Tomatidine Lowers Inflammation in Skeletal Muscle Myobundles

Tomatidine Attenuates Inflammatory Responses to Exercise-Like Stimulation in Donor-derived Skeletal Muscle Myobundles

Authors

Maddalena Parafati*, Tushar Sanjay Shenoy, Zon Thwin, Mauro Parlaecchio, Siobhan Malany

Abstract

Donor-derived myobotes offer a pre-clinical model for studying muscle biology, the effects of exercise-like electrical stimulation, and assessing drug efficacy and toxicity. We engineered a 3D muscle microphysiological system from myoblasts isolated from vastus lateralis of young and older adults. Over a three-week differentiation process, we applied two cycles of low frequency electrical stimulation daily for seven days generating functional, mature myobundles, as confirmed by gene expression profiling. Both young- and old-derived myobundles showed synchronous contraction in response to electrical stimulation, however, the contraction magnitude was reduced in old-derived myobundles compared to young-derived muscle atrophy and promote skeletal muscle hypertrophy. Bioinformatic analyses revealed that infusion of tomatidine during electrical stimulation modulated the IL-6/JAK/STAT3 pathway. The contraction magnitude decreased in the young-derived myobundles treated with tomatidine compared to vehicle-treated controls, while no significant difference was observed in the old-derived myobundles. Secretome analysis revealed age-related changes in secreted proteins linked to inflammation and extracellular matrix remodeling. Notably, tomatidine attenuates the inflammatory and extracellular matrix remodeling responses in the myobundles triggered by electrical stimulation, partially preventing the secretion of proinflammatory proteins. This intervention strategy helps balance muscle adaptation and repair, while limiting excessive proinflammatory responses. Our microphysiological system provides a valuable platform for investigating signaling pathways involved in muscle function, and pharmacological responses, advancing the understanding of age-related muscle biology.

Keywords

donor-derived myobundles; muscle-on-a-chip; E-Stim-induced contraction; transcriptome; secretome; inflammation; tomatidine.

Introduction

The physiological effects of physical exercise are recognized for their beneficial impact on both the cardiovascular and musculoskeletal systems serving as preventive and therapeutic strategies for cardiovascular disease, diabetes, and obesity. Repeated exercise induces an adaptive and transient response, including the release of cytokines, known as myokines, which play a critical role in the prevention and treatment of various chronic diseases. Inflammation, often associated with tissue damage, is also a trigger of exercise-induced stress response, facilitating repair processes.

A critical process in skeletal muscle adaptation to exercise involves remodeling of the extracellular matrix (ECM), essential for effective muscle contraction, force transmission, and matrix reorganization. Two enzymes families, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are key regulators of ECM turnover. MMP-2 expression is decreased during low-intensity exercise. Among the four TIMP isoforms, TIMP-1 and -2 are significantly increased in response to exercise-like stimulation. In parallel to ECM remodeling, myokines such as interleukin (IL)-6, IL-8, CC chemokine ligand 2 (CCL2), and tumor necrosis factor-alpha (TNF-α) not only mediate inflammatory responses but also contribute to long-term adaptive responses to exercise training.

While tomatidine has shown promise in mitigating inflammation and muscle atrophy, its role in modulating cytokine secretion and ECM remodeling enzymes in response to electrical stimulation remains unexplored. Our study aims to address this gap by investigating anti-inflammatory effects of tomatidine on muscle contraction, remodeling enzymes and gene expression using phenotypic analysis, genome-wide transcriptomics, and quantitative secretome analyses in young- and old-derived myobundles.

Methods

MUSCLE BIOPSY AND PARTICIPANTS CELL ISOLATION

Cell isolation was performed as previously described. Briefly, vastus lateralis biopsies were obtained from young active (YA, age 21-40 years) and old sedentary (OS, age 65-80 years) volunteers, who gave informed consent to participate in this study (n = 5), at the Translational Research Institute at AdventHealth (Orlando, Florida).

CELL CULTURE AND MYOBLAST IMMUNOMAGNETIC CELL SORTING PROCEDURE

Myogenic precursors were thawed and then cultured on T150 flasks coated with collagen I (Rat Tail Collagen I, 0.1 mg/mL, ibidi) at 6,000 cells/mL and expanded in Skeletal Muscle Growth medium (Promocell, Heidelberg, Germany) for 3 days to promote myoblast proliferation. Tissue chips were perfused using a syringe pump at a constant flow rate of 1 mL/h, at 37°C and 5% CO2. Myoblasts were then harvested and expanded in Skeletal Muscle Growth medium with 0.1 mg/mL Primocin (InvivoGen, San Diego, CA) at 125 mL/min every 6 h for 6 days followed by stage 2 maintenance medium (MEM-α, 0.5% (v/v) ITS, 2% (v/v) B27, 20 mM HEPES, pH 7.3 and, 0.1 mg/mL Primocin) at 125 mL/min every 6 h for an additional 6 days to promote maturation of myotubes.

ELECTRIC PULSE STIMULATION AND REAL-TIME RECORDING IMAGES ACQUISITION AND ANALYSIS

Differentiated myotubes were stimulated on day 14 with electrical pulses: 3 V/mm 2 ms and 2 Hz every 12 h for seven days. On day 21, contractions were recorded at 10 fps for 40 s in an environmentally controlled Cytation C10 high content imager (Agilent, Biotek). Myotube contractility was quantified by digital image correlation (DIC). Reference images were taken before and during stimulation. 4x images are acquired with a resolution of 1224 × 904 pixels (12.5 × 143 mm). The region of interest (ROI) included the entire image excluding the micro post. DIC was performed using motion analyzer GOM correlate software (Zeiss, Germany) to determine displacement of the engineered myobundles. The reference image (the first frame prior to application of electrical stimulation) is compared to each test image (with E-Stim applied) in the sequence. After the analysis, the DIC values are saved as a raw EXCEL file that stores the numerical displacement info for each grid at individual frames. Each frame represents a timepoint and subsequent frames are plotted as time on the X-axis. The average pixel value across the rows and columns in the ROI is converted to microns (1.75 µm/pixel) and plotted on the Y-axis as displacement to generate a graph of displacement vs. time.

TOTAL RNA EXTRACTION AND RNA SEQUENCING ANALYSIS

Total cellular RNAs are isolated from myobundles using RNeasy kits, as described by the manufacturer (Qiagen). RNA was subjected to DNase treatment (Qiagen). In addition to control samples, transcript abundance was quantified using RSEM (RSEM v1.3.1), and genes with insufficient average counts were excluded from further statistical analysis. Differential expression analysis was performed using the DESeq2 package, with an FDR-corrected P-value threshold of 0.05. The results were further filtered to extract transcripts showing a 2.0-fold change (log2FC) in either direction.

COLLECTION OF CONDITIONED MEDIA AND HUMAN INFLAMMATORY CYTOKINE AND ECM ARRAYS

Myobundles were perfused with ethanol (EtOH, %) and tomatidine (5 µM) simultaneously with the onset of electrical stimulation (E-Stim) and continued for 7 days. At the conclusion of E-Stim, serum-free conditioned media were collected, centrifuged at 3000 rpm for 20 min at 4°C in presence of 10 µg/mL phosphatase inhibitor cocktail (Thermo Fisher Scientific) and 10 µg/mL protease inhibitor cocktail (Sigma) and stored at -80°C until use. The human inflammatory antibody array including 40 proteins (AAH-INF-3, RayBioTech, Norcross, GA) was used to screen for pro-inflammatory cytokines and ECM enzymes as per manufacturer’s instructions. 1.5 mL culture supernatant was concentrated to 800 µL using protein concentrators with a molecular weight 3 kDa cut-off (Thermo Fisher scientific). Briefly, the membranes were blocked for 30 min at room temperature (RT) and then incubated overnight (O/N) at 4°C with concentrated conditioned raw media. Subsequently, the membranes were washed 3x with wash buffer I and 2x with wash buffer II. The washed membranes were then loaded with biotinylated cocktail and incubated for 2h at RT followed by subsequent wash steps and loaded with horseradish peroxidase (HRP)-streptavidin cocktail for 2 h at RT. Following further washing, chemiluminescence detection solution was applied for 1 min and specific protein signals were detected using the ChemiDoc™ Touch Imaging System (BioRad). The individual spots were quantified using imageJ software (https://imagej.nih.gov/ij/) and expressed as relative quantity.

DATA AVAILABILITY

All data generated during this study are either included in the manuscript or are available at the Gene Expression Omnibus (GEO) database (GSE296683). Precursor cells from patient biopsy samples were obtained from AdventHealth Orlando through a Material Transfer Agreement to the University of Florida with restrictions for sharing with a third party.

Results

TOMATIDINE TREATMENT MODULATES CONTRACTILE FUNCTION IN DONOR-DERIVED MYOBUNDLES

We previously demonstrated muscle contraction in tissue-engineered human skeletal myobundles through electrical stimulation (E-Stim). In this study, we applied a similar electrical stimulation regime. On day 14, differentiated myotubes, under mechanical tension, were exposed to two 30-min E-Stim cycles per day followed by a 12 h recovery period. The stimulation protocol was continued for seven days, and by day 21, the donor-derived myobundles displayed highly aligned myotubes oriented along the direction of the applied electric field. Prior to nucleic acids extraction, myotubes underwent a final round of electrical stimulation, and contractile movement was recorded by confocal imaging. Using digital image correlation (DIC), we observed a significant increase in contractility magnitude and synchronicity of vehicle-treated donor-derived myobundles compared to previous 5-day E-Stim regime.

Specifically, young active (YA)-derived myobundles showed average contraction displacement of 15 ± 2.8 µm, while old sedentary (OS)-derived myobundles exhibited an average displacement of 8.7 ± 0.9 µm. In the absence of electrical stimulation, both groups were compared to baseline measured displacement. In tomatidine (Tom) treated myobundles during E-Stim, a significant reduction in contraction displacement (10.5 ± 1.3 µm) was observed in the younger cohort. In contrast, the older cohort did not show a significant difference in contraction compared to vehicle treated (7.8 ± 0.6 µm) (Figure 1A). Our data show that muscle twitch kinetics are age-dependent. Tomatidine exposure is associated with reduced variability in both cohorts.

TOMATIDINE INCREASED GENES ASSOCIATED WITH IL-6/JAK-STAT SIGNALING PATHWAY IN YOUNG E-STIM MYOBUNDLES

Tomatidine has been shown to exhibit protective effects against the release of pro-inflammatory cytokines/chemokines and to mitigate skeletal muscle atrophy both in vivo and in vitro. In our study, treatment of YA-derived E-Stim myobundles revealed 215 increased and 219 decreased DEGs compared to E-Stim matched control. In contrast, treatment in OS-derived myobundles revealed a significantly smaller number of DEGs, with only 12 increased and 2 decreased DEGs compared to ground controls. GSEA analysis based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database highlighted a significant enrichment of DEGs involved in responses to tomatidine, particularly those associated with the activation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway (Figure 2C).

The JAK/STAT signaling pathway is a critical mediator that transmits extracellular signals to the nucleus, initiating the transcription of genes involved in several biological activities. In skeletal muscle, this pathway plays a central role in regulating skeletal muscle mass, repair, and the pathogenesis of muscle-related diseases. Activation of the JAK/STAT pathway can yield opposite effects on muscle differentiation and repair. In our study, we observed upregulation of several STAT3 target genes, including the leptin receptor (LEPR) (1.2-fold increase), Wnt family member 5A (WNT5A) (0.9-fold increase), Toll-like receptor 3 (TLR3) (1.6-fold increase), and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) (2.3-fold increase). In addition, the suppressor of cytokine signaling (SOCS) 3 (1.3-fold increase), an inducible negative feedback inhibitor of cytokine signaling, was also upregulated. These results are consistent with an overall increase in STAT3 target genes, which includes a modest upregulation of myeloid differentiation primary response 88 (MyD88) (0.6-fold increase). MyD88 plays a key role in promoting skeletal muscle growth in vivo and mediating STAT3 phosphorylation.

A marked 5-fold increase in IL-6 and 1.6-fold increase in IL-8/CXCL8 mRNA expression was observed in YA-derived myobundles when compared to the old. Consistent with the mechanical changes associated with E-Stim-induced exercise and myogenesis, both contracting myobundles exhibited in the conditioned media the presence of tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1, -2). The analysis showed a 2- and 1.3-fold increase in TIMP-1 and TIMP-2 protein secretion, respectively, by OS-derived myobundles when compared with the young in the control conditions. Interestingly, the current results show that the expressions of pro-enzymes and activated forms of MMPs (1, 2, 3, 8, 9, 10 and 13) were not detectable by the array antibodies.

E-STIM-INDUCED CONTRACTION DRIVES MYOKINES SECRETION, WHILE TOMATIDINE INHIBITS THEIR RELEASE

Although we identified activation IL-6/JAK/STAT3 signaling pathway, the underlying mechanism remained unclear. Therefore, we aimed to further investigate the role of IL-6/JAK/STAT3 signaling pathway in inflammation. To validate pathway activation, donor-derived myobundles were analyzed post E-Stim using inflammatory cytokine and ECM antibody arrays to quantify up to 40 cytokines and 10 ECM remodel enzymes, respectively, secreted into the conditioned media of donor-derived myobundles after the final E-Stim cycle.

Despite a marked increase in IL-6 mRNA, the corresponding IL-6 protein remained detectable but were not elevated. A soluble form of the IL-6 receptor was also secreted by the donor-derived myobundles. Additionally, we found marked increases in two chemokines, monocyte chemoattractant protein-1 (MCP-1/CCL2) and IL-8/CXCL8, in the secretome of both YA- and OS-derived contracting myobundle. In particular, we observed similar levels of IL-8 and increased CCL2 by 1.6-fold in YA-derived myobundles when compared to the old. Consistent with the mechanical changes associated with E-Stim-induced exercise and myogenesis, both contracting myobundles exhibited in the conditioned media the presence of tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1, -2). The analysis showed a 2- and 1.3-fold increase in TIMP-1 and TIMP-2 protein secretion, respectively, by OS-derived myobundles when compared with the young in the control conditions. Interestingly, the current results show that the expressions of pro-enzymes and activated forms of MMPs (1, 2, 3, 8, 9, 10 and 13) were not detectable by the array antibodies.

Discussion

Understanding muscle cell-autonomous mechanisms underlying age-related muscle inflammation following exercise is critical to prevent muscle injury, chronic inflammation, attenuating inflammatory responses that disrupt muscle regeneration and adaptive remodeling. Compared to traditional 2D models, 3D bioengineered skeletal muscle derived from cultured myobundles offers significant advantages in vitro. This study highlights the age-related anti-inflammatory effect of tomatidine in muscle.

Myobundles embedded in a 3D matrix allow for extended culture time, myotubes maturation and enhanced contractility. In this study, we used 3D donor-derived engineered myobundles to model inflammation-induced adaptation during low-frequency intermittent E-Stim and to investigate the anti-inflammatory effects of tomatidine on muscle function, comprehensive gene signatures by RNA-sequencing, and the secretome.

Previously, we demonstrated that donor-derived myobundles exhibited hallmarks of muscle aging, providing an in vitro platform to study structural and genetic changes as well as contractile responses of YA- and OS-derived skeletal muscle to exercise-like E-Stim. E-Stim is well established for inducing myogenic differentiation and myobundle hypertrophy and is used to examine exercise effects on muscle cell biology and function. We showed that a 7-day E-Stim regime resulted in synchronized, age-dependent increases in contraction displacement between YA- and OS-derived myobundles. E-Stim significantly increased skeletal muscle-specific structural proteins and pro-myogenic transcription factors involved in muscle differentiation. Additionally, we revealed muscle-autonomous activation of IL-6/JAK/STAT3 signaling, a key mediator of adaptive remodeling during E-Stim.

Exercise-induced regenerative inflammation promotes tissue repair. Skeletal muscle functions as a secretory organ, releasing bioactive molecules such as myokines and extracellular vesicles-derived factors, to modulate immune responses. Exercise alters cytokine secretion, promoting muscle regeneration, reducing inflammation, preventing atrophy and promoting neo-angiogenesis. Muscle fiber’s anti-inflammatory effects are largely attributed to the release of signaling molecules that modulate inflammation for non-muscle cells, thereby reducing their burden. In this study, we examined the specific effects of tomatidine on muscle contractility in donor-derived myobundles. We also demonstrated an anti-inflammatory mechanism during muscle exercise adaptation involving potential crosstalk with IL-6/JAK/STAT3 pathway. When tightly regulated, this pathway may protect against inflammation and contribute to the improvement of skeletal muscle repair and remodeling following exercise.

Author contributions:

Conceptualization, M.P. and S.M.; Investigation and methodology, M.P. and S.M.; Isolation and amplification of CD56+ human myoblasts, M.P., S.T. & Z.T.; Cell seeding and culturing experiments, M.P.; Bioinformatic and antibody arrays analysis: M.P.; Digital Image correlation analysis, M.P.; Project administration, S.M.; Providing reagents and materials, S.M.; Writing – original draft and preparation, M.P.; Funding acquisition, S.M. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest Statement:

S. Malany is a member of the board at Micro-gRx, INC. All other authors declare they have no conflicts of interest with the contents of this article.

Funding Statement:

This study was supported by the National Institutes of Health National Center for Advancement of Translational Sciences (5UG3T2002598 to S.M.) and the Center for the Advancement of Science in Space (CASIS) user agreement #UA-2019-011 to the University of Florida and University of Florida Prosper Bridge Fund (M.P.).

Acknowledgments:

The Authors thank the scientific program manager Dr. Lucie Low for advocating the NIH Tissue Chips program at the National Center for Advancing Translational Sciences (NCATS). We thank Austin Hinkle for helping with creating microfluidic devices as an intern at Micro-gRx. We thank the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida NextGen Sequencing Core (RRID:SCR_019152) and Alberto Riva at UFLICBR Bioinformatics Core.

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  30. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  31. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  32. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  33. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  34. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  35. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  36. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  37. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  38. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  39. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  40. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  41. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  42. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  43. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  44. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  45. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  46. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  47. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  48. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  49. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  50. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  51. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  52. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  53. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  54. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  55. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  56. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  57. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  58. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  59. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  60. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  61. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  62. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  63. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  64. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  65. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  66. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  67. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  68. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  69. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  70. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  71. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  72. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  73. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  74. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  75. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  76. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  77. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  78. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  79. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  80. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  81. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  82. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  83. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  84. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  85. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  86. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  87. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  88. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  89. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  90. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  91. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  92. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  93. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  94. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  95. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  96. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  97. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  98. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  99. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  100. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  101. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  102. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  103. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  104. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  105. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  106. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  107. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  108. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  109. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  110. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  111. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  112. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  113. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  114. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  115. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  116. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  117. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  118. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  119. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  120. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  121. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  122. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  123. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  124. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  125. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  126. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  127. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  128. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  129. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  130. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  131. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  132. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  133. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  134. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  135. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  136. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  137. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  138. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  139. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  140. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  141. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  142. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  143. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  144. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  145. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  146. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  147. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  148. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  149. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  150. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  151. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  152. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  153. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  154. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  155. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  156. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  157. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  158. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  159. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  160. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  161. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  162. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  163. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  164. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  165. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  166. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  167. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  168. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  169. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  170. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  171. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  172. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  173. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  174. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  175. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  176. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  177. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  178. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  179. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  180. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  181. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  182. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  183. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  184. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  185. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  186. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  187. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  188. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  189. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review. Front. Physiol. 10, 1550 (2020).
  190. Jiao, A. et al. Regulation of skeletal myotube formation and alignment by nanotypographically controlled cell-secreted extracellular matrix. J Biomedical Materials Res 106, 1543–1551 (2018).
  191. Lesnak, J. B., Berardi, G. & Sluka, K. A. Influence of routine exercise on the peripheral immune system to prevent and alleviate pain. Neurobiology of Pain 13, 100126 (2023).
  192. Khodabukus, A. et al. Electrical stimulation increases hypertrophy and metabolic flux in tissue-engineered human skeletal muscle. Biomaterials 198, 259–269 (2019).
  193. Kim, J.-H., Yu, S.-M. & Son, J. W. Human Tissue-Engineered Skeletal Muscle: A Tool for Metabolic Research. Endocrinol Metab 37, 408–414 (2022).
  194. Dennis, R. G. & Kosnik, I. P. E. EXCITABILITY AND ISOMETRIC CONTRACTILE PROPERTIES OF MAMMALIAN SKELETAL MUSCLE CONSTRUCTS ENGINEERED IN VITRO. In Vitro Cell Dev Biol Anim 36, 327 (2000).
  195. Ebrahim, M. et al. De novo revertant fiber formation in a 3D culture model of human muscular dystrophy skeletal muscle. The Journal of Clinical Investigation 127, 2247–2260 (2017).
  196. Dessaigne, F., Schleder, C., Perruchot, M.-H. & Rouger, K. 3D in vitro models of skeletal muscle: myosphere, myobundle and bioprinted muscle construct. Vet Res 52, 72 (2021).
  197. Carraro, E., Rossi, L., Maghin, E., Canton, M. & Piccoli, M. 3D in vitro Models of Pathological Skeletal Muscle: Which Cells and Scaffolds to Elect? Front. Bioeng. Biotechnol. 10, 941623 (2022).
  198. Mankhong, S. et al. Experimental Models of Sarcopenia: Bridging Molecular Mechanism and Therapeutic Strategy. Cells 9, 1385 (2020).
  199. Parafati, M. et al. Human skeletal muscle tissue chip autonomous payload reveals changes in fiber type and metabolic gene expression due to spaceflight. npj Microgravity 9, 77 (2023).
  200. Pedrotty, D. M. et al. Engineering skeletal myoblasts: roles of three-dimensional culture and electrical stimulation. American Journal of Physiology-Heart and Circulatory Physiology 288, H1620–H1626 (2005).
  201. Huang, Y.-C., Dennis, R. G. & Baar, K. Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation. American Journal of Physiology-Cell Physiology 291, C11–C17 (2006).
  202. Flabiami, M. et al. Muscle Differentiation and Myotubes Alignment Is Influenced By Micropatterned Surfaces and Exogenous Electrical Stimulation. Tissue Engineering Part A 15, 2447–2457 (2009).
  203. Hurley, B. F., Hanson, E. D. & Sheaff, A. K. Strength Training as a Countermeasure to Aging and Chronic Disease: Sports Medicine 41, 1203–1220 (2011).
  204. Lee, J. H., Kimura, H., Hoshi, T. & Ozawa, K. MyD88 plays a key role in LPS-induced inflammation during recovery from exercise. Journal of Applied Physiology 122, 559–570 (2017).
  205. Mathur, N. et al. Exercise as a Mean to Control Low-Grade Systemic Inflammation. Mediators of Inflammation 2008, 109502 (2008).
  206. Severinsen, M. C. K. & Pedersen, B. K. Muscle–Organ Crosstalk: The Emerging Roles of Myokines. Endocrine Reviews 41, 594–609 (2020).
  207. Suzuki, K. Cytokine Response to Exercise and Its Modulation. Antioxidants 7, 17 (2018).
  208. Oishi, Y. & Manabe, I. Macrophages in inflammation, repair and regeneration. International Immunology 30, 511–528 (2018).
  209. Cerqueira, E., Marinho, D. A., Neiva, H. P. & Lourenço, O. Inflammatory Effects of High and Moderate Intensity Exercise—A Systematic Review.
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