Children with cerebral palsy (CP) have muscle growth impairments (muscle contractures), altered walking patterns and show markers of inflammation. During muscle repair macrophages coordinate with muscle stem cells-satellite cells (MuSC), which have previously been shown to be altered in abundance and function in children with CP. We investigated: 1) if macrophage populations in contractured muscles of children with CP are similar to typically developing (TD) children with a chronic ACL-tear, and 2) if macrophages, capillaries, MuSC, myonuclei, centrally nucleated fibers were associated with each other, indicative of repair. Thirty-six subjects participated in this study (CP: 11.2 ±0.7 years, 18M/12F, TD: 13.5 ±0.8 years, 3M/3F). Muscle biopsies were obtained during surgical correction for muscle contractures-adductors/ gastrocnemius (CPCon), or vastus lateralis (TD-ACL and CP NonCon). Muscle cross-sections were immunohistochemically labeled for total, anti-inflammatory (M2) macrophages, capillaries, myofiber boundaries, while MuSC abundance, activation and proliferation information were used from a prior study. Macrophage subpopulations in CP Con were similar to TD-ACL muscles. Within CPCon there were positive associations between total, M1 macrophages, and MuSC content (r= 0.54, r=0.70, p<0.05, respectively), but not in the CP NonCon muscles. Centrally-nucleated fibers, myonuclear abundance and MuSC content were also positively associated with each other only in the CPCon muscles (r=0.65, r=0.46, r=0.66, p<0.05, respectively). In TD-ACL injured muscles similar associations were seen between macrophages and MuSC, central nucleation and myonuclear abundance. Collectively, our data suggest that contractured muscles in children with CP may be in a state of repair, similar to ACL-injured TD children.

To report a family with autosomal-dominant chronic progressive external ophthalmoplegia due to a novel truncating pathogenic variant in RRM2B and to show the challenges facing clinicians in diagnosing rare neuromuscular diseases. Four family members were examined. Muscle biopsy, mitochondrial DNA analysis, next generation sequencing, and targeted mitochondrial gene panel followed by Sanger sequencing and complementary deoxyribonucleic acid analysis were performed. A novel heterozygous RRM2B truncating variant c.968_972del p.(Phe323*) was identified. complementary deoxyribonucleic acid analysis showed expression of both RRM2B alleles. The proband was initially misdiagnosed as myasthenia gravis. Based on the phenotype and family history, chronic progressive external ophthalmoplegia was suspected and confirmed by finding of a novel RRM2B variant. The detection of another truncating pathogenic variant in exon 9 of RRM2B further supports this exon as mutation hot spot and underlines the role of the C-terminal highly conserved amino acids for the interaction of the p53R2 dimer with the R1 dimer.

Evaluating efficacy of clinical tools in determining causes of recurrent rhabdomyolysis.

The efficacy of oral administration of leucine and phenylalanine tracers to measure MyoPS (LEUMyoPS and PheMyoPS, respectively) in response to varying anabolic stimuli was investigated. Participants were randomized to a rested-fasted (FAST), rested-fed (FED), or exercise-fed (EXFED) condition. FED and EXFED consumed a mixed carbohydrate and AA beverage enriched with L-[1-13C]leucine (25%) and L-[ring-2H5]phenylalanine (30%) at rest or after a bout of resistance exercise, while FAST consumed only the equivalent tracer dose. Blood samples were obtained every 30 min for 300 min to determine tracer precursor enrichment with muscle biopsies obtained before and at 120 and 300 min after tracer ingestion to determine MyoPS by the precursor-product method. LEUMyoPS over 300 min was greater (p < 0.01) in EXFED (0.090 ± 0.024%/h; mean ± SD) and FED (0.067 ± 0.028%/h) compared to FAST (0.024 ± 0.015%/h). PHEMyoPS over 300 min was greater (p < 0.01) in EXFED (0.128 ± 0.034%/h) and FED (0.098 ± 0.020%/h) compared to FAST (0.056 ± 0.012%/h). There was a positive correlation (r = 0.81, p < 0.0001) between LEUMyoPS and. Oral essential amino acid tracer ingestion can be used as an alternative method to detect changes in MyoPS in response to mixed macronutrient feeding and feeding plus exercise and may be an option for the study of human muscle protein synthesis where intravenous isotope administration is logistically difficult or prohibitively expensive.

Integration of multiple omics reveals key targets and cellular mechanisms for intervention in sarcopenia.

The study aimed to investigate the effects of 5 weeks of post-exercise cold-water immersion (CWI) following high-intensity interval training (HIIT) sessions on the satellite cell pool, muscle content of inflammatory markers, muscle expression of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), maximal oxygen uptake (V̇O2max), and running performance. Sixteen healthy males completed baseline assessments, including muscle biopsies, a graded exercise test for V̇O2max determination, and a constant work-rate (CWR) running test to assess time to task failure (TTF). Participants were ranked according to V̇O2max and randomly allocated to either a training-only control group (n = 7) or a CWI group (n = 9), which underwent CWI (11.2°C ± 0.2°C for 15 min) following each HIIT session. The HIIT program consisted of three weekly sessions 5-8 × 2-min bouts at 95% V̇O2max. At the end of weeks four and five, all participants repeated the same sequence of assessments. Training increased V̇O2max values, TTF at CWR, satellite cell pool, PGC-1α content, and induced changes in muscle morphology (connective tissue), as indicated by a main effect of time (p ≤ 0.031); none of the analyzed variables showed a main effect of condition (p ≥ 0.098) or interaction (p ≥ 0.088). No significant alterations were observed in inflammatory markers over time (p ≥ 0.395) and condition (p ≥ 0.115). In conclusion, 5 weeks of post-exercise CWI following HIIT did not influence the satellite cell pool, muscle inflammation status, muscle PGC-1α content, muscle morphological, V̇O2max, or running performance.

PERM1 has been identified as a key regulator of muscle energy metabolism, contractile function, and mitochondrial biogenesis. To investigate the impact fasting and acute and chronic high-intensity exercise on p38MAPK, pCaMKII, PGC-1α, ERRα, and PERM1 and on PERM1 target genes (CKMT2, GLUT4, and SIRT3) in human skeletal muscle. We performed secondary analyses of muscle biopsy samples from two previously published studies, and from one unpublished study. Muscle biopsies were analyzed from the following protocols: 1) nine men pre-, during, and post- an 8 hour fast with or without two hours of arm ergometer exercise; 2) nine men and eight women pre- and 3 hours post- acute high-intensity interval cycling exercise (HIIE); and 3) eleven men and eight women pre- and post- a 6-week period of high-intensity interval training (HIIT) or non-exercise control. We used RT-PCR and western blotting to determine the mRNA and protein levels, respectively. Immunolabelling, microscopy, and subcellular fractionation were also performed to assess PERM1 cellular localization. Fasting did not induce detectable changes in the PERM1-related pathways. HIIE significantly increased p-p38MAPK (p<0.05, d=1.27) protein, and PERM1 (p<0.05, d=0.781) and PGC-1α (p<0.05, d=1.51) mRNA. Six weeks of HIIT increased the protein levels of PERM1 isoform 2 (p<0.05, ƞ2=0.168) and CKMT2 (p<0.05, ƞ2=0.226). PERM1 was localized in the perinuclear region and enriched in the mitochondria. Our results suggest that only some components of PERM1-related pathways are preserved in human skeletal muscle, highlighting the importance of future studies examining PERM1 function in humans.

This pilot clinical study aimed to develop and validate standardized manufacturing and quality control procedures for autologous skeletal muscle-derived (SkM-MSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs), and to explore the feasibility, safety and preliminary efficacy of periurethral injection of these products in women with stress urinary incontinence (SUI). Twenty-six diagnosed with SUI were enrolled and allocated to receive either SkM-MSCs or BM-MSCs. Autologous MSCs were isolated from skeletal muscle or bone marrow biopsies, expanded under Good Manufacturing Practices (GMP), and subjected to rigorous quality control assessments, including identity, genetic stability, viability, potency, and sterility. In this pilot study, ten million MSCs were injected periurethrally under local anesthesia, and participants were followed for 12 months post-treatment. Eleven SkM-MSCs and nine BM-MSCs final products met all quality criteria and were administered. One participant from the SkM-MSCs lost the follow-up. MSC therapies were well tolerated, with no long-term adverse effects or tumor formation observed. In the SkM-MSCs group, the proportion of women with a positive cough test decreased significantly from 100% to 40% (p = 0.010). In the BM-MSCs group, modest improvements were seen but did not reach statistical significance. Overall, improvements in both pad test outcomes and quality of life measures among participants were observed, though not uniformly significant. The study was discontinued before reaching its intended sample size due to limited efficacy, logistical challenges, and financial constraints. Autologous MSC‑based therapy for SUI was feasible and showed an acceptable short‑term safety profile in this pilot research setting; however, clinical efficacy remained modest. The manufacturing and quality control methodology is reproducible in specialized cell processing centers, but its application should remain confined to clinical research conducted in compliance with current regulations governing human cell therapy. Future studies with optimized cell products, refined delivery strategies and adequately powered, randomized designs are required before any potential translation to routine clinical practice can be considered.

Exploring the role of chemerin in skeletal muscle phenotype in those with kidney disease.

Muscle fiber type composition is a sports performance determinant, but its use in sports practice is hampered by the invasive muscle biopsy. We aimed to determine the best non-invasive method or combination of such methods to estimate muscle fiber type composition. We hypothesized that methods measuring different fiber type-specific characteristics, i.e., metabolism, power, contractility and endurance, would complement each other. Forty young, healthy and recreationally-to-competitively trained participants (20 men) underwent non-invasive tests and four muscle biopsies: two in the vastus lateralis and two in the gastrocnemius medialis. The average relative area occupied by type II fibers across the biopsies served as the criterion measure (multi-muscle FTarea%). On average, this was calculated based on the fiber type of ~5200 fibers and the cross-sectional area of ~1200 fibers per participant. Participants performed 30-m sprints, squat jumps, a Wingate with blood lactate measurements, isometric and isokinetic knee extensions, a maximal ramp incremental cycling exercise and underwent peripheral electrical stimulation of the quadriceps and a proton magnetic resonance spectroscopy scan to measure muscle carnosine. Multi-muscle FTarea% ranged from 21.2 to 60.1%. Many parameters were significantly associated with multi-muscle FTarea%, but post-Wingate blood lactate concentration showed the strongest correlation (r=0.67; p<0.001). A multiple linear regression model with sex, post-Wingate blood lactate, peak knee extension torque at 300°.s -1 scaled to upper leg lean mass, average vastus lateralis and soleus carnosine and singlet contraction time as predictors could explain 68% of the multi-muscle FTarea% variance, confirming our hypothesis. The proposed model which combines non-invasively measured parameters of metabolism, power and contractility can provide a reasonably accurate estimate of vastus lateralis and gastrocnemius medialis fiber type composition.

Evaluation of the immunohistochemical and histological differences of the human cremaster muscle in retractile and undescended testis.

Critical illness myopathy (CIM) is a prevalent cause of intensive care unit-acquired weakness (ICUAW). It is associated with prolonged mechanical ventilation and increased mortality and frequently evolves into postintensive care syndrome with long-term cognitive and physical impairments. Although several molecular mechanisms have been implicated in CIM, including muscle atrophy and altered contractility, its exact etiopathogenesis is not fully understood. Evidence suggests that activation of the NLRP3 inflammasome and upregulation of atrogenes significantly contribute to this condition. In murine models of sepsis and denervation, which mimic CIM, NLRP3 activation induces muscle atrophy. Physical therapy, including enhanced mobilization supported by servo-assisted devices, a nonpharmacological intervention, can regulate inflammation, reduce IL-1β and NLRP3 levels, and improve mitochondrial and autophagy processes; however, whether it reduces NLRP3 activation and alleviates atrophy in human CIM remains uncertain. We will conduct a prospective, randomized, controlled, open-label study during the early ICU stay. Adults at risk of CIM (n = 16) will be randomized 1:1 to conventional physical therapy or to conventional therapy plus enhanced PT (an additional servo-assisted mobilization device, MOTOmed letto®) twice daily for 60min over seven consecutive days. An additional ICU control group without CIM (n = 8) will be included for biomarker analyses. At baseline and on day 7, blood and vastus lateralis muscle biopsies will be obtained for histologic, molecular, and transcriptomic analyses. The primary outcomes are as follows: (1) NLRP3 inflammasome priming and activity (protein and gene expression by Western blot, RT-qPCR, and ELISA) and (2) indices of muscle atrophy (fiber diameter, atrogenes expression, the myosin/actin ratio, and sarcomere organization by immunofluorescence and transmission electron microscopy). The secondary outcomes include strength and function (MRC-SS, FSS-ICU), feasibility and safety, microarray-based transcriptomic profiling, and associations between biochemical/molecular markers and CIM diagnosis by clinical assessment and ultrasound. We hypothesize that enhanced PT (in addition to conventional care) reduces NLRP3 activation and muscle atrophy in critically ill adults. This translational trial combines clinical assessments integrating muscle biopsies and blood measures to elucidate CIM pathogenesis and assess PT intervention. These findings may identify therapeutic targets and inform early mobilization strategies in ICU practice. ClinicalTrials.gov NCT07017517. Registered on 26 May 2025.

Age-related muscle mass is driven by a reduction in insulin sensitivity partly mediated by reduced amino acid and anabolic signalling kinetics. Insulin activates Akt-mTORC1 signalling in skeletal muscle, with inositol hexakisphosphate kinase 1 (IP6K1) shown to inhibit this signalling pathway in pre-diabetic humans. We aimed to compare muscle and plasma IP6K1 in young vs older adults and the possible role of IP6K1 in the anabolic response to protein and protein plus resistance exercise (RE). Nine young (24.9 ± 0.4years) and nine older (66.2 ± 0.5years), moderately active adults received primed continuous infusions of L-[ring-2H5]phenylalanine in basal and postprandial state. Blood and muscle biopsy samples were collected prior to and following ingestion of 25g whey protein with or without knee extension exercise to examine skeletal muscle protein signalling and whole-body phenylalanine kinetics. Young adults had greater plasma IP6K1 at all time points. Older adults had reduced muscle IP6K1 at 120min post-exercise. Muscle IP6K1 decreased 240min postprandially in young adults compared with basal and there was no effect of exercise in either group. Older adults presented with reduced plasma and muscle IP6K1 in both postprandially and post-RE states, as well as reduced phenylalanine rate of disappearance for the same comparisons. IP6K1 may be involved in the reduction in amino acid metabolism, and the insulin-mediated response to protein and RE.

Introduction: Physical training stimulates mitochondrial biogenesis and protein synthesis in skeletal muscle. If the training load is too demanding and prolonged, an overtraining syndrome may develop. Reported overtraining symptoms include recurrent respiratory infections, sleep disturbances, with potential alterations at the skeletal muscle level such as soreness, and atrophy. The aim of the present study was to investigate skeletal muscle (mal)adaptations in response to overtraining, using a translational approach combining human data and an in vitro model. Methods: Electrical stimulation was applied to C2C12 myotubes to mimic endurance exercise. For the simulated training (s-T), stimulation was applied once daily for three days. In the simulated overtraining (s-OT), stimulation was applied three times daily for three days. Proteomic analysis, mitochondrial respiration and imaging were performed in the cellular model. In humans, knee extensor neuromuscular function at rest and in response to a standardized exercise was assessed in overtrained vs. well trained athletes. Muscle biopsies were also collected to assess mitochondrial respiration. Results: In s-T, we observed beneficial adaptations, including myotube hypertrophy, increased expression of myosin heavy chain proteins, and enhanced mitochondrial respiration. In contrast, s-OT showed myotube atrophy, reduced myosin heavy chain content, and impaired mitochondrial respiration. Consistent with these respiratory findings, proteomics analysis revealed a decreased abundance of proteins involved in the mitochondrial respiratory chain in s-OT. Interestingly, mitochondria appeared aggregated in s-OT myotubes, suggesting possible impairments in mitochondrial quality control. Live-cell imaging revealed possible autophagy alterations in the s-OT condition but not in s-T. Human data - neuromuscular function and muscle samples - are being processed and results will be presented during the conference. Conclusions: Our in vitro model provides a molecular platform for further investigations of skeletal muscle maladaptations to overtraining. The integrative approach will help to clarify the molecular mechanisms and ameliorate the clinics of muscle maladaptations triggered by excessive exercise in humans.

Autoantibody internalization has been implicated in autoimmune disease pathogenesis, yet its mechanisms, and generality across different diseases, cell types, and affected tissues remain poorly defined. Using bulk RNA sequencing, we identified reproducible, autoantibody-specific transcriptomic signatures consistent with autoantigen dysfunction in muscle biopsies from patients with anti-Mi2 dermatomyositis and anti-PM/Scl scleromyositis across independent cohorts. Electroporation of purified patient IgG into primary cultures of healthy cells was sufficient to induce the corresponding transcriptomic programs in vitro . Direct immunofluorescence demonstrated immunoglobulin internalization into subcellular compartments matching the localization of the autoantigen in different affected tissues. Spatial transcriptomic analyses revealed that antibody-secreting cells translocated cytoplasmic material (i.e., immunoglobulin RNA) into adjacent affected cells expressing autoantibody-specific transcripts. The disease-specific transcripts were present not only in muscle fibers, but also in other cells, including macrophages, endothelial cells, and fibroblasts. Autoantibody-induced transcriptomic programs were associated with cell damage and autoantibody-specific reactive inflammatory programs, including activation of type I interferon and TGF-ß1 signaling in anti-Mi2 dermatomyositis and activation of type II interferon in anti-PM/Scl scleromyositis. Antibody internalization was also observed in different tissues from patients with other autoimmune diseases, including anti-U1RNP mixed connective tissue disease, anti-Ku overlap syndrome, and anti-Scl70 systemic sclerosis. Together, these findings establish autoantibody internalization as a shared pathogenic mechanism across diverse autoimmune diseases, providing a unifying framework for conditions driven by autoantibodies against intracellular antigens.

Skeletal muscle metabolic and physical capacities are influenced by both genetics and load status and decline with age. Recent advances in sequencing have detailed cell types at unprecedented detail; yet these approaches do not scale to adequately model human muscle physiological heterogeneity. We produced a powerful resource for ageing studies, including consistent deep transcriptomic profiles of 1,675 human muscle biopsies (∼28,000 genes per profile) and multiple single-cell spatial transcriptomic technologies. We present several novel models of tissue ageing. Five Quantitative network models (QNMs), built using >40 trillion calculations and 930 human muscle transcriptomes, modelled aging and the influence of load status. Additional differential expression (DE) signatures for atrophy, hypertrophy and cardio-respiratory adaptation were integrated with single-cell RNAseq and cell-specific bulk profiles to reveal cell-enriched modules and the topology of human skeletal aging. Rapamycin transcriptomes from cultured muscle and endothelial cells, along with in vivo signatures for insulin resistance and sex, were integrated into these analyses. We show that >3,000 genes are DE with muscle age (equally up and down); that a novel pre-frailty signature in elderly subjects has a remarkably strong overlap with the response of healthy muscle during experimental atrophy and that the hypertrophy signature in elderly muscle, but not young muscle, opposes the age-regulated transcriptome. We report that non-responders for hypertrophy or gains in cardio-respiratory capacity have highly distinct genome-level response to exercise. QNM revealed cell-specific processes in endothelial cells and fibroblasts, including novel interactions between insulin sensitivity, age and senescence. From two hundred and eighty-six hub genes consistent in both young and old muscle network models, 27% had known roles in muscle biology, while of the top 50 hub genes (45% protein coding), 80% were newly linked to human muscle biology, including ARHGAP4, CEP131 and IFITM10 and many short- and long-noncoding RNAs. Many genes demonstrated extreme changes in topology in old muscle, such as the neddylation and aging linked gene, DCUN1D5. GeoMX-based spatial muscle fibre-type profiling (57 regions), along with Xenium (8 regions) and Merscope (54 regions) single-cell spatial technologies located key aging, frailty and load-responsive genes to individual cell types and provided novel insight into the location of autocrine/paracrine secreted factors such as GDNF, while IL6 was located to rare endothelial cells. A machine-learning model ranked the factors most associated with the topological changes with age. This prioritised network features over DE signatures, highlighting positive correlating edges to down-regulated genes during atrophy, genes up-regulated by Rapamycin and both positive and negative correlating insulin sensitivity features, along with gene hub status, best explained muscle ageing. Genome level modelling produced an independently validated transcriptomic 'age clock' and found it to be invariant to muscle load status in people >50y, while we revealed novel interactions between gene length and age. Release of an unprecedented level of consistently aligned genomic data, along with QNMs with >7,000 searchable modules, provides a powerful resource for the aging research communities.

Background: Blood flow restriction (BFR) allows exercise at lower external load with similar or greater improvements compared with traditional training in non-trained individuals. However, the effect in well-trained competitive athletes is unclear. The aim of this study was to compare effort-matched high-intensity interval training (HIIT) microcycles performed with or without BFR on endurance performance and muscular adaptations in well-trained cyclists. Methods: 17 well-trained cyclists (31 ± 9 years; VO2max: 67 ± 6ml×kg-1×min-1) were randomized to groups performing five HIIT sessions (6 x 5 min intervals with 2.5 min of recovery) with (BFR) or without (HIIT) thigh cuffs occluding the legs. VO2max, power output at 4 mmoL/L blood lactate (LT4), mean power output during 5-minute maximal cycling (MPO5min), percentage of VO2max used at LT4 (%VO2max @LT4), haemoglobin mass and blood volume (BV) were assessed. Muscle biopsies from m. vastus lateralis evaluated muscle cross-sectional area (CSA), capillaries, citrate synthase (CS), Hydroxyacyl-Coenzyme A dehydrogenase (HADH) and cytochrome c oxidase 4 (COX4). Results: The BFR group trained at a 42% lower power output than HIIT group (177±3 W vs 307±8 W, respectively, p<0.01), but with no differences in heart rate or rate of perceived exertion. Both groups improved MPO5min by ⁓4%, with no changes in LT4, VO2max, haemoglobin mass and BV. HIIT showed a significant reduction in CSA for type 2 muscle fibers compared with BFR, whereas no changes were found in the other muscle analyses. Conclusions: BFR applied during a 6-day interval microcycle provides similar performance gains as traditional HIIT in well-trained cyclists.

Calsequestrin-1 (CASQ1)-related myopathy is a rare skeletal muscle disorder caused by mutations in CASQ1 gene, which encodes a major calcium-buffering protein of the sarcoplasmic reticulum (SR). It is characterized histopathologically by tubular aggregates or optically empty vacuoles, predominantly affecting type II muscle fibers. In this study, we report two unrelated Chinese patients presenting with late-onset, slowly progressive muscle weakness, fatigue, and myalgia. Both had mildly to moderately elevated serum creatine kinase levels. Muscle biopsies revealed typical optically empty vacuoles primarily in type II fibers. Whole-exome sequencing identified an identical heterozygous CASQ1 variant, c.730G > C (p.Asp244His), located at a highly conserved residue. In vitro expression of the mutant CASQ1 in HeLa cells confirmed its aggregation tendency, suggesting impaired protein folding or calcium handling. Immunofluorescence revealed abnormal aggregation of CASQ1 protein around the edge of vacuoles, co-localized with SQSTM1/p62, and the endoplasmic reticulum (ER) stress marker PERK. Our findings support the pathogenic role of the p.Asp244His variant and provide further insights into CASQ1-related myopathy in Asian populations.

To investigate the mechanisms governing energy and redox balance in skeletal muscle, we developed a computational model describing the coupled biochemical reaction network of glycolysis and mitochondrial oxidative phosphorylation (OxPhos) in fast-twitch oxidative glycolytic (FOG) muscle fibres. The model was identified against dynamic in vivo recordings of phosphocreatine (PCr), inorganic phosphate (Pi) and pH in rodent hindlimb muscle and verified against independent data from in vivo experiments and muscle biopsies. Step response testing reveals that mass action kinetics in combination with feedback control are sufficient to accomplish myoplasmic ATP homeostasis over a 100-fold range of ATP turnover rates. This vital emergent property of the metabolic model is associated with intermediary metabolite dynamics typical of a second-order underdamped system, which has been previously reported for the glycolytic pathway. Lactate dehydrogenase (LDH) knockout simulations suggest that the contribution of the LDH reaction to redox balance is more fundamental to muscle function than its role in counteracting myoplasmic acidification across the physiological range of ATP demands in this myofibre phenotype. Furthermore, LDH knockout simulations confirm that mitochondrial uptake of myoplasmic NADH and H+ in and by itself is sufficient to maintain redox balance and proton balance over ATP turnover rates in the range of mitochondrial ATP synthesis. We conclude that aerobic lactate production in working muscles is a by-product of the metabolic flexibility of FOG myofibres afforded by expression of high levels of LDH and OxPhos enzymes to support continual myoplasmic redox balance and ATP synthesis under conditions of high-intensity mechanicalwork. KEY POINTS: Feedback regulation suffices to accomplish myoplasmic ATP homeostasis over a 100-fold range of ATP turnover. Second-order underdamped behaviour is predicted to arise as a generic trait of the ATP metabolic network in mammalian cells. Aerobic lactate is a by-product of the metabolic and functional flexibility. LDH's role in maintaining redox balance is more important than its role in counteracting cellular acidification.

Tubulinopathies encompass a spectrum of disorders resulting from variants in genes encoding α- and β-tubulins, the key components of microtubules. While previous studies have linked de novo or dominantly inherited TUBA4A missense variants to neurodegenerative phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, spastic ataxia, and recently, an isolated congenital myopathy, the full phenotypic and genotypic spectrum of TUBA4A-related disorders remains incompletely characterised. In this multi-centre study, we identified one previously reported and 12 novel TUBA4A missense variants in 31 individuals from 19 unrelated families. Remarkably, individuals in 17 families presented with a myopathy without any CNS involvement or history of such disease. In the remaining two families, we observed probands with cerebellar ataxia and epilepsy accompanying proximal and axial muscle weakness along with protein aggregation. The coexistence of neuromuscular and neurodegenerative features with protein aggregation defines a multisystem proteinopathy. These two families thus establish the first association between TUBA4A and multisystem proteinopathy. Our cohort exhibited diverse genotypes and inheritance patterns: four families demonstrated autosomal dominant transmission through heterozygous variants in TUBA4A, three probands had recessive inheritance due to homozygous variants, while the respective heterozygous carriers were asymptomatic; five probands carried de novo variants, and nine probands with heterozygous variants were classified as sporadic cases. Clinical phenotypes ranged from mild to severe myopathy, predominantly affecting the axial and paraspinal muscles. We observed a range of disease onset, from congenital to late adulthood. Creatine kinase levels were variable, ranging from normal to highly elevated. Cardiac function remained preserved across the cohort. Muscle biopsies showed heterogenous myopathic changes, including myofibre size variation, nemaline bodies, core-like regions, and internal nuclei. Immunohistochemical analysis revealed protein accumulations positive for TDP-43 (n=2), p62 (n=5), and TUBA4A (n=6). Complementary in silico and in vitro investigations suggested that the identified TUBA4A variants cause significant protein abnormalities and may differentially impact microtubule dynamics. Correlation analyses integrating clinical severity, variant location, and mechanistic readouts further demonstrated that domain specificity within TUBA4A influences both the pattern of muscle involvement and the extent of microtubule disruption. Our findings establish myo-tubulinopathies as distinct clinical entities, encompassing both primary myopathies and multisystem proteinopathies with muscle involvement. This study broadens the phenotypic and genotypic spectrum of TUBA4A-related disorders beyond autosomal dominant or de novo mechanisms and neurodegenerative presentations. These results underscore the importance of considering TUBA4A variants in the differential diagnosis of axial myopathies and multisystem proteinopathies, regardless of central nervous system (CNS) involvement.