TNF-α-induced contractile dysfunction in three-dimensional engineered muscle

Three-dimensional engineered muscle (3D-EM) provides a physiologically relevant model for examining skeletal muscle function. Tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine elevated in chronic conditions such as sarcopenia and cachexia, has been linked to muscle weakness. However, the mechanism underlying this effect remains unclear. In this study, we used a 3D-EM system to evaluate the direct impact of TNF-α on muscle contractile force. 3D-EM was produced by seeding C2C12 myoblasts with type I collagen on a culture device, followed by 15 days of differentiation. Constructs were then treated with TNF-α for 48 or 72 h, and contractile output was measured during electrical pulse stimulation. Immunohistochemical analysis and RNA sequencing (RNA-seq) with subsequent enrichment analysis were conducted to assess tissue structure and transcriptomic changes. After 48 h, TNF-α reduced contractile force by 60 %, and after 72 h, by 90 % relative to controls. Immunohistochemistry showed myotube atrophy accompanied by loss of fast-twitch fibers. RNA-seq combined with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated suppression of extracellular matrix, sarcomere organization, and calcium signaling pathways. These results suggest that TNF-α reduced force generation in 3D-EM by impairing extracellular matrix integrity, sarcomeric structure, and calcium-dependent contraction mechanisms, with preferential effects on fast-twitch fibers. Overall, this study offers mechanistic insight into the basis of sarcopenia and demonstrates the utility of 3D-EM as a model of cytokine-induced muscle weakness.