Exercise is well known to enhance insulin sensitivity in skeletal muscle, yet the underlying mechanisms remain incompletely understood. We have previously shown that neutrophil recruitment contributes to contraction-induced GLUT4 translocation and local myokine induction, but whether these immune cells also participate in the post-exercise increase in insulin sensitivity has been unclear. Here using GLUT4-EGFP transgenic mice and sciatic nerve-mediated in situ contraction of the hindlimb, with analyses focused on extensor digitorum longus (EDL) muscle, we demonstrate that neutrophil recruitment and subsequent formation of neutrophil extracellular traps (NETs) are crucial for the well-known post-exercise increase in insulin sensitivity. Two-photon imaging revealed that NET-like cell-free DNA (cfDNA) structures persisted for hours after contraction, formingspatially confined perivascular immunometabolic nichesalong the capillary meshwork. Strikingly insulin-stimulated GLUT4 translocation waspreferentially enriched at these NET-rich sites, whereas DNase-mediated NET degradation eliminated cfDNA signals andabolished the contraction-induced enhancement of GLUT4 translocation, glucose uptake and attenuated AS160 (T642) phosphorylationunder low-dose insulin. Our findings demonstrate that neutrophils are essential components of the mechanism underlying enhanced post-exercise insulin sensitivity involving, at least in part, the local formation of NETs. These NET-governed immunometabolic niches constitute a structural and spatial framework underlying the exercise-induced acute improvement of insulin-responsive metabolic efficiency in skeletal muscle. KEY POINTS: Neutrophil extracellular traps (NETs) establish spatially confined immunometabolic niches that are indispensable for the post-exercise increase in insulin sensitivity. High-resolution imaging revealed that insulin-stimulated GLUT4 translocation is markedly enhanced predominantly in NET-rich perivascular regions, indicating a spatially restricted mechanism of post-exercise insulin sensitization. DNase-mediated degradation of NETs abolished this enhancement, establishing their essential role in local insulin-responsive GLUT4 translocation. These NETs are formed by neutrophils rapidly recruited to skeletal muscle after contraction and deposited along the capillary network.

Obesity impacts the histological features and modifies the apelin levels in different skeletal muscles of aging female wistar rat.

Eccentric muscle contractions occur when muscles actively lengthen, acting as brakes that dissipate energy and stabilise joints. When actively stretched, muscle force rises in two phases: an initial steep increase (phase 1), followed by a slower, sustained rise (phase 2). The temperature sensitivity of this response is poorly understood, despite its relevance for musculoskeletal models that often rely on data collected at non-physiological temperatures. We studied active lengthening contractions in mouse extensor digitorum longus muscle at 17°C, 27°C and 37°C. Force development in both phases was temperature sensitive. Phase 1 stiffness decreased at higher temperatures, consistent with faster ATP-dependent cross-bridge detachment, and contributions from mechanical strain-dependent detachment. In phase 2, stiffness increased with temperature, consistent with stronger and faster titin-actin interactions. The transition between phases (muscle ‘give’) varied with temperature and may reflect lower temperatures delaying cross-bridge detachment and engagement of the parallel elastic elements. Together, these findings highlight the intrinsic tuning of muscle mechanics, with potential implications for susceptibility to muscle damage under different thermal conditions, and provide a foundation for the development for more accurate musculoskeletal models.

Burn injury dysregulates protein signaling in type 1 and type 2 muscles in rats.

BackgroundDuchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats.MethodsCRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses.ResultsMDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition.ConclusionsMDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13395-026-00419-4.

Evidence suggests that muscle activity can affect muscle carnosine, but the results are mixed. To address this question, we investigated muscle carnosine under two extremes of the muscle activity-inactivity spectrum. Forty-five male Wistar rats were divided into three groups: immobilization (n = 16), SHAM control (n = 14), and immobilization + exercise (n = 15). In the immobilized groups, one side was submitted to a sciatic nerve sectioning surgery, with the opposite side being submitted to a SHAM control surgery, creating four experimental conditions: denervated (DEN), SHAM active control (SHAM), denervated + exercise (DEN + Ex), and SHAM + exercise (SHAM + Ex). The immobilization period was 12 wk, and the swimming training period was 10 wk (4 times per week, up to 30 min per session). The tibialis anterior (TA) and soleus muscles from both sides were assessed for carnosine and anserine contents, total histidine-dipeptides (HCDs), cross-sectional fiber area (CSA), and fiber type distribution. Contractile function was determined ex vivo in the extensor digitorum longus, and the expression of the Carns1, Cndp2, and TauT genes was determined with real-time polymerase chain reaction in TA. Physical inactivity drastically reduced muscle mass, contractile function, and fiber CSA. Long-term postdenervation muscle paralysis reduced muscle carnosine and anserine content, which was not dependent on diet, age, sex, or fiber type. This demonstrates that muscle inactivity is a strong modulator of muscle HCD content, at least under extreme conditions. Gene expression was not significantly altered in any of the experimental conditions. Exercise training, on the other hand, did not affect muscle HCDs and may be a less potent regulator of muscle HCD content.NEW & NOTEWORTHY This study demonstrated that an extreme model of muscle inactivity in rats (i.e., 12 wk of hindlimb paralysis following denervation) resulted in a substantial decline in muscle carnosine and anserine, which occurred irrespective of fiber type shift. Conversely, exercise training had no effect on histidine-dipeptide content. These findings, along with previously published studies, reinforce the notion that muscle inactivity is an important modulator of histidine-dipeptide homeostasis in skeletal muscle.

Duchenne muscular dystrophy (DMD) is a genetic muscular disease characterized by progressive muscle degeneration. p16 is expressed in skeletal muscles and induces cellular senescence in a rat model of DMD, whereas its ablation enhances muscle regeneration. However, the mechanism underlying this phenomenon remains unclear. This study aimed to elucidate the mechanism for p16-induced DMD exacerbation. RNA-seq analysis revealed p16-dependent upregulation of cytokine gene expression in DMD rat skeletal muscles, which also altered the systemic blood cytokine profile. Furthermore, the effect of an altered humoral environment on muscle regeneration was assessed using the transplanted extensor digitorum longus muscle. Regeneration of grafted muscles from wild-type rats was suppressed in DMD rats but was significantly improved by p16 ablation. Notably, p16 was expressed in the myofibers of DMD rats, and enzymatically isolated myofibers from DMD rats also showed p16-dependent cytokine expression. Thus, cytokines secreted by senescent-like myofibers mediate the anti-regenerative niche in DMD rats, uncovering a novel mechanism for disease progression and potential therapeutic targets.

Exercise elicits a spectrum of metabolic and inflammatory responses that are crucial for skeletal muscle adaptation and overall health, particularly in the context of metabolic diseases, yet the contribution of prostanoid signalling to these processes remains unclear. We hypothesised that exercise-induced thromboxane production enhances skeletal muscle glucose uptake and improves whole-body glucose control. Plasma prostanoids were quantified in men and women with normal glucose tolerance or type 2 diabetes before, immediately after and 3 h after a single bout of exercise. Cyclooxygenase (COX-2) transcript levels were evaluated in human skeletal muscle, whole blood, peripheral blood mononuclear cells and skeletal muscle-resident immune cells. Metabolic and transcriptomic effects of thromboxane receptor activation were analysed in mouse C2C12, rat L6 and human primary skeletal muscle cells. Glucose tolerance in vivo was assessed following i.p. administration of the thromboxane receptor agonist I-BOP in male and female mice. Tissue-specific glucose uptake was quantified by measuring radiolabelled 2-deoxyglucose incorporation during an IVGTT. Acute exercise increased plasma thromboxane B₂ concentrations and skeletal muscle mRNA levels of PTGS2 (encoding COX-2) selectively in monocyte/macrophage populations. In skeletal muscle cells, the thromboxane receptor agonist I-BOP increased glucose uptake in a dose-dependent manner up to 2.5-fold within 4 h and enhanced glycogen synthesis by 430%. Transcriptomic and signalling analysis revealed activation of protein kinase A and cytoskeletal remodelling pathways linked to GLUT4 trafficking. In vivo, I-BOP improved glucose tolerance in male mice in a dose-dependent manner, without altering insulin levels. Thromboxane receptor stimulation increased glucose uptake in extensor digitorum longus muscle by 43%. Importantly, thromboxane receptor activation preserved its glucose-lowering efficacy in diet-induced obese male mice. Exercise induces skeletal muscle-derived thromboxane production through macrophage-specific COX-2 activation. Thromboxane receptor stimulation enhances glucose uptake and glycogen storage via cytoskeletal remodelling, partially mimicking the acute exercise transcriptomic response. In vivo, thromboxane receptor activation improves glucose tolerance and skeletal muscle glucose uptake, with preserved efficacy in obesity. These findings identify thromboxane signalling as a previously unrecognised immunometabolic axis linking inflammation to glucose regulation and highlight the thromboxane receptor as a potential therapeutic target for metabolic disease.

Obesity has been increasingly recognized not only as a metabolic disorder but also as a condition that impairs neuromuscular function, including strength relative to body mass. This translational study investigated whether obesity affects both force generation and contraction‐relaxation dynamics. In control (CN) and diet‐induced obese (OB) male mice, contractile properties of isolated extensor digitorum longus (EDL) and soleus (SOL) muscles were assessed in vitro. In parallel, plantar flexor performance was assessed in 25 normal‐weight (CN) and 25 class I obese (OB) sedentary men through maximal voluntary isometric contractions and a dynamic calf raise test. OB mice exhibited lower specific force and slower rates of force development and relaxation in both EDL and SOL (p < 0.05). In men, the lower rate of torque development and prolonged relaxation kinetics of the plantar flexors (p < 0.05), combined with a higher body mass to maximal voluntary isometric torque ratio (p < 0.05), contributed to slower calf raise phases in OB compared to CN men (p < 0.05). These findings reveal that obesity not only has a negative impact on the muscle force generating capacity but also induces slower muscle contractile kinetics.

Myoblasts autonomously govern myofiber-type specification of newly formed myotubes through autocrine-paracrine-dependent manners mediated by multipotent modulators. Netrin-1, which is particularly produced in myoblasts isolated from the extensor digitorum longus (EDL; fast-twitch myofiber-abundant) rather than the soleus (slow-twitch myofiber-abundant), and netrin-4, which is abundantly expressed during myogenic differentiation initiation, stimulate the synthesis of fast-type myosin heavy chain (MyHC) isoforms. However, the mechanisms by which netrin-1 and netrin-4 promote fast-twitch myotube formation remain unclear. Here, we investigated the roles of netrin receptors, uncoordinated-5 homologues (UNC5A, -B, -C, and -D), deleted in colorectal cancer (DCC), and the DCC paralog (neogenin) during myogenic differentiation, focusing on fast-twitch myotube formation. We confirmed that UNC5A, UNC5B, UNC5C, and neogenin synthesis patterns in EDL myoblasts showed no marked differences compared with those in soleus myoblasts. Notably, UNC5A knockdown severely inhibited fast-twitch myotube formation compared with other receptor knockdown treatments and significantly reduced the synthesis of fast-type MyHC isoforms. Additional treatment with recombinant netrin-1 or netrin-4 induced fast-type MyHC mRNA expression; however, this effect was suppressed by UNC5A knockdown. These findings revealed that UNC5A is involved in fast-twitch myotube formation via netrin ligands, highlighting an autonomous fast-type myofiber commitment system within myoblasts.

Congenital split foot/hand is a rare limb anomaly. Although various surgical techniques have been described, detailed gross anatomical studies of soft tissue adaptation, particularly in the foot, are extremely rare. This study presents a detailed anatomical description of a case of severe bilateral split foot. A comprehensive dissection was performed on the lower limb of a 64-year-old male donor with bilateral split foot/hand. Radiographic evaluation classified the deformity as Blauth type IV, characterized by the absence of the lateral cuneiform bone and severe hypoplasia/aplasia of the second and third metatarsals. Significant changes were revealed in the musculotendinous apparatus. The key finding was a unique tendon loop passing through the central cleft, formed by the tendon of the extensor digitorum longus and connecting with the tendons of the flexor digitorum longus and flexor hallucis longus. This study presents the first detailed macroscopic anatomical description of split foot, demonstrating that this congenital anomaly involves complex, structured tendon and muscle adaptations that extend beyond skeletal deficiencies alone. The discovery of a persistent tendon loop-previously reported only once in split hand-indicates asynchronous development of skeletal and soft tissue structures. These findings should be taken into account for surgical planning, emphasizing the need to identify and manage such abnormal soft tissue structures during reconstructive procedures.

First study to characterize the proteomic alterations induced by β-Hydroxy-β-Methylbutyrate in combination with Dexamethasone. Evaluation of β-Hydroxy-β-Methylbutyrate in a severe glucocorticoid-induced muscle atrophy model. Analysis focused on white (fast-twitch) skeletal muscle, which is preferentially affected by glucocorticoid-induced atrophy. Although some effects of glucocorticoids are necessary for metabolic responses during stress, chronic exposure to high levels of these hormones can cause side effects such as hyperglycemia, insulin resistance, and muscle atrophy. Elevated glucocorticoid levels are observed in several clinical conditions, and their exogenous administration, such as high-dose dexamethasone treatments, is included in many clinical protocols. In this context, the supplement β-Hydroxy-β-methylbutyrate (HMB) emerges as a promising approach to counteract side effects its anticatabolic action. Therefore, the aim of this study was to evaluate the protein profile in the muscles and liver of rats treated with HMB in response to a model of severe muscle atrophy induced by dexamethasone. A total of 24 male Wistar rats, 60 days old, were used and distributed into the following groups: (1) Placebo Experimental Group (PEG), n = 8, treated only with saline solution; (2) Dexamethasone Experimental Group (DEG), n = 8, treated with intraperitoneal Dexamethasone injection (1 mg/kg/day); and (3) Dexamethasone + HMB Experimental Group (DEHG), n = 8, treated with intraperitoneal Dexamethasone injection (1 mg/kg/day) and gavaged with HMB (0.3 g/kg/day). After 10 days of treatment, the animals were euthanized for collection of the Soleus, Extensor Digitorum Longus (EDL) muscles, and liver, followed by protein processing and identification. In the Soleus muscle, when comparing DEG vs. PEG, proteins such as Myosin heavy chain 2, Myosin-4, Myosin-6, and Myosin-7 showed decreased expression in DEG compared to PEG. Comparing PEG vs. DEHG also showed a decrease in myosin expression in DEHG, but only Myosin-6 and Myosin-7 were reduced among the identified isoforms. In the EDL muscle, both DEG and DEHG showed reduced levels of all identified myosin subtypes. However, when comparing DEG and DEHG, the only myosin isoform preserved in DEHG compared to DEG was Myosin heavy chain 2. In the liver, dexamethasone treatment impaired glucose uptake, altered the Cytochrome P450 system, and reduced mitochondrial function, as reflected by reduced ATP levels. In conclusion, dexamethasone induced the loss of contractile proteins (myosins) in both muscles. The HMB dosage used in this study partially prevented myosin loss in the Soleus muscle but did not inhibit myosin loss in the EDL muscle. In the liver, dexamethasone induced changes in several proteins related to glucose metabolism; however, HMB was unable to attenuate dexamethasone-induced hepatic glycogenolysis.

Hypokalemic periodic paralysis (HypoKPP) is an ion channelopathy causing episodic skeletal muscle weakness triggered by hypokalemia. Reduced inward rectifier K+ (Kir) channel activity contributes to membrane depolarization and paralysis, suggesting that pharmacologic activation of muscle K+ channels may restore excitability. The Kv7 channel agonist retigabine previously mitigated low-K+ weakness in HypoKPP models. Here, we tested whether this effect persists under conditions producing sustained, severe weakness. Extensor digitorum longus (EDL) muscles from mice homozygous for the SCN4A R669H mutation were studied by isometric twitch force recording at 34°C, with or without 20 U/L insulin. Weakness was induced by reducing extracellular K+ to 1.0 mM. Retigabine (10 μM) was applied to the bath, and twitch force was analyzed by paired or unpaired t tests (n = 4-7 per group). Baseline twitch force in 4.75 mM K+ was ~50% lower in HypoKPP than wild-type muscle (p = 0.01). Force declined further after 1 h in 4.75 mM K+ (p = 0.016) and was completely lost at 1.0 mM K+ with insulin. Retigabine significantly reduced loss of force at both 4.75 and 1.0 mM K+ (p = 0.047 and p = 0.007, respectively). Kv7 channel activation by retigabine preserved contractile force even during sustained depolarization from severe hypokalemia. These findings extend prior work and support development of K+ channel agonists as a therapeutic approach for HypoKPP.

Postmenopausal women have an increased risk for age-related conditions like sarcopenia, osteoporosis and type 2 diabetes. Here we examined the effects of low-dose lithium (Li) supplementation, a well-known glycogen synthase kinase 3 (GSK3) inhibitor, on ovariectomized (OVX) mice, focusing on muscle strength and endurance, bone mineral density (BMD) and insulin sensitivity. 28-week-old female sham and OVX C57BL/6J mice were divided into three groups: sham, OVX and OVX-Li, with the latter receiving 50mg/kg/day of Li in their drinking water for 8weeks. Li supplementation enhanced isometric specific force and fatigue resistance of the soleus and extensor digitorum longus (EDL) muscles. Potential cellular mechanisms underlying these benefits include enhanced Ca2+ uptake, lowered oxidative stress, increased expression of select mitochondrial markers and a blunted muscle transcriptomic response to OVX surgery with Li supplementation. OVX mice exhibited lower BMD than sham controls; however Li supplementation restored BMD levels. Finally Li supplementation yielded modest improvements in insulin tolerance. In conclusion our findings highlight the advantages of low-dose Li for musculoskeletal and metabolic health in OVX female mice. KEY POINTS: Ovariectomy surgery compromises musculoskeletal and metabolic health in female mice. Here, we explored the potential benefits of low-dose lithium (Li) treatment. Li improved muscle isometric force production and fatigue resistance. RNA-Seq demonstrated that Li bluntedthe effects of OVX surgery. Li supplementation increased bone mineral density and insulin tolerance.

S-ketamine is recognized as a rapid-acting antidepressant, exerting its effects primarily through activation of the mTOR signaling pathway in the brain, which plays a key role in neuroplasticity. Given the shared molecular mechanisms between brain and skeletal muscle, we investigated whether S-ketamine can also modulate regulatory proteins involved in muscle protein synthesis (MPS) and muscle protein breakdown (MPB) in skeletal muscle. Adult female Flinders Sensitive Line rats received a single intraperitoneal injection of S-ketamine (20mg/kg) or saline, and soleus and extensor digitorum longus (EDL) muscles were collected two hours post-injection for protein analysis using Western blot. S-ketamine significantly increased phosphorylated mTOR (p-mTORSer2448) in both soleus and EDL, while total ULK1 protein expression was elevated in soleus. These findings suggest that S-ketamine can stimulate mTOR-related signaling in skeletal muscle, potentially enhancing MPS, although the activation was limited to specific signaling proteins. The results provide novel insights into the peripheral effects of S-ketamine beyond the central nervous system, highlighting the potential relevance for skeletal muscle physiology and anabolic regulation. Future studies are warranted to determine the temporal dynamics of these effects, the dose-dependence, and the impact of repeated administration on muscle hypertrophy. Overall, this study expands understanding of S-ketamine's systemic actions and raises new questions regarding its potential as a modulator of skeletal muscle protein metabolism.

Duchenne muscular dystrophy (DMD) is a X-linked progressive genetic muscle disorder. A clinical report showed significant improvements of motor function in patients with dystrophinopathy who received heart transplantation with immunosuppressant tacrolimus (TCL) treatment. This preliminary animal study aimed to clarify whether TCL administration can suppress muscle inflammation and improve muscle strength in a CRISPR/Cas9-generated DMD rat model. Eleven-week-old male DMD rats were treated with either TCL (0.5 mg/kg, twice daily, p.o.) or vehicle for 14 days. After the medication, forelimb grip strengths were measured and systemic inflammation was analyzed by whole blood counts and splenic flow cytometry (CD45, CD3, CD4, CD8, and CD11b). Histopathological changes in the tibialis anterior, soleus, and extensor digitorum longus muscles were evaluated. No significant differences were observed in body weight or grip strength between the TCL-treated and vehicle groups. Furthermore, TCL treatment did not significantly alter the proportions of splenic T-cell subsets or myeloid cells. Histopathological analysis revealed persistent myofiber necrosis, inflammatory infiltration, and fibrosis in both groups, without significant improvements in the TCL group. These findings indicate that a short-term TCL administration for DMD rats is insufficient to yield functional or pathological improvements. Although this preliminary study did not demonstrate therapeutic efficacy, our results provide a baseline knowledge of immunosuppressant for future investigations to optimize dosage, timing, and intervention duration using animal DMD model.

Along- and cross-muscle fiber shear moduli in skeletal muscle.

Duchenne muscular dystrophy (DMD) is one of the most severe forms of inheritable muscular dystrophies, caused by a genetic mutation resulting in the loss of dystrophin. Dystrophin loss initiates a cascade of negative mechanistic changes in skeletal muscle, such as disrupted protein homeostasis and mitochondrial dysfunction. Recent evidence suggests the leucine metabolite, β-hydroxy-β-methylbutyrate (HMB), may improve physical function in DMD boys and improve aspects of the dystrophic phenotype in preclinical mdx mice. HMB has been shown to modulate protein turnover and mitochondrial function, both of which are dysregulated in DMD. Therefore, this study examined the effect of 3-wk of HMB supplementation (0.75 mg/g/day via drinking water), starting at 3-wk of age in mdx mice. HMB-treated mdx mice exhibited increased full-body grip strength and holding impulse, compared with mdx controls. HMB treatment also increased normalized muscle mass of the fast-twitch extensor digitorum longus (EDL) muscle, which coincided with increased average fiber size and improved absolute/specific in vitro force production. Moreover, HMB-treated EDL muscles displayed increased mitochondrial complex II succinate dehydrogenase activity, alongside upregulated markers of mammalian target of rapamycin complex 1 (mTORC1) signalling (p70S6K1 and 4EBP1 phosphorylation), suggestive of increased protein synthesis. Finally, muscle fibers isolated from HMB-treated mdx mice showed improved mitochondrial efficiency that was associated with increased maximal respiration, spare respiratory capacity, and ATP synthesis. This study is the first to show HMB-induced improvements on in vitro and in vivo measures of mdx skeletal muscle force production that are coupled with improved mitochondrial function, suggesting that HMB may be a viable treatment option for DMD.NEW & NOTEWORTHY This is the first study to examine the effect of HMB in 3-wk-old mdx mice undergoing extensive muscle damage and regeneration both in vivo and in vitro. HMB treatment increased voluntary grip strength and holding impulse, while elevating force production of isolated mdx EDL muscles, which were associated with improved muscle mass, muscle fiber size, and succinate dehydrogenase activity. Finally, these improvements coincided with increased markers of mTORC1 signalling, mitochondrial respiration, and ATP production.

Skeletal muscle is a structurally organized and functionally diverse organ composed of heterogeneous myofiber types and supporting non-myocyte populations that act in concert to generate force, regulate metabolism, and maintain systemic homeostasis. Myopathies occur in many different diseases, but the mechanisms that drive these muscle pathologies are still largely unknown, partly because conventional approaches cannot link histopathological features to molecular states at single-fiber resolution. To address this challenge, we brought histopathology and spatial transcriptomics together by applying high-resolution Seq-Scope technology to a rodent model of mTORC1 hyperactivation, which produces diverse pathological alterations within individual myofibers. Cross-sections from extensor digitorum longus (EDL) and soleus (SOL), two muscles with distinct fiber-type compositions, were profiled to determine how transcriptome changes are linked to histopathological outcomes. Our analyses reveal that mTORC1 hyperactivation elicits distinct, fiber-type–dependent pathological programs. Type I and IIa fibers, abundant in SOL but scarce in EDL, were largely resistant to mTORC1-induced pathology, exhibiting only minimal morphological alterations and no fiber type-specific responses beyond those commonly observed throughout the tissue. In contrast, type IIx fibers, shared between both muscles, diverged into opposing fates: in SOL, they underwent abnormal enlargement driven by sustained growth signaling, cytoskeletal remodeling, and impaired proteostasis with defective autophagy; whereas in EDL, they developed basophilia characterized by lipid-supported respiration fueling excessive ribonucleotide synthesis and RNA accumulation. Within the same muscle, type IIb fibers displayed striking heterogeneity with discrete transcriptional states encompassing canonical stress responses, oxidative metabolic activation, and developmental reprogramming. In parallel, non-myocytic populations, including activated macrophages and fibroblasts, accumulated preferentially in SOL, forming a fibrotic microenvironment supporting inflammation, tissue remodeling and hypertrophy. Taken together, these findings reveal that sustained mTORC1 signaling disrupts muscle homeostasis through distinct metabolic and structural routes, directly linking histopathological phenotypes to their molecular states at single-fiber resolution.

Flexion tests are commonly used in equine locomotion examinations to identify underlying locomotor issues, yet their neuromuscular effects remain poorly understood. Response variability raises concerns about their clinical value in lameness assessments and pre-purchase evaluations. Primarily, to investigate the effect of full-limb flexion tests on static (flexed position) and dynamic (subsequent trot-up) muscle activity. Secondarily, to assess their effect on locomotion asymmetry during trotting. In vivo experiments. Sixteen warmblood horses were randomly assigned to one of four groups (n = 4) for surface electromyography of selected limbs. Forelimb region muscles included Splenius muscle, Triceps brachii caput longus muscle, Longissimus dorsi muscle, Extensor digitorum communis muscle, and Ulnaris lateralis muscle, while hindlimb region muscles included Gluteus medius muscle, Biceps femoris muscle, Semitendinosus muscle, Extensor digitorum longus muscle, and Flexor digitorum profundus muscle. Electromyographic data were side-normalised, and kinematic data from inertial measurement units assessed asymmetry in head, withers, and pelvis. Each limb underwent a 60-s full-limb flexion test, followed by straight-line trotting. Muscle activity in static and dynamic phases was compared to baseline using one-way repeated measures ANOVA, and two-way repeated measures ANOVA with statistical parametric mapping, respectively. Kinematic variables were compared using a linear mixed-effect model. No significant differences in normalised muscle activity were observed in fore- or hindlimb region muscles during flexion or subsequent trotting. However, pelvic displacement increased during the suspension phase of the ipsilateral limb, with significant vertical displacement (MaxDiff) after left (8.4 mm ± 2.4 SE [standard error], p = 0.002) and right (8.1 mm ± 2.4 SE, p = 0.004) hindlimb flexion test. Only full-limb flexion tests were investigated, limiting generalisability to distal or carpal/tarsal tests. Flexion force was not standardised. We found no evidence that flexion tests affect equine neuromuscular functioning in the muscles we investigated.