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A better understanding of the cellular and molecular mechanisms that are involved in skeletal muscle adaptation to exercise is fundamentally important to take full advantage of the enormous benefits that exercise training offers in disease prevention and therapy. The aim of this study was to elucidate the transcriptional signatures that distinguish the endurance-trained and untrained muscles in young adult males (24 ± 3.5 years). We characterized baseline differences as well as acute exercise-induced transcriptome responses in vastus lateralis biopsy specimens of endurance-trained athletes (ET; n = 8; VO2max, 67.2 ± 8.9 mL/min/kg) and sedentary healthy volunteers (SED; n = 8; VO2max, 40.3 ± 7.6 mL/min/kg) using microarray technology. A second cohort of SED volunteers (SED-T; n = 10) followed an 8-week endurance training program to assess expression changes of selected marker genes in the course of skeletal muscle adaptation. We deciphered differential baseline signatures that reflected major differences in the oxidative and metabolic capacity of the endurance-trained and untrained muscles. SED-T individuals in the training group displayed an up-regulation of nodal regulators of oxidative adaptation after 3 weeks of training and a significant shift toward the ET signature after 8 weeks. Transcriptome changes provoked by 1 h of intense cycling exercise only poorly overlapped with the genes that constituted the differential baseline signature of ETs and SEDs. Overall, acute exercise-induced transcriptional responses were connected to pathways of contractile, oxidative, and inflammatory stress and revealed a complex and highly regulated framework of interwoven signaling cascades to cope with exercise-provoked homeostatic challenges. While temporal transcriptional programs that were activated in SEDs and ETs were quite similar, the quantitative divergence in the acute response transcriptomes implicated divergent kinetics of gene induction and repression following an acute bout of exercise. Together, our results provide an extensive examination of the transcriptional framework that underlies skeletal muscle plasticity.

Skeletal muscle tissue engineering aims at generating biological substitutes that restore, maintain or improve normal muscle function; however, the quality of cells produced by current protocols remains insufficient. Here, we developed a multifactor-based protocol that combines adenovector (AdV)-mediated MYOD expression, small molecule inhibitor and growth factor treatment, and electrical pulse stimulation (EPS) to efficiently reprogram different types of human-derived multipotent stem cells into physiologically functional skeletal muscle cells (SMCs). The protocol was complemented through a novel in silico workflow that allows for in-depth estimation and potentially optimization of the quality of generated muscle tissue, based on the transcriptomes of transdifferentiated cells. We additionally patch-clamped phenotypic SMCs to associate their bioelectrical characteristics with their transcriptome reprogramming. Overall, we set up a comprehensive and dynamic approach at the nexus of viral vector-based technology, bioinformatics, and electrophysiology that facilitates production of high-quality skeletal muscle cells and can guide iterative cycles to improve myo-differentiation protocols.

Background Critical illness myopathy (CIM) is associated with severe skeletal muscle wasting and impaired function in intensive care unit (ICU) patients. The mechanisms underlying CIM remain incompletely understood. To elucidate the biological activities occurring at the transcriptional level in the skeletal muscle of ICU patients with CIM, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using RNA sequencing (RNA-seq). We also compared the skeletal muscle gene signatures in ICU patients with CIM and genes perturbed by mechanical loading in one leg of the ICU patients, with an aim of reducing the loss of muscle function. Methods RNA-seq was used to assess gene expression changes in tibialis anterior skeletal muscle samples from seven critically ill, immobilized, and mechanically ventilated ICU patients with CIM and matched control subjects. We also examined skeletal muscle gene expression for both legs of six ICU patients with CIM, where one leg was mechanically loaded for 10 h/day for an average of 9 days. Results In total, 6257 of 17,221 detected genes were differentially expressed (84% upregulated; p  < 0.05 and fold change ≥ 1.5) in skeletal muscle from ICU patients with CIM when compared to control subjects. The differentially expressed genes were highly associated with gene changes identified in patients with myopathy, sepsis, long-term inactivity, polymyositis, tumor, and repeat exercise resistance. Upstream regulator analysis revealed that the CIM signature could be a result of the activation of MYOD1, p38 MAPK, or treatment with dexamethasone. Passive mechanical loading only reversed expression of 0.74% of the affected genes (46 of 6257 genes). Conclusions RNA-seq analysis revealed that the marked muscle atrophy and weakness observed in ICU patients with CIM were associated with the altered expression of genes involved in muscle contraction, newly identified E3 ligases, autophagy and calpain systems, apoptosis, and chaperone expression. In addition, MYOD1, p38 MAPK, and dexamethasone were identified as potential upstream regulators of skeletal muscle gene expression in ICU patients with CIM. Mechanical loading only marginally affected the skeletal muscle transcriptome profiling of ICU patients diagnosed with CIM.

Introduction: Circadian rhythms influence muscle metabolism and gene expression, suggesting that training time-of-day may shape hypertrophic and molecular adaptations. Evidence in trained individuals, however, remains limited. Objective: To compare the effects of morning versus evening resistance training on hypertrophy, performance, endocrine markers, sleep, and skeletal-muscle transcriptomics in bodybuilders. Methodology: In a randomized parallel-group trial, 112 trained males were assigned to 12 weeks of supervised training either in the morning (07:00–09:00) or evening (17:00–19:00). Primary outcome was change in vastus lateralis CSA (MRI). Secondary outcomes included lean mass (DXA), strength, hormones, sleep (actigraphy), chronotype, and RNA-seq profiling. Results: Evening training produced a greater VL-CSA increase (+8.2% vs +6.0%; p=0.04) and stronger induction of mTOR- and ribosome-related transcriptional pathways (FDR < 0.05). Chronotype moderated hypertrophic responses. Discussion: Training time influenced phenotypic and molecular adaptations, with evening sessions eliciting broader anabolic signaling. Conclusion: Evening resistance training yields modestly greater hypertrophy and distinct transcriptomic responses; aligning training with chronotype may enhance outcomes. Introducción: Los ritmos circadianos influyen en el metabolismo muscular y la expresión génica, lo que sugiere que el momento del día en que se entrena puede determinar las adaptaciones hipertróficas y moleculares. Sin embargo, la evidencia en individuos entrenados aún es limitada. Objetivo: Comparar los efectos del entrenamiento de resistencia matutino versus vespertino sobre la hipertrofia, el rendimiento, los marcadores endocrinos, el sueño y la transcriptómica del músculo esquelético en culturistas. Metodología: En un ensayo aleatorizado de grupos paralelos, 112 hombres entrenados fueron asignados a 12 semanas de entrenamiento supervisado, ya sea por la mañana (07:00–09:00) o por la tarde (17:00–19:00). El resultado primario fue el cambio en el área de sección transversal (CSA) del vasto lateral (RM). Los resultados secundarios incluyeron la masa magra (DXA), la fuerza, las hormonas, el sueño (actigrafía), el cronotipo y el perfil de ARN mediante secuenciación (RNA-seq). Resultados: El entrenamiento vespertino produjo un mayor incremento en el área de sección transversal del músculo vasto lateral (+8,2 % frente a +6,0 %; p = 0,04) y una inducción más potente de las vías de transcripción relacionadas con mTOR y los ribosomas (FDR < 0,05). El cronotipo moduló las respuestas hipertróficas. Discusión: El horario de entrenamiento influyó en las adaptaciones fenotípicas y moleculares, y las sesiones vespertinas provocaron una señalización anabólica más amplia. Conclusión: El entrenamiento de resistencia vespertino produce una hipertrofia ligeramente mayor y respuestas transcriptómicas distintas; alinear el entrenamiento con el cronotipo podría mejorar los resultados. Introdução: Os ritmos circadianos influenciam o metabolismo muscular e a expressão génica, sugerindo que o horário do treino pode determinar adaptações hipertróficas e moleculares. No entanto, as evidências em indivíduos treinados são ainda limitadas. Objectivo: Comparar os efeitos do treino de resistência matutino versus vespertino sobre a hipertrofia, o desempenho, os marcadores endócrinos, o sono e a transcriptómica do músculo esquelético em fisiculturistas. Metodologia: Num ensaio clínico randomizado de grupos paralelos, 112 homens treinados foram alocados a 12 semanas de treino supervisionado, no período da manhã (07:00–09:00) ou no período da noite (17:00–19:00). O desfecho primário foi a alteração da área de secção transversa (AST) do vasto lateral (RM). Os desfechos secundários incluíram massa magra (DXA), força, hormonas, sono (actigrafia), cronotipo e perfil de RNA por sequenciação (RNA-seq). Resultados: O treino noturno resultou num maior aumento da área de secção transversal do músculo vasto lateral (+8,2% vs. +6,0%; p = 0,04) e numa indução mais potente das vias de transcrição relacionadas com o mTOR e os ribossomas (FDR < 0,05). O cronotipo modulou as respostas hipertróficas. Discussão: O horário do treino influenciou as adaptações fenotípicas e moleculares, com as sessões noturnas a desencadearem uma sinalização anabólica mais abrangente. Conclusão: O treino de resistência noturno produz uma hipertrofia ligeiramente maior e respostas transcriptómicas distintas; alinhar o treino com o cronotipo pode melhorar os resultados.

Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. We investigated nuclear heterogeneity and transcriptional dynamics in the uninjured and regenerating muscle using single-nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers. This revealed distinct nuclear subtypes unrelated to fiber type diversity, previously unknown subtypes as well as the expected ones at the neuromuscular and myotendinous junctions. In fibers of the Mdx dystrophy mouse model, distinct subtypes emerged, among them nuclei expressing a repair signature that were also abundant in the muscle of dystrophy patients, and a nuclear population associated with necrotic fibers. Finally, modifications of our approach revealed the compartmentalization in the rare and specialized muscle spindle. Our data identifies nuclear compartments of the myofiber and defines a molecular roadmap for their functional analyses; the data can be freely explored on the MyoExplorer server ( https://shiny.mdc-berlin.de/MyoExplorer/ ). The transcriptional programs of nuclei in the muscle syncytium were assumed to be homogenous except at the neuromuscular and myotendinous junctions. Here, using single-nucleus transcriptomics, the authors reveal a previously unrecognized diversity and dynamics of myonuclear transcriptional programs.

Muscle atrophy and functional decline (sarcopenia) are common manifestations of frailty and are critical contributors to morbidity and mortality in older people 1 . Deciphering the molecular mechanisms underlying sarcopenia has major implications for understanding human ageing 2 . Yet, progress has been slow, partly due to the difficulties of characterizing skeletal muscle niche heterogeneity (whereby myofibres are the most abundant) and obtaining well-characterized human samples 3 , 4 . Here we generate a single-cell/single-nucleus transcriptomic and chromatin accessibility map of human limb skeletal muscles encompassing over 387,000 cells/nuclei from individuals aged 15 to 99 years with distinct fitness and frailty levels. We describe how cell populations change during ageing, including the emergence of new populations in older people, and the cell-specific and multicellular network features (at the transcriptomic and epigenetic levels) associated with these changes. On the basis of cross-comparison with genetic data, we also identify key elements of chromatin architecture that mark susceptibility to sarcopenia. Our study provides a basis for identifying targets in the skeletal muscle that are amenable to medical, pharmacological and lifestyle interventions in late life. The Human Muscle Ageing Cell Atlas provides a series of integrated cellular and molecular explanations for sarcopenia and frailty development in advanced ages.

Tissue regeneration requires coordination between resident stem cells and local niche cells 1 , 2 . Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity 3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing 4 ) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life. A lifetime cartography of in vivo senescent cells shows that they are heterogeneous. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing and arrests stem cell proliferation and tissue regeneration.

Leucine has gained recognition as an athletic dietary supplement in recent years due to its various benefits; however, the underlying molecular mechanisms remain unclear. In this study, 20 basketball players were recruited and randomly assigned to two groups. Baseline exercise performance—assessed through a 282-foot sprint, free throws, three-point field goals, and self-rated practice assessments—was measured prior to leucine supplementation. Participants were then given a functional drink containing either leucine (50 mg/kg body weight) or a placebo for 28 days. After supplementation, the same exercise performance metrics were reassessed. Following leucine supplementation, biceps brachii muscle tissue from both groups was collected for transcriptome sequencing and qPCR verification. Our results suggested that leucine supplementation significantly improved 282-foot sprint performance, reducing times from 17.4 ± 0.9 to 16.2 ± 0.9 seconds in the leucine group, compared to minimal changes in the control group (from 17.3 ± 0.9 to 17.1 ± 0.8 seconds; P = 0.034). For other exercise performance metrics, no significant differences were observed (P > 0.05); however, trends toward improvement were noted. Transcriptomic analysis revealed 3,658 differentially expressed genes (DEGs) between the two groups. These DEGs were enriched in pathways related to immune response (P < 0.0001), positive regulation of cytokine production (P < 0.0001), and neutrophil extracellular trap formation (P < 0.0001), among others. Weighted Gene Co-expression Network Analysis (WGCNA) identified a module (turquoise) strongly associated with muscle growth, with DEGs in this module enriched in cytoskeletal pathways in muscle cells. Gene expression changes ( α-tubulin , β-tubulin , CK18 , CK8 , vimentin , cofilin , gelsolin , profilin , MAP1 , MAP2 , MAP4 , E-cadherin , and N-cadherin ) were verified by qPCR. In summary, leucine supplementation improved exercise performance, particularly by significantly reducing sprint times and showing trends of improvement in other performance metrics, including three-point field goals, free throws, and self-rated well-being. Identified DEGs enriched in pathways related to immune response, cytokine production, and cell adhesion. WGCNA highlighted a key module associated with muscle growth, enriched in cytoskeletal pathways. qPCR validation confirmed the upregulation of cytoskeleton-related genes, supporting the transcriptomic findings. These results suggest that leucine enhances muscle adaptation by regulating cytoskeletal dynamics, providing molecular insights into its role in improving athletic performance.

The capacity to regenerate skeletal muscle after injury requires a complex and well-coordinated cellular response, which is challenged in aged skeletal muscle. Here, we unravel the intricate dynamics of elderly human skeletal muscle regeneration by combining spatial, temporal, and single cell transcriptomics. Using spatial RNA sequencing ( n  = 3), we profile the expression of human protein-coding genes in elderly human skeletal muscle biopsies before as well as 2-, 8-, and 30-day post injury (NCT03754842). Single Cell-Spatial deconvolution analysis highlights monocytes/macrophages and fibro-adipogenic progenitors (FAPs) as pivotal players in human muscle regeneration. By utilizing flow cytometry ( n  = 9) and cell sorting we confirm the increased cellular content and activity during regeneration. Spatial correlation analysis unveils FAPs and monocytes/macrophages co-localization and intercellular communication, mediated by complement factor C3. Immunostaining confirms C3 expression in FAPs and FAP secretion of C3, suggesting a role in phagocytosis of necrotic muscle cells. Finally, functional assays demonstrate C3’s impact on human monocyte metabolism, survival and phagocytosis, unveiling its involvement in skeletal muscle regeneration. These insights elucidate the FAP-macrophage interplay in aged human muscle with perspectives for future therapeutic interventions to reduce the age-induced decline in regenerative capacity. Successful skeletal muscle regeneration involves a complex and finely tuned inter-cellular response. Here, by using spatial transcriptomics, the authors identify an intercellular communication axis between fibro-adipogenic progenitors and macrophages to enhance macrophage-mediated tissue repair.

Cellular senescence is a hallmark of organismal aging but how it drives aging in human tissues is not fully understood. Here we leverage single nucleus multiomics to profile senescence in mononucleated cells of human skeletal muscle and provide the first senescence atlas. We demonstrate the intra- and inter-populational transcriptomic and epigenomic heterogeneity and dynamics of cellular senescence. We also identify commonalities and variations in senescence-associated secretory phenotypes (SASPs) among the cells and elucidate SASP mediated cellular interactions and niche deregulation. Furthermore, we identify targetable SASPs and demonstrate the possibility of using Maraviroc as a pharmacological senotherapeutic for treating age-associated sarcopenia. Lastly, we define transcription factors that govern senescence state and SASP induction in aging muscle and elucidate the key function and mechanism of JUNB in SASP activation. Altogether, our findings demonstrate the prevalence and function of cellular senescence in skeletal muscle and identify a novel pharmacological intervention for sarcopenia. This study leverages single-nucleus multiomics to map cellular senescence atlas in aging human skeletal muscle and uncovers potential targets and senotherapeutics for treating age-associated sarcopenia.