Fatiguing Exercise Reduces Cellular Passive Young’s Modulus in Human Vastus Lateralis Muscle

Abstract Previous studies demonstrated that acute, exercise-induced fatigue transiently reduces whole-muscle stiffness. Because reduced muscle stiffness at fatigue may contribute to increased injury risk and impaired contractile performance, the present study seeks to elucidate potential intracellular mechanisms underlying these reductions. To that end, cellular passive Young’s Modulus was measured in single, permeabilized muscle fibers from healthy, recreationally-active males and females. Eight volunteers (4 male, 4 female) completed unilateral, repeated maximal voluntary knee extensions until fatigue, after which percutaneous needle biopsies were performed on the fatigued (F) and non-fatigued (NF) Vastus Lateralis muscles. Muscle samples were processed for mechanical assessment and separately for imaging and phosphoproteomics. Single fibers were passively (pCa 8.0), incrementally stretched to 156% of the initial sarcomere length to assess Young’s Modulus, calculated as the slope of the resulting stress-strain curve at short (strain = 1.00-1.24 %Lo) and long (strain = 1.32-1.56 %Lo) fiber lengths. Titin phosphorylation was assessed by liquid chromatography followed by high-resolution mass spectrometry (LC-MS). Passive modulus was significantly reduced by fatigue at short and long lengths in male, but not female, participants. Fatigue increased phosphorylation of four serine residues located within the elastic region of titin and reduced phosphorylation at one serine residue but did not impact active tension nor sarcomere ultrastructure. Collectively, these results suggest muscle fatigue reduces cellular passive modulus in young males, but not females, concurrent with altered titin phosphorylation. These results provide mechanistic insight contributing to the understanding of sex-based differences in soft tissue injury and falls risk. Key Points Summary Previous studies have shown that skeletal muscle stiffness is reduced following a single bout of fatiguing exercise. Lower muscle stiffness at fatigue may increase risk for soft-tissue injury, however, the underlying mechanisms of this change are unclear. Our findings show that fatiguing exercise reduces passive Young’s modulus in skeletal muscle cells from males but not females, suggesting that intracellular proteins contribute to reduced muscle stiffness with fatigue in a sex-dependent manner. The phosphorylation status of the intracellular protein titin is modified by fatiguing exercise in a way that may contribute to altered muscle stiffness after fatiguing exercise. These results provide important mechanistic insight that may help explain why biological sex impacts risk for soft tissue injury in with repeated or high intensity mechanical loading in athletes and falls risk in older adults. New and Noteworthy Muscle fatigue has previously been shown to reduce musculotendinous stiffness, but the underlying mechanisms remain unclear. Our study presents novel evidence of fatigue-induced reductions in passive cellular Young’s Modulus in skeletal muscle from males, but not females, in conjunction with fatigue-induced alterations in titin phosphorylation. Collectively, these results suggest that intracellular mechanisms including titin phosphorylation may contribute to altered skeletal muscle stiffness following fatiguing exercise, and that this response is mediated by biological sex.