Molecular and Mitochondrial Responses to Exercise in Human Skeletal Muscle Myotubes

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Recent Advances in Nutritional Physiology: A Muscle-centric Perspective (University of Exeter, UK) (2026) Proc Physiol Soc 75, SA08

Research Symposium: Molecular and Mitochondrial Responses to Exercise in Human Skeletal Muscle Myotubes

John Noone1, Reichelle Yeo2, Maria Pino2, Yifei Sun3, Giovanna Distefano2, Mark Campbell4, Katie Whytock2, Cheehoon Ahn2, Polina Krassovskaia5, Donghai Zheng5, Filip Jevtovic5, Cynthia Stowe6, Shannon Emilson6, Eric Ravussin7, Nicolas Musi8, Kim Huffman9, Nicolas Broskey5, Bryan Bergman10, Martin Walsh3, Bret Goodpaster2, Joseph Houmard5, Lauren Sparks2

1University of Limerick Ireland, 2AdventHealth Translational Research Institute, Florida USA, 3Icahn School of Medicine at Mount Sinai, New York USA, 4Department of Radiology, University of Washington, Seattle, Washington USA, 5Department of Kinesiology, East Carolina University, Greenville, North Carolina USA, 6Department of Biostatistics and Data Science, Wake Forest University School of Medicine, Winston-Salem, North Carolina USA, 7Pennington Biomedical Research Center, Baton Rouge, Louisiana USA, 8Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California USA, 9Duke University School of Medicine, Durham, North Carolina USA, 10Anschutz Medical Campus, University of Colorado, Aurora, Colorado USA

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Introduction: Exercise training improves skeletal muscle mitochondrial energetics and insulin sensitivity, contributing to enhanced metabolic health and reduced all-cause mortality. Skeletal muscle stem cells may retain exercise-induced adaptations through persistent epigenetic, transcriptional and metabolic programming. Whether habitual physical activity and exercise training induce long-term cell-autonomous adaptations in skeletal muscle energetics and molecular regulation remains unclear.

Aim: To determine whether habitual physical activity and exercise training imprint persistent cell-intrinsic adaptations in human skeletal muscle stem cell-derived myotubes and skeletal muscle fibres.

Methods: Human skeletal muscle cells (HSkMCs) were isolated from vastus lateralis biopsies collected as part of the MoTrMyo ancillary study within the Molecular Transducers of Physical Activity Consortium (MoTrPAC). MoTrMyo recruited 601 extensively phenotyped participants (habitually active (HA), n=101; sedentary (SED), n=500). Of these subjects, baseline analyses were performed in a selection of habitually active (HA) endurance exercisers (HA-EE), habitually active (HA) resistance exercisers (HA-RE), sedentary individuals (SED) and sedentary individuals with obesity (OS-SED). CD56⁺ myoblasts were immunopurified, differentiated into myotubes and assessed using high-resolution respirometry under carbohydrate (CHO)- and fatty acid-supported (FAO) conditions, with and without a 24-hour free fatty acid (FFA) challenge. RNA sequencing and DNA methylation analyses were performed in untreated myotubes. Complementary intervention studies assessed the effects of 12 weeks of endurance or resistance exercise training on mitochondrial energetics in HSkMCs (MoTrMyo) and permeabilized skeletal muscle fibre bundles (MoTrEnergetics) from previously sedentary participants. Statistical analyses used linear models adjusting for known covariates, with significance accepted at p<0.05.

Results: HA-EE HSkMCs demonstrated enhanced carbohydrate- and fatty-acid-supported mitochondrial respiratory capacity compared to HA-RE and sedentary groups (SED and OS-SED), alongside greater metabolic flexibility following FFA challenge. Differential gene expression between individual groups was modest; however, comparison of habitually active (HA; HA-EE + HA-RE) and sedentary (SED + OS-SED) participants identified 208 differentially expressed genes. Pathway analyses demonstrated enrichment of RHO GTPase cycling, RAC signaling, myogenesis, muscle contraction, vascular development and cellular differentiation. Integrative RNA sequencing and DNA methylation analyses identified coordinated transcriptomic-epigenetic relationships involving genes and promoter CpG methylation associated with RHO GTPase signaling (DEF6 promoter CpG1389), cell adhesion (F11R promoter CpG1434), protein tyrosine phosphatase signaling (PTPN20 promoter CpG16413), extracellular matrix organization (AEBP1 promoter CpG14036), peptide processing (LVRN promoter CpG14684) and Ras signaling (TBC1D10C promoter CpG19048). WGCNA identified positive associations between mitochondrial respiration and gene co-expression modules. Complementary intervention studies demonstrated that 12 weeks of endurance or resistance training increased mitochondrial respiratory capacity in both primary HSkMCs (MoTrMyo) and permeabilized skeletal muscle fibres (MoTrEnergetics), with increased Complex II-supported respiration following both interventions.

Discussion: These findings demonstrate that habitual physical activity and exercise training induce persistent adaptations in skeletal muscle energetics evident in isolated HSkMCs and intact skeletal muscle fibres. Habitual exercise was associated with coordinated transcriptomic and epigenetic signatures linked to mitochondrial energetics, cytoskeletal remodeling and cellular differentiation, while intervention studies confirm that exercise training enhances mitochondrial respiratory capacity across multiple experimental models. Collectively, these findings support the concept that habitual physical activity imprints a persistent, cell-autonomous skeletal muscle phenotype and provide mechanistic insight into how regular exercise promotes long-term metabolic health.

Where applicable, experiments conform with Society ethical requirements.

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