Skeletal Muscle

Nemaline myopathy is a severe inherited skeletal muscle disorder characterized by profound muscle weakness, impaired mobility, and early mortality in severe cases. Despite the devastating clinical burden, no disease-modifying therapies are currently available. Nemaline myopathy-causing genes either encode sarcomeric structural proteins or regulators of sarcomeric protein stability, resulting in primary defects in sarcomere structure and function. Recessive mutations in KLHL40 cause one of the most severe forms of nemaline myopathy, frequently resulting in perinatal lethality. Given its loss-of-function mechanism, KLHL40-associated nemaline myopathy is an attractive candidate for gene replacement therapy. We therefore evaluated the therapeutic potential of systemic AAV9-mediated KLHL40 gene replacement in a mouse model of severe nemaline myopathy. A single systemic administration of an AAV9 vector encoding human KLHL40 during early disease produced durable rescue of survival, skeletal muscle pathology, motor performance, and contractile function in KLHL40-deficient mice. Therapeutic benefit was accompanied by restoration of sarcomere organization and normalization of myofiber size without detectable long-term histopathological abnormalities in major organs. Collectively, our findings provide a strong preclinical foundation for developing AAV9-mediated KLHL40 gene replacement as a treatment for severe KLHL40-associated nemaline myopathy.

Previously exercise trained skeletal muscle is more growth-responsive to retraining. Using a murine training-detraining-retraining approach and multi-omics, we find that Pecam1 gene expression is lower, but capillarization is appreciably higher in previously trained (preconditioned) relative to naive trained control muscle. Greater capillarity could be permissive for accelerated hypertrophic adaptation. Exercise preconditioned myonuclei feature differential promoter CpG regulation in genes related to Wnt signaling. These epigenetic alterations with retraining align with our prior observations of methylation changes within the same pathway after a longer period of chronic training, suggesting a more rapid response due to preconditioning. Methylome-transcriptome integration and single myonucleus RNA-sequencing expose the polyamine metabolism enzyme Smox as a target that relates to heightened hypertrophic adaptability with retraining. Smox induction is sufficient to cause hypertrophy in aligned myotubes cultured on a stiffness-tuned substrate along with a growth-supportive transcriptional program. Integration of our multi-omics data suggests that Smox regulates repression of Ddit4/Redd1 (an inhibitor of mTORC1 signaling) after retraining. Smox may govern a favorable muscle fiber growth environment in previously trained muscle by sensitizing anabolic potential through polyamine metabolism. A lower adaptive threshold mediated by Smox could contribute to myometaplasticity, or a change to how subsequent muscle adaptations are made.

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion in the 3' untranslated region of the DMPK gene that results in the expression of expanded CUG RNA which trap splicing factors resulting in widespread alternative splicing dysregulation. A panel of these splicing events, or splicing index (SI), is currently used to measure splicing dysregulation in DM1 patients. Despite significant interest in splicing, little is known about its progression over time in DM1. The objective of this study was to evaluate the changes in SI over 3 years in a well-phenotyped cohort of DM1 participants and identify factors contributing to these changes. RNA was extracted from vastus lateralis (VL) biopsies of 20 DM1 participants taken 3 years apart and the SI score was measured using targeted RNA sequencing. Baseline SI scores correlated with percent predicted muscle strength of the ankle dorsiflexors, knee extensors and hand grip. On average, SI scores worsened over 3 years, but the progression varied markedly among participants. Adult/juvenile phenotypes and lower baseline SI scores were among the factors associated with higher progression of SI scores over time. These findings emphasize the importance of integrating clinical phenotype and baseline molecular severity when interpreting longitudinal SI change, particularly in the context of clinical trials.

Nutrients and oxygen are sensed within the muscle to control growth and disruption of either signal is sufficient to lead to muscle atrophy. While nutrient limitation is sensed via a conserved transcriptional atrophy program (commonly referred to as atrogenes) dictated via the Forkhead box O (FoxO) transcription factors, how low oxygen promotes muscle loss remains unknown. Accordingly, the downstream mechanisms that initiate muscle loss when oxygen and nutrients are limiting are only partly understood. Here, we find Hypoxia Inducible Factor (HIF), the master regulator of our adaptation to low oxygen, is necessary and sufficient to mediate muscle loss under hypoxia in mice. RNA sequencing in skeletal muscle isolated from starved or hypoxic mice identifies Decidual Protein Induced by Progesterone 1 (Depp1), which is induced in skeletal muscle when nutrients or oxygen is limiting via FoxO1 and HIF activation, respectively. Whole body Depp1 loss in mice reduces muscle loss under fasting and hypoxia and skeletal muscle Depp1 overexpression is sufficient to mediate muscle atrophy. Mechanistically, Depp1 localizes to the mitochondria and is necessary to control autophagy activation and mitochondrial degradation in skeletal muscle. Taken together, our studies nominate Depp1 as a new atrogene necessary for muscle loss under multiple atrophy scenarios involving FoxO and HIF.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multisystem disorder. Its hallmark feature, post-exertional malaise (PEM), is a delayed and prolonged worsening of symptoms following even minor exertion. As PEM commonly includes muscle pain, weakness, exercise intolerance and impaired recovery, skeletal muscle dysfunction may contribute to its pathophysiology, but the underlying molecular processes remain poorly understood. We generated induced pluripotent stem cell-derived skeletal muscle myotubes from individuals with ME/CFS (n = 6) and healthy controls (n = 7) to investigate disease-associated transcriptomic alterations and identify potential therapeutic candidates. Myogenic progenitor cells from both groups differentiated into elongated, multinucleated myotubes expressing titin and myosin heavy chain, while qPCR profiling of myogenic differentiation markers further supported successful differentiation. Gene-level analysis identified three differentially expressed genes at an FDR-adjusted p < 0.05: ERVH48-1, CDX4 and SLC2A14. Isoform-switching analysis identified 16 genes with significant differential isoform usage, including CACNA1H, MIEF2, SMAD2 and CHD4. Gene set enrichment analysis identified broader pathway-level alterations involving RNA processing, protein synthesis, proteostasis, mitochondrial quality control, metabolism and muscle-related processes. Library of Integrated Network-based Cellular Signatures Phase 2 (LINCS2) analysis further prioritised 14 compounds associated with mitochondrial metabolism, glucose and lipid regulation, PPAR signalling, calcium and excitatory signalling, neuroimmune activity and cellular stress responses. Overall, ME/CFS iPSC-derived myotubes showed modest gene-level differences alongside broader isoform- and pathway-level alterations, supporting their use for investigating skeletal muscle biology and prioritising candidates for functional validation.

Individuals with fibrodysplasia ossificans progressiva (FOP), a rare genetic disorder caused by mutations in the bone morphogenetic protein receptor ACVR1 (also known as ALK2), experience progressive and severely debilitating endochondral heterotopic ossification (HO). Previous studies have identified fibro-adipogenic progenitors (FAPs) as a major cellular source of HO in FOP mouse models. Here, we used mRNA sequencing of FAPs isolated from injured muscle of FOP mice to capture the earliest gene expression changes associated with the transformation of muscle tissues into bone. Whereas the transcriptomes of mutant and wild-type FAPs were indistinguishable prior to injury and at 1 day post injury, dramatic dysregulation of gene expression was observed at 3 and 5 days after injury. Surprisingly, gene expression changes suggested early specification of osteochondral lineages by 3 days post injury, approximately 3 to 4 days before the appearance of histologically identifiable cartilage. We identified dysregulation of genes encoding components of major signaling pathways, cell cycle regulators, extracellular matrix components, and hypoxia response mediators, and showed that these gene expression changes are temporally associated with abnormal population growth of mutant FAPs. We further demonstrated that treatment of FOP mice with either an anti-activin A monoclonal antibody or the ACVR1 kinase inhibitor BLU-782 (IPN60130; fidrisertib) normalized the expression of the majority of dysregulated FAP genes. However, the relative efficacy of the drugs differed, and distinct sets of overlapping genes were normalized by the treatments. The substantial transcriptional changes caused by these drugs in FAPs from normally regenerating muscle were also enumerated and described. These findings define early transcriptional events associated with pathogenic FAP reprogramming and provide a molecular framework for understanding the shared and distinct effects of activin A and ACVR1 inhibition.

Duchenne muscular dystrophy (DMD) is an X-linked disorder mainly affecting young boys characterized by repeated cycles of myofiber injury/regeneration and chronic muscle inflammation. Although glucocorticoids can slow disease progression, their potential long-term adverse effects highlight the need for new steroid-sparing therapies for DMD. Annexin A1 (ANXA1) is a glucocorticoid-induced pro-resolving protein that signals primarily through formyl peptide receptor 2 (FPR2). We investigated whether the ANXA1 mimetic peptide Ac2-26 could reduce inflammation, stimulate tissue regeneration, and improve skeletal muscle function in muscular dystrophy. 3-week-old dystrophin-deficient male mdx mice were randomized to receive daily intraperitoneal injections of vehicle control, deflazacort (1 mg/kg/day), or Ac2-26 (1 mg/kg/day) for a duration of 5-weeks. Body composition, skeletal muscle mass, contractile function, and tissue histopathology/immune-cell infiltration were assessed at 8-weeks of age. Direct effects on myogenesis were also examined in vitro using primary D2-mdx myoblasts. ANXA1 was expressed predominantly by myeloid cells rather than resident dystrophic muscle cells. Both deflazacort and Ac2-26 markedly reduced intramuscular inflammation and increased myofiber size. However, Ac2-26 produced broader anabolic and functional benefits, increasing body-weight gain, lean body mass, hindlimb muscle weights, and absolute contractile force. Ac2-26 treatment also directly promoted myogenesis of dystrophic myoblasts in vitro, while pharmacological FPR2 blockade impaired myotube development. Overall, Ac2-26 mimicked the anti-inflammatory effects of deflazacort while providing superior benefits for body weight and composition, skeletal muscle growth, myofiber regeneration, and absolute muscle strength. These findings support therapeutic targeting of the ANXA1-FPR2 axis as a potential safe and effective steroid-sparing immunomodulatory strategy for DMD.

Loss-of-function variants in SNUPN, encoding the nuclear import factor Snurportin-1 (SPN1) required for spliceosomal small nuclear ribonucleoprotein (snRNP) transport, cause a recently described form of limb-girdle muscular dystrophy (LGMD). However, the role of SPN1 in skeletal muscle homeostasis remains poorly understood, in part due to the lack of a suitable in vivo model. Here, we generated a zebrafish snupn loss-of-function model that recapitulates key features of the skeletal muscle phenotype observed in patients. Mutant larvae developed severe locomotor impairment by 6 days post-fertilization (dpf), accompanied by sarcomeric disorganization and impaired muscle fiber integrity. Transcriptomic profiling at 6 dpf revealed widespread alternative splicing and transcriptional dysregulation, with prominent alterations in extracellular matrix and basement membrane components, together with upregulation of stress- and inflammation-associated genes. Notably, these late-stage abnormalities were preceded by disruption of the muscle progenitor population at 2 dpf, with reduced Pax7 progenitor abundance and myogenic gene expression together with altered muscle differentiation and organization. Together, these findings identify SPN1 as a key regulator of skeletal muscle homeostasis linking RNA processing to extracellular niche integrity and myogenic progenitor maintenance. This zebrafish model provides an in vivo platform for dissecting LGMD-associated disease mechanisms and developing therapeutic strategies aimed at restoring muscle function and regenerative capacity.

Background Muscle wasting, systemic inflammation, and functional decline are highly prevalent and detrimental in patients with advanced-stage non small cell lung cancer (NSCLC). Methods: In this cross sectional study, we investigated NSCLC associated muscle remodeling by analyzing skeletal muscle biopsies from patients with NSCLC (n = 18) and matched controls (n = 18) using quantitative proteomics, histology, fluorescence-activated cell sorting, gene expression profiling, and high resolution respirometry. Findings: NSCLC muscle was characterized by type II muscle fiber atrophy, greater collagen deposition, and redistribution of lipids to the extracellular matrix (ECM), together with remodeling of the inflammatory, immune, ECM and mitochondrial proteome. Additionally, mitochondrial respiratory capacity and morphology were altered in patients with NSCLC, which was associated with increased oxidative stress and dysregulated calcium handling. Concomitantly, we detected STAT3 activation and immune cell alterations, which may negatively impact skeletal muscle health in patients with NSCLC. Finally, we identified a shift in fibro-adipogenic progenitors (FAPs), favoring the CD90 subtype. Mechanistically, conditioned media from patient-derived FAPs reduced myotube width in vitro, uncovering a novel mechanism by which altered paracrine signaling from the muscle resident stromal compartment drives atrophy in cancer cachexia. Interpretation: These findings provide human evidence that altered FAP composition, mitochondrial homeostasis, calcium handling, and immune cell landscape accompany muscle wasting in NSCLC, which may inform therapeutic strategies to preserve skeletal muscle health in patients with cancer.

Store-operated calcium entry (SOCE) is a ubiquitous calcium (Ca2+) signalling mechanism that replenishes endoplasmic and sarcoplasmic reticulum Ca2+ stores. We previously identified STIM1L, a skeletal muscle-enriched STIM1 splice variant that enables rapid SOCE activation in human myotubes. To determine its physiological role in vivo, we generated a constitutive knockout mouse model that selectively abolishes STIM1L expression while preserving the canonical STIM1 isoform. Stim1l-KO mice developed normally and exhibited no overt abnormalities, but both sexes showed reduced voluntary wheel-running activity, suggesting neuromuscular perturbation. Skeletal muscle deprived of STIM1L did not show any histological alterations or changes in fibre- type and size. Likewise, force-frequency relationships and fatigue resistance measured ex vivo in isolated extensor digitorum longus and soleus muscles were indistinguishable from control muscles. This lack of difference may be explained by the increased expression of STIM1 in several skeletal muscles and suggests that changes in the voluntary running activity of Stim1l-KO mice may arise from STIM1L loss in non-muscular tissues, including the central nervous system. Notwithstanding, primary myotubes derived from Stim1l-KO mice displayed a marked delay in SOCE activation kinetics, whereas SOCE amplitude remained comparable to controls. This was consistent with a compensatory STIM1 upregulation maintaining overall Ca2+ entry capacity. Interestingly, electrically evoked Ca2+ transients in intact Stim1l-KO flexor digitorum brevis fibres exhibited significantly faster decay kinetics than controls. This revealed another role of STIM1L in Ca2+ handling in skeletal muscle that appears independent of its contribution to SOCE activation. Together, these findings establish STIM1L as a regulator of SOCE activation kinetics and indicate an additional role in myofibre Ca2+ clearance, while suggesting that compensatory upregulation of canonical STIM1 may preserve muscle structure and contractile function in its absence in mice.

Aged skeletal muscle has decreased ability to rebound from physiological stressors such as disuse atrophy. We previously observed that older adults during recovery following muscle disuse were characterized by rapid skeletal muscle immune cell expansion, cellular senescence and collagen deposition, responses that were biased toward older women. Here we used single-nucleus RNA sequencing and complementary in vitro muscle primary cell experiments in young (YF; 22[plusmn]3y; n=8) and older females (OF; 66[plusmn]5y; n=9) to investigate age-mediated cellular function and intercellular communication during recovery from disuse atrophy. Compared with YF, OF exhibited a markedly greater transcriptional response at 7d-recovery (YF: 1,548 DEGs, OF: 7,999 DEGs), driven primarily by slow- (YF: 96 DEGs, OF: 984 DEGs) and fast-twitch myonuclei (YF: 194 DEGs, OF: 1,234 DEGs), satellite cells (YF: 548 DEGs, OF: 2,784 DEGs) and FAPs (YF: 326 DEGs, OF: 2,102 DEGs). Satellite cells from OF demonstrated collagen signatures including elevated THBS1 expression and enrichment of TGF-[beta] signaling. Concurrently, OF FAPs exhibited increased expression of fibroblast activation marker ADAMTS14. Furthermore, NicheNet analyses unmasked altered FAP-satellite cell communication in OF. Complementary In vitro experiments revealed that myogenic progenitor cells collected at 7d-recovery from OF (vs YF) donors displayed impaired myogenic differentiation and a cellular senescence-associated phenotype, while fibroblasts exhibited greater myofibroblast-like activation. Conditioned media derived from OF fibroblasts at 7d-recovery further increased cellular senescence and impaired myogenic potential (vs YF fibroblast conditioned media). Collectively, these findings suggest that recovery from disuse atrophy in older females is characterized by altered FAP-satellite cell communication and intrinsic function which may contribute to poor muscle remodeling.

Background: Insufficient oxygen (hypoxia) or complete lack of oxygen (anoxia) causes cellular dysfunction and death in terrestrial animals. As a result, animals have developed homeostatic strategies, including changes in movement strategies to escape and survive stressful environments. Caenorhabditis elegans respond to changes in oxygen by increasing the frequency of reversals (backward movement) and increasing locomotor speed; these behaviors enable them to escape unfavorable environments and move to more suitable ones. The molecular and neural mechanisms underlying behavioral responses to low-oxygen environments are incomplete. Neuropeptides regulate C. elegans behaviors by modifying neural circuit function in response to hypoxia. Loss of function in C. elegans neuropeptide nlp-40 and its receptor aex-2 increased survival to anoxia [1]. However, the cells in which NLP-40 and AEX-2 function for anoxia, as well as the mechanisms underlying responses to anoxia, remain elusive. We hypothesized that NLP-40 and AEX-2 regulate anoxia by altering escape behaviors. Results: We found that neuropeptide NLP-40 functions in cholinergic neurons and AEX-2 functions in cholinergic or GLR-1 interneurons or motor neurons to regulate susceptibility to anoxia. We found that loss of either nlp-40 or aex-2 decreased locomotor activity and reversal frequency early during 48 hours of anoxia, suggesting that nlp-40 and aex-2 are required for the proper execution of these behaviors. We found that neuronal knockdown of nlp-40 recapitulated the behaviors observed in nlp-40 mutant animals and increased survival; however, restoring aex-2 function in glr-1 interneurons or motor neurons was sufficient to rescue behaviors to wild-type and restore vulnerability to anoxia. Conclusions: Overall, our results suggest that NLP-40 and AEX-2 likely function in cholinergic or glr-1 interneurons or motor neurons to regulate escape behaviors during anoxia. Escape behaviors are part of the homeostatic response and are typically protective in the wild; however, in our paradigm, we show that loss of either nlp-40 or aex-2 reduces escape responses and confers protection against 48 hours of anoxia. Collectively, these results highlight a new role for NLP-40 and AEX-2 in anoxia and shed light on mechanisms underlying the adaptive responses to anoxia.

Longitudinal muscle growth occurs through the addition of sarcomeres in series, a process termed serial sarcomerogenesis. To investigate contraction mode dependence of sarcomerogenesis in Sprague-Dawley rats (n=36, 14 to18 weeks, ~468g), we compared the effects of isometric, eccentric, and concentric training (4 to 6 weeks; 3 times/week) on morphological and mechanical adaptations of the plantar flexors. Isometric training was performed at a joint angle corresponding to the plateau nearing the descending limb of the force length relationship, eccentric contractions began at a shorter muscle length and lengthened to this same joint position, and concentric contractions started at this lengthened position but then actively shortened. Owing to the rapid regulation of serial sarcomere number the 4-and 6-week timepoints were collapsed. While force output increased across all training modes (+16%; p<0.05), longitudinal muscle growth was regulated by contraction type. Both isometric and eccentric training were highly effective at increasing serial sarcomere number when optimal loading conditions (high forces at longer lengths) are provided (+7-9%; p<0.05). Concentric training, even when initiated at long lengths with high forces was not an effective stimulus for longitudinal remodelling (+2%; p>0.05). Therefore, these findings identify the mechanical loading environment associated with isometric and eccentric training as a key driver of serial sarcomerogenesis.

How perennial, postmitotic multinucleated tissues, such as skeletal myofibers, maintain their identity and transcriptional adaptation to homeostatic perturbations through adult life is an outstanding question. To address this issue, we investigated the consequences of loss of 3D-genome architecture in skeletal muscles by generating myofiber-specific Ctcf-deficient (CtcfmKO) mice. CtcfmKO mice did not exhibit muscular phenotype at birth but spontaneously developed a severe myopathy. Integrated analysis of snRNAseq, ATACseq and promoter-capture Hi-C revealed both common and fiber-type specific patterns of dysregulated gene expression associated with alterations in chromatin accessibility and promoter-based interactions in Ctcf-deficient myonuclei at distinct stages of myopathy development. Decreased chromatin accessibility at promoters and changes in their connectivity with distal elements were observed across all myonuclei as a direct consequence of Ctcf deficiency at early stages and associated with downregulation of genes implicated in myofiber contraction and anabolism, metabolism, adhesion and neuromuscular transmission. Conversely, at later stages, upregulation of genes leading to persistent activation of ER stress/UPR and catabolism resulted from global reconfiguration of chromatin structure and connectivity, partly as indirect consequence of Ctcf deficiency. Notably, type-IIB myonuclei exhibited specific alterations in gene expression that culminated in loss of fiber-type identity and ectopic expression of inflammatory genes. These results reveal a requirement of Ctcf for maintenance of fiber-type identity and transcriptional adaptation in vivo, through multilayered control of 3D genome integrity. They also indicate an unprecedented association between Ctcf deficiency in myofibers and susceptibility to develop myopathies, whereby Ctcf dispensability for developmental myogenesis confers vulnerability to develop myopathic syndromes.

Age-related skeletal muscle atrophy differs between upper and lower body muscles and may also differ between lower limb muscles, but these muscle-specific patterns remain poorly characterised. We acquired axial 3T magnetic resonance images from sex-balanced samples of thirty young (24+/-4 years) and thirty older adults (73+/-4 years). Muscle volume of seventeen lower limb muscles/compartments was quantified using manual segmentation and expressed as absolute volume, relative to body mass, and as a percentage of total muscle volume. Absolute muscle volume was lower in older adults in 13 out of 17 muscles compared to young, with substantial heterogeneity in the effect sizes (d=0.01-3.36). Age group * muscle interaction contrasts revealed muscle-specific age-related differences in volume. Three of the quadriceps muscles demonstrated greater age-related atrophy compared to several other muscles (e.g. vastus lateralis differing from 14 of the other 16 muscles; d[&ge;]1.54). Conversely, the anterior and deep posterior compartments of the shank and biceps femoris short head showed significantly smaller age-related atrophy than several other muscles. These interaction effects were exacerbated when expressed in relative terms. Consequently, three of four shank compartments, gluteus maximus and adductor magnus represented a greater proportion of total muscle volume in older adults, whereas the vastus lateralis, vastus intermedius, rectus femoris, semitendinosus, gracilis, and iliopsoas contributed a smaller proportion in older adults. These findings highlight the variability in age-related leg muscle atrophy in a large mixed sex cohort, resulting in a redistribution of muscle volume in the lower limb.

Tangential flow filtration (TFF) is an effective method for scalable EV isolation. Here, we describe and validate a reference protocol for TriPort-TFF, a laboratory-assembled TFF system featuring a three- port reservoir design enabling sample circulation, retentate return, and diafiltration. Constructed from individually sourced, commercially available components with detailed assembly and operating parameters, TriPort-TFF can be built at a lower cost than commercially integrated TFF platforms. We evaluated the system using conditioned media (CM) from human MDM cultured with EV-depleted human serum, human cerebral organoids in serum-free medium, and mouse BMDM cultured with EV-depleted foetal bovine serum (FBS) unstimulated or LPS-stimulated. The system reproducibly isolated EVs from all three sources, and biological replicates of BMDM showed distinct proteomic clusters between LPS-stimulated and unstimulated conditions. The requirement for subsequent size-exclusion chromatography (SEC) after TFF depended on the sample serum-presence. In serum-containing CM, TFF alone co-concentrated serum proteins alongside EVs, subsequent SEC improved the particle-to- protein ratio, reduced inter-replicate variability and improved proteomic depth. TFF alone in serum- free organoid CM already yielded higher particle-to-protein ratios than TFF-only preparations from serum-containing CM, with SEC further improving this ratio. TriPort-TFF offers an accessible system for EV-isolation that can be combined with SEC for diverse sample types.

Fibro-adipogenic progenitors (FAPs) have emerged as central regulators of the skeletal muscle homeostasis and the muscle microenvironment. However, the role of FAPs in the context of exercise-induced muscle hypertrophy remains largely unexplored. Here, we utilized six weeks of blood flow restricted resistance exercise (BFRRE) to study cellular adaptations within the muscle microenvironment accompanying muscle hypertrophy in healthy older individuals. Using flow cytometry, we characterized global changes of key cell populations within the skeletal muscle microenvironment, including FAPs, muscle stem cells (MuSCs), and immune cells. BFRRE induced significant enlargement of both FAPs and MuSCs, consistent with cellular adaptation to exercise. Notably, exercise shifted the FAP pool toward an increased predominance of the CD90high FAP phenotype, without altering total FAP abundance. In addition to matrix and collagen-related genes, transcriptional analysis of genes associated with secretory proteins revealed enrichment of promyogenic factors in CD90high versus CD90low FAPs. Conditioned media experiments of freshly isolated FAPs demonstrated that CD90high FAPs promote myotube growth in vitro compared to CD90low counterparts, suggesting that this phenotypic shift may facilitate muscle hypertrophy. In parallel, BFRRE increased the proportion of pro-inflammatory (CD11c+) macrophages within the skeletal muscle niche, highlighting a dynamic immune response during adaptation. Finally, we show identified possible link between pro-inflammatory macrophages and FAPs, as TNF markedly reduced the proliferation of human primary FAPs ex vivo, suggesting that macrophage-derived signals may attenuate excessive FAP expansion during tissue remodelling. Together, these findings provide new insight into how the remodelling of the cellular niche may support muscle hypertrophy in response to exercise. The coordinated expansion and phenotypic remodeling of FAP and immune cell populations may represent an important mechanism through which exercise supports hypertrophy in older individuals.

Objective: Mitochondrial oxidative capacity in striated muscle is governed by the PGC-1/ERR transcriptional axis, which controls not only steady-state respiration but the capacity to expand energy production when demand rises. Whether this reserve function requires dedicated downstream effectors, and whether its loss is detectable at rest, remains unclear. We asked whether Perm1, a striated muscle-specific PGC-1/ERR-induced regulator, is required cell-autonomously for cardiac bioenergetic reserve. Methods: We generated cardiomyocyte-specific Perm1 knockout mice using alphaMHC-nCre, complemented by an adult tamoxifen-inducible alphaMHC-MerCreMer model, and assessed cardiac development, structure, and function longitudinally through 12 months of age in both sexes. We evaluated mitochondrial function by high-resolution respirometry, transmission electron microscopy, and molecular profiling. We tested energetic capacity under load by treadmill exercise with tissue ATP quantification and by beta-adrenergic stimulation of cultured neonatal cardiomyocytes. Results: Perm1 deletion did not perturb embryonic cardiac development, and neither constitutive nor adult-onset deletion impaired cardiac structure, systolic function, or diastolic function through 12 months. Despite this preserved resting phenotype, mitochondrial respiration was reduced by 20 - 25% across complex I- and complex I+II-supported conditions, without changes in citrate synthase activity, mtDNA copy number, or OXPHOS subunit abundance. Instead, the MICOS components IMMT (MIC60) and CHCHD3 (MIC19) were selectively reduced, and mitochondrial subpopulations shifted toward fewer subsarcolemmal and more interfibrillar mitochondria, with unchanged total mitochondrial density. Functionally, Perm1-deficient mice of both sexes showed reduced treadmill capacity at 6 and 12 months and failed to maintain cardiac ATP during exercise at a submaximal workload, while skeletal muscle ATP and plasma lactate were unaffected. This deficit was cardiomyocyte-autonomous: beta-adrenergic stimulation increased ATP content in control cardiomyocytes, but Perm1 knockdown reduced this response. Conclusions. Perm1 is a cardiomyocyte-intrinsic determinant of bioenergetic reserve, dispensable for resting cardiac function but required to expand ATP production under increased demand. These findings identify a mode of metabolic regulation that is invisible to resting functional phenotyping.

Maternal hyperglycemia can affect offspring development and metabolism, but how its effects on skeletal muscle change during postnatal life remains unclear. We investigated skeletal muscle phenotypes in male offspring of streptozotocin-induced hyperglycemic mice at 4 and 8 weeks of age, representing early post-weaning and later postnatal stages, respectively. Maternal hyperglycemia resulted in lower offspring body and skeletal muscle weights at both time points. Myofiber cross-sectional area was reduced without a reduction in total myofiber number, indicating impaired myofiber growth rather than myofiber loss. At 4 weeks, skeletal muscle showed type I/IIA hybrid myofibers and increased expression of fetal, myogenic, and proliferation-associated genes. This immature profile was not maintained at 8 weeks; instead, the transcriptional profile shifted toward reduced mitochondrial and oxidative metabolism. DNA methylation changes at 4 weeks showed limited correspondence with transcriptional alterations. Taken together, maternal hyperglycemia impaired postnatal myofiber growth at both time points, with distinct molecular profiles at 4 and 8 weeks.

Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.