prior to normalization. Seven poor-quality arrays were excluded (none from Control, DMD, or LGMD groups). Remaining arrays were processed by robust multi-array average (RMA) normalization, and differential expression was assessed by limma with Benjamini-Hochberg FDR correction. ResultsMYORG was significantly downregulated in DMD (log2 fold-change [logFC] = -0.93, adj.P<0.001), LGMD2A (logFC = -0.82, adj.P<0.01), LGMD2B (logFC = -1.01, adj.P<0.01), and LGMD2I (logFC = -1.03, adj.P<0.01) in GSE3307. STRADB was significantly reduced in DMD (logFC = -0.33, adj.P<0.05) and showed a near-significant trend in LGMD2I (logFC = - 0.42, adj.P = 0.061). MYORG downregulation in DMD was independently replicated in GSE38417 (logFC = -1.40, adj.P<0.001) and GSE1007 (logFC = -0.80, adj.P<0.001). STRADB was also significantly downregulated in GSE38417 DMD (logFC = -0.45, adj.P<0.001). Deoxygalactonojirimycin, an iminosugar and an FDA/EMA-approved pharmacological chaperone (migalastat/Galafold) for Fabry disease, has been reported to be a specific molecular interactor that stabilizes MYORG protein in skeletal muscle. ConclusionsThis multi-dataset study further supports the role of MYORG and STRADB as activity-sensitive muscle genes that are robustly downregulated in DMD and LGMD. The pharmacological interaction between migalastat and MYORG provides a mechanistically grounded rationale for investigating this approved agent as an adjunct therapy in muscular dystrophies, in combination with the existing standard of care. This also supports active investigation of iminosugar analogs to target MYORG as potential therapeutics for improving skeletal muscle function in dystrophies, frailty, and sarcopenia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a life-saving treatment for hematologic malignancies, but long-term survivors present with lower muscle mass and functional capacity. In adult HSCT survivors 10-20 years after treatment, single nucleus RNA sequencing uncovered elevated XRRA1 expression levels in all muscle nuclei populations, which was retained in primary muscle stem cell cultures. HSCT survivors were characterized in vivo by impaired neuromuscular innervation that associated with muscle weakness, and lower muscle stem cell neurotrophic action. Despite these impairments, the molecular and physiological responses to heavy resistance training (HReT) were preserved in HSCT survivors, as demonstrated in a pre-registered clinical trial (ClinicalTrials.gov: NCT04922970). After 12 weeks of HReT, gains in muscle mass and strength were similar in HSCT survivors and healthy controls. In addition, we observed that [~]9% of muscle-resident immune cells persist into adulthood and that bone marrow derived cells do not adopt alternative cell fates in muscle tissue, resolving long-standing questions in human muscle biology. Together, these findings uncover molecular mechanisms of HSCT sequelae in muscle nuclei and muscle stem cells, which, importantly, can at least partly be overcome by mechanical loading. Given the growing population of HSCT survivors and the multitude of benefits of HReT for all organ systems, our findings support the importance of HReT in this population to promote healthspan. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/26351644v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@14322d1org.highwire.dtl.DTLVardef@a30589org.highwire.dtl.DTLVardef@c07930org.highwire.dtl.DTLVardef@544b02_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundSkeletal muscle represents around 40% of total human body weight and exhibits remarkable plasticity. It can hypertrophy, atrophy, or regenerate in response to changes in activity, nutrient availability, or injury. The main component of striated muscle, the myofiber, is a post-mitotic, multinucleated cell that contains the muscles contractile unit, the sarcomere. The myonuclei within these fibers are specialized and differ in terms of gene expression and localization. Adult muscles also contain various other cell types, including adult muscle stem cells (MuSCs), macrophages, fibro-adipogenic progenitors (FAPs), and endothelial cells. MuSCs are central to muscle plasticity, and are capable of activation, proliferation, differentiation, and fusion to form new myofibers during regeneration, or to fuse with existing myofibers during hypertrophy. Muscle hypertrophy and myofibers enlargement involve increased protein synthesis and reduced protein degradation, as well as myonuclear accretion following satellite cell activation. Multiple signaling pathways, such as the mTOR pathway and the RhoA/SRF mechanotransduction pathway, are involved in these processes. MethodsWe performed single-nucleus RNA sequencing (snRNA-seq) on plantaris muscles of adult mice, comparing samples 7 days after hypertrophy induction (overload, 7OV) to non-hypertrophied controls (Ctl). RNAscope experiments on isolated myofibers identified the heterogeneity of myonuclei along the myofiber. ResultsSnRNA-seq analysis revealed a previously unknown population of myonuclei (UM). UM-Ctl, which is present only in the Ctl condition, and UM-7OV, only in the 7OV condition. These myonuclei are localised at the tips of myofibres. Furthermore, we determined that UM-7OV are not newly fused myonuclei from activated satellite cells. Trajectory analyses suggest that UM-Ctl transition into UM-7OV during hypertrophy, returning to a near-basal homeostatic state after 21 days of overload (21OV). Gene expression analysis showed that UM-Ctl and UM-7OV have distinct gene expression profiles compared to other myonuclei and respond differently to hypertrophy. ConclusionOur findings suggest the existence of a specific population of myonuclei with unique localization and gene expression profiles, which play distinct roles at baseline and during hypertrophy. These results highlight the differential properties of myonuclei in the myofiber and their potential specific functions in muscle homeostasis and adaptation.

Colorectal cancer (CRC) cachexia induces skeletal muscle dysfunction, impeding quality of life and worsening cancer prognosis. Multiple preclinical models, including the widely used mouse model of subcutaneous inoculation with the C26 colorectal carcinoma cell line, have been developed to study the biological mechanisms of CRC cachexia and elucidate potential new treatments. It has been proposed that a distinct cell line of the same origin, namely CT26, is relatively non-cachexic. However, studies evaluating the relative potential of C26 and CT26 cells to induce cancer cachexia in parallel have been limited. The differences in the biological mechanisms by which C26 and CT26 impact skeletal muscle mass and function have also not been fully elucidated. In the current study, we investigated the differential capacity of C26 and CT26 to induce cancer cachexia using both an in vitro cancer-muscle cell co-culture and an in vivo syngeneic mouse model. Our results show that both C26 and CT26 cells induced significant atrophy of murine C2C12 skeletal myotubes. In the mouse model, while C26 and CT26 both reduced skeletal muscle mass and fat mass, only C26 tumors led to loss of body weight and impaired skeletal muscle force output. We further show that C26 tumor-bearing mice exhibit greater muscle inflammation than CT26 tumor-bearing mice. In addition, mice bearing C26 and CT26 tumors showed differential regulation of the innate immune responses and muscle protein turnover. Overall, our data suggests that although both C26 and CT26 cells do exhibit cachexic effects, C26 cells induce greater loss in body weight, fat mass, skeletal muscle mass, and physical function via promoting chronic inflammation and deregulating protein balance of skeletal muscle.

BackgroundThe maintenance of skeletal muscle health plays a pivotal role in prolonging both the lifespan and healthspan. However, muscle mass and strength exhibit significant declines with age. Ageing is associated with a reduced muscle protein synthesis response to key anabolic stimuli, including the androgen hormone testosterone, termed anabolic resistance. Testosterone enacts its anabolic effects in muscle through androgen receptor (AR) mediated pathways. Emerging evidence suggests that AR availability may represent a rate-limiting factor in androgen signalling, with AR saturation occurring below physiological testosterone levels in some tissues. Prior research in rodents has reported age-related reductions in AR expression, suggesting changes in AR protein content may constitute a key component of anabolic resistance. However, reports of the effects of age on the human skeletal muscle AR are inconclusive and limited by small sample sizes. Therefore, this study aimed to characterise age-related changes in expression of the AR, its regulators and downstream target genes in human skeletal muscle. MethodsWe developed and used a novel R-based pipeline, MetAR, to perform reproducible meta-analyses of publicly available bulk RNA-Seq datasets from NCBI GEO and investigate associations between target gene expression and variables of interest without the need for high-performance computing. Eligible datasets included skeletal muscle samples from healthy adult males aged [&ge;]18 years, with an age range of [&ge;] 10 years and sample size [&ge;] 6. Raw counts data were downloaded, appraised and TMM normalised. Dataset-level associations between age and target gene expression were assessed using linear and generalised additive models (GAMs). Random-effects meta-analyses were performed, and heterogeneity, publication bias and leave-one-out sensitivity assessed. ResultsSixteen skeletal muscle bulk RNA-seq datasets (n = 364; age 18-92 years) were eligible for inclusion in the meta-analyses. AR expression was negatively associated with age ({beta} = -0.006 log2 TMM-CPM per year, p < 0.001) corresponding to a 4.4% decrease in expression per decade. Age was also associated with a significant reduction in expression of various regulators of AR stability, transcriptional activity and nuclear transport. Additionally, steroidogenic enzymes and key downstream targets of the AR, including genes encoding for key structural proteins and mitochondrial function were negatively associated with age. ConclusionsCollectively, these findings suggest a multi-faceted age-associated remodelling of AR expression, signalling and nuclear transport that may contribute to the development of anabolic resistance and consequent age-associated muscle loss.

The adult skeletal muscle regenerates robustly upon injury, but this regenerative capacity rapidly declines with age. In this study, we identify the lanthionine synthetase C-Like (LanCL) proteins, mammalian homologs of the bacterial peptide cyclase LanC, as positive regulators of muscle regeneration in middle-aged mice. In a barium chloride-induced injury model, we found the protein levels of LanCL1 and LanCL2 to increase during an early phase of regeneration in middle-aged (12-month-old) but not young adult (4-month-old) mice. Utilizing a mouse line lacking all three LanCL proteins (LanCL triple KO or LTKO), we examined a potential role of LanCL in injury-induced muscle regeneration. Consistent with an age-dependent function of LanCL, we observed a delayed regeneration of the tibialis anterior (TA) muscle after injury, as reflected by reduced sizes of regenerating myofibers in middle-aged (but not young) LTKO compared to age-matched WT mice. Although the pool size of quiescent satellite cells (Pax7+) was comparable between 12-month-old LTKO and WT muscles without injury, the number of Pax7+ cells was significantly higher in regenerating LTKO muscles at day 5 after injury, accompanied by drastically decreased numbers of MyoD+ and MyoG+ cells, as well as increased numbers of proliferating cells. In addition, we detected elevated expression of pro-inflammatory cytokines in regenerating LTKO muscles, while the number of macrophages was similar comparing LTKO and WT muscles. Taken together, our observations suggest that in aging muscles LanCLs are important for proper timing of inflammation resolution and regeneration upon injury. New & NoteworthyPhysiological roles of the mammalian homologs of bacterial LanC, LanCLs, are poorly understood. Our work uncovers a function of LanCLs in post-injury regeneration of aging skeletal muscles. Middle-aged LanCL triple KO mice displayed a delay in satellite cell differentiation and regenerative myofiber formation, as well as persistent inflammatory cytokine expression, suggesting that LanCLs may have an age-dependent role in modulating inflammation in the injured muscles to facilitate regeneration.

BackgroundAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron (MN) loss, muscle atrophy and paralysis. Although traditionally considered a MN-specific disease, accumulating evidence supports a crucial contribution of skeletal muscle pathology to disease onset and progression. Except for specific mutations, to date there is no effective treatment for ALS. FOXO transcription factors regulate programs of atrophy, metabolism and stress response in skeletal muscle, and their inhibition has shown beneficial effects in cellular and Drosophila models of ALS. MethodsIn this study, we investigated whether pharmacological FOXO inhibition (iFOXO) could modify disease progression and muscle pathology in female hSOD1G93A mice. Mice received daily oral administration of iFOXO starting at presymptomatic (P50; n=5 per group) or symptomatic (P90; n=9 mice per group) stages until end-stage. Body weight was monitored longitudinally, and motor performance was evaluated using grip strength and hanging-wire tests. Tibialis anterior and soleus muscles, representing fast- and slow-twitch muscles respectively, were analyzed by histology and immunofluorescence to assess fiber atrophy, fibrosis, lipid accumulation, satellite cell pool and fiber type composition. Quadriceps muscles (n=3 per group) were used for RNA-seq analysis. ResultsWhile histological analyses revealed severe fiber atrophy and increased fibrosis in hSOD1G93A mice, satellite cell numbers were preserved or mildly increased in a muscle and treatment onset dependent manner. iFOXO treatment did not improve motor performance, survival or attenuate muscle atrophy. Transcriptomic profiling indicated that genotype was the predominant driver of gene expression changes, while iFOXO produced only subtle, treatment onset dependent effects on pathways related to oxidative stress responses, mitochondrial function and adaptive metabolism. ConclusionOverall, FOXO inhibition alone showed limited therapeutic benefit in the hSOD1G93A ALS mouse model. These findings highlight the dominant influence of ALS driven molecular alterations over pharmacological modulation and emphasize the need for combinatorial therapeutic strategies targeting multiple disease mechanisms, including those preserving nerve health.

Up to 50% of adults with colorectal cancer (CRC) are at risk of progressive involuntary loss of skeletal muscle mass and function known as cachexia. Available options to prevent and treat cachexia in cancer survivors are currently limited. Long-chain polyunsaturated fatty acids (LC-PUFAs) and their bioactive metabolites, termed specialized pro-resolving lipid mediators (SPMs), promote the resolution of inflammation and support muscle growth and repair. However, prior studies of cachexia have mainly focused on fish oil supplements, and it is not fully understood how different individual omega-3 (n-3) and omega-6 (n-6) LC-PUFAs mediate CRC-induced muscle wasting. In addition, the crosstalk between cancer cells, the host immune system, and skeletal muscle cells in response to LC-PUFA treatments remains unclear. This study aimed to examine the effects of n-3 and n-6 LC- PUFAs on CRC-induced muscle wasting and the underlying cellular and molecular mechanisms involved. Using murine C2C12 skeletal muscle cells and CT26 colorectal carcinoma cells, we investigated the impacts of LC- PUFAs including arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3), and docosahexaenoic acid (DHA, 22:6n-3) on CT26-induced muscle cell wasting in the presence or absence of lipoxygenase (LOX) inhibitors such as NDGA or BLX-3887. We also examined the lipidomic profile of C2C12-CT26 co-cultures in response to individual LC-PUFA treatments. Our results suggest that LC- PUFAs including ARA, EPA, DHA, and DPA each individually protect against CRC-induced muscle cell wasting in vitro, and these protective effects are dependent on 15-LOX activity. Furthermore, we found that C2C12-CT26 co-culture produced mature SPMs in response to individual PUFA treatments. Taken together, this study suggests that individual n-3 and n-6 LC-PUFAs can mitigate CRC-associated cachexia primarily by producing 15-LOX- derived bioactive lipid mediators.

Regeneration of skeletal muscle preserves muscle mass and function, which decline with age. Here, we sought to identify long noncoding (lnc)RNAs involved in skeletal muscle myogenesis and potentially relevant to muscle aging. Cross-sectional analysis of skeletal muscle transcriptomes from healthy 22-through 89-year-old individuals revealed lncRNA LANCL1-AS1 among the top declining transcripts. Conversely, LANCL1-AS1 increased robustly during skeletal myogenesis and promoted myogenic differentiation in culture. Affinity pulldown by ChIRP followed by mass spectrometry revealed that LANCL1-AS1 associated with the mitochondrial protein LRPPRC, enhancing the formation of the chaperone complex LRPPRC-SLIRP, which maintains longer poly(A) tails of mitochondrial (mt-)mRNAs and stabilizes mt-mRNAs. Importantly, while myoblasts from old rhesus monkey muscle expressed lower levels of LANCL1-AS1 and mt-mRNAs, and displayed lower mitochondrial activity than young monkey myoblasts, overexpressing LANCL1-AS1 in old myoblasts restored mitochondrial activity and myogenesis. We propose that the age-associated reduction in LANCL1-AS1 contributes to impaired mitochondrial function and reduced myogenic capacity in aging skeletal muscle.

BackgroundEstrogen-related receptor gamma (ERR{gamma}) drives an exercise mimicking aerobic gene program in the skeletal muscle that could be beneficial in aging. We have investigated the effect of chronic ERR{gamma} activation on minimizing sarcopenia. MethodsExperiments were performed in muscle specific ERR{gamma} transgenic (TG) mice and wild type (WT) littermates, at young (4-5 months) and old (24-26 months) age. In the skeletal muscle, global gene expression changes, as well as myofiber histological changes in fiber type, size, vascular supply and neuromuscular junction (NMJ), and mitochondrial content were measured. Functional analysis was performed using in vivo muscle contraction assay. Exercise fitness was measured using treadmill sprint and endurance test. Gene and protein expression was measured using QPCR and Westerns, respectively. ResultsERR{gamma} activates a pan-ERR aerobic program in the skeletal muscle to increase expression of 574 genes including ERR, mitochondrial homeostasis (e.g. Mfn1, Opa1, Drp1, Fis1, and Tfam), vascularization (e.g. Vegfa, Angpt1, Fgf1), and neuromuscular junction (NMJ) (e.g. Nrp1, Aspa, Ptprm, Cxcr4), simultaneously suppressing the expression of atrophy related genes (e.g. Atrogin1, Traf6, Nedd4, Myd88, p21). ERR{gamma} increases mitochondrial content [Mitochondrial area: old TG vs. WT, 2.00 fold; young TG vs. WT, 1.32 fold], oxidative capacity [NADH-TR activity: old TG vs. WT, 1.20 fold; young TG vs. WT, 1.22 fold] and myofiber type [2a: old TG (687{+/-}258) vs. WT (252{+/-}71); young TG (797{+/-}168) vs. WT (440{+/-}76); 2x: old TG 1348{+/-}87 vs. WT 976{+/-}219; young TG 1131{+/-}135 vs. WT 936{+/-}84; 2b: old TG (798{+/-}103) vs. WT (1628{+/-}148); young TG (967{+/-}133) vs. WT (1623{+/-}189)], and capillarity [capillary-to-myofiber ratio: old TG (3.25{+/-}0.19) vs. WT (2.41{+/-}0.16); young TG (3.41{+/-}0.21) vs WT (2.59{+/-}0.2)] and [NMJ number [old TG (67{+/-}8) vs. WT (40{+/-}9); young TG (77{+/-}11) vs WT (77{+/-}7)], mitigating age-related loss of NMJ and myofiber cross-sectional area [old TG (1570{+/-}147{micro}m2) vs. WT (1692.5{+/-}208{micro}m2) WT; young TG (1828.15{+/-}132.8{micro}m2) vs. WT (2109.7{+/-}296.8{micro}m2)]. ERR{gamma} overexpression preserves muscle contractility with aging [Fatigue resistance: 22.72% reduction in force in old vs. young WT; 3.11% reduction in force between old vs. young TG]. Furthermore, ERR{gamma} maintains exercise fitness in old mice [Running: old TG (2964.52{+/-}405m) vs. old WT (910.75{+/-}6034m); young TG (2232.43{+/-}193.64m) vs. young WT (1366.76{+/-}60.76m)]. ConclusionsERR{gamma} drives a pan-ERR and counter sarcopenic gene program enhancing oxidative myofiber type, mitochondrial content, vasculature, and NMJ in aging muscle. Consequently, ERR{gamma} minimizes myofiber atrophy, preserves contractility, and improves exercise fitness in old mice. Therefore, ERRs are potential translational targets for combating sarcopenia.

Cancer-related fatigue is the most common and persistent symptom in breast cancer, with fatigue reported up to 10 years post-diagnosis. Unlike many cancers, fatigue in breast cancer often arises during early-stage disease in the absence of cachexia. While many factors contribute to fatigue, the direct contribution of cancer-associated skeletal muscle pathology remains poorly understood. Here we analyzed pectoralis major muscle biopsies from individuals with non-metastatic breast cancer and non-cancer controls. Using single-fiber morphometry and spatial transcriptomics, we identified fiber-type-specific structural alterations and spatially localized transcriptional reprogramming within the muscle microenvironment. Single-fiber morphometry revealed selective atrophy of oxidative type I and type IIa muscle fibers, while glycolytic type IIx fibers were relatively preserved. Concordant spatial transcriptomic profiling revealed suppression of oxidative metabolic programs, evidence of mitochondrial dysfunction, and spatially localized catabolic signaling originating from intramuscular adipocytes. This study introduces an integrated framework for profiling skeletal muscle architecture and spatially localized gene expression in surgically obtained muscle biopsies and represents the first application of spatial transcriptomics to human skeletal muscle from individuals with cancer. These findings demonstrate structural and metabolic remodeling of skeletal muscle in non-metastatic breast cancer and suggest targeting muscle metabolism represents a promising therapeutic strategy for cancer-related fatigue.

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.

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) have become defining therapies in the management of type 2 diabetes and obesity. Despite recent interest in the effects of GLP-1RA therapy on skeletal muscle, their influence on muscle repair after injury remains largely untested. Because GLP-1RA use is common in populations at heightened risk for diminished regenerative capacity, a critical unanswered question is whether GLP-1R agonism supports muscle regeneration or alters the normal course of recovery after injury. Using intramuscular glycerol injection as an adipogenic injury model, we assessed whether semaglutide, a widely prescribed GLP-1RA, alters the balance between myogenesis and adipogenesis during regeneration. Surprisingly, semaglutide treatment markedly increased the formation of intramuscular adipose tissue (IMAT) and inhibited the growth of regenerated fibers. These effects were injury-dependent, as uninjured muscle showed no detectable differences in IMAT or myofiber size. Together, these findings identify a previously underappreciated context in which GLP-1RA therapy may adversely affect muscle quality.

BackgroundCancer cachexia (CC) is characterized by skeletal muscle atrophy and reduced strength, partly linked to dysfunction of muscle stem cells (MuSCs) and alterations in their niche. Although exercise may mitigate muscle loss, its effects in CC remain debated and its feasibility is often limited in advanced patients. Neuromuscular electrical stimulation (NMES) offers a promising alternative, by promoting MuSC proliferation and fusion, increasing muscle size and macrophage content in healthy muscle. This study investigated whether NMES, initiated at tumor onset, could improve MuSC regulation and its niche while limiting muscle atrophy and weakness in a tumor-bearing mouse model. MethodsTen-week-old male BALB/c mice were subcutaneously injected with C26 tumor cells or PBS. Tumor-bearing mice were divided into NMES-treated (C26 NMES) and non-stimulated controls (C26). NMES consisted of six sessions (two series of three consecutive daily sessions separated by one rest day), starting seven days post-inoculation when tumors became visible. Each session was delivered at a submaximal intensity corresponding to 15% of maximal strength. Muscle mass, myofiber size, strength and cellular composition were assessed. ResultsMuscle mass was decreased by 13% in C26 mice as compared to PBS controls, while C26 NMES mice showed a [~]7% improvement over C26 mice. Mean myofiber size decreased similarly in both tumor-bearing groups as compared to PBS controls (-12-14%). However, NMES reduced the proportion of small myofibers (400-600 {micro}m{superscript 2}) as compared to C26 mice. Maximal torque loss was less severe in C26 NMES mice (-28%) than in C26 mice (-34%). As compared with PBS mice, C26 mice exhibited increased MuSC proliferation (+97%) but reduced differentiation (-61%), as indicated by fewer myogenin-positive cells. NMES normalized MuSC proliferation, restored myogenin-positive cell number, and enhanced MuSC fusion, reflected by an increased number of PCM1-positive myonuclei (+8-11%). NMES also modulated inflammation, reducing neutrophils (-42%) and increasing macrophages (+35%), through the proliferation of CD169-positive resident macrophages (+106%). In vitro, macrophages exposed to C26 muscle extracts showed elevated pro-inflammatory markers (COX2 and TNF-; +21% and +16%) as compared to PBS controls. This effect was abolished with extracts from C26 NMES muscles. Additionally, C26 extracts reduced the expression of anti-inflammatory markers by macrophages (CD206 and IL-10; -23%), whereas NMES restored their levels to those of controls. ConclusionNMES-induced mild contractile activity is an effective stimulus for preserving muscle strength and mass, improving MuSC regulation, and modulating muscle inflammation in a mouse model of CC.

Background & aims: Autophagy activation is a promising strategy to counteract age-related cellular dysfunction. While preclinical studies suggest dietary protein restriction can induce autophagy via mTORC1 inhibition, direct human evidence using dynamic, flux-based measurements remains limited. The aim of this study was to determine whether a low protein diet could modulate autophagic flux in humans. Methods: We conducted a randomized crossover trial in which 74 healthy adults were randomized to receive two 4-week interventions of either average-protein (20% energy) or reduced-protein (10% energy) diets prescribed to maintain calculated energy balance, separated by a 4-week washout period. The primary outcome was autophagic flux measured in whole blood using a validated assay that preserves PBMCs in their physiological environment during lysosomal inhibition. Secondary outcomes included metabolic markers, body composition, and self-reported health metrics. Results: Sixty-three participants completed both interventions (mean {+/-} SD age 29.5 {+/-} 7.2 yrs; BMI 24.0 {+/-} 3.2 kg/m2). Reducing protein intake did not alter autophagic flux (adjusted mean difference: -8.46 ng LC3B-II/mg protein/h; 95% CI: -24.06 to 7.14; p = 0.28). Metabolomic profiling confirmed effective dietary separation, with lower circulating urea following reduced protein intake. Small differences in body weight and muscle mass were observed, while fat mass was unaffected. Fasting glucose, insulin, lipids, blood pressure, and quality of life did not differ between the two diets. Conclusions: Moderate protein restriction does not increase basal autophagy in circulating immune cells of healthy adults, suggesting protein reduction alone, without caloric deficit, may be insufficient to activate autophagy in human blood.

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

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