Predicting myosin heavy chain isoform from postdissection fiber length in human skeletal muscle fibers.

Experimental techniques in single human skeletal muscle cells require manual dissection. Unlike other mammalian species, human skeletal muscle is characterized by a heterogeneous mixture of myosin heavy chain (MHC) isoforms, typically used to define "fiber type," which profoundly influences cellular function. Therefore, it is beneficial to predict MHC isoform at the time of dissection, facilitating a more balanced fiber-type distribution from a potentially imbalanced sample. Although researchers performing single fiber dissection report predicting fiber-type based on mechanical properties of fibers upon dissection, a rigorous examination of this approach has not been performed. Therefore, we measured normalized fiber length (expressed as a % of the length of the bundle from which the fiber was dissected) in single fibers immediately following dissection. Six hundred sixty-eight individual fibers were dissected from muscle tissue samples from healthy, young adults to assess whether this characteristic could differentiate fibers containing MHC I ("slow" fiber type) or not ("fast" fiber type). Using receiver operator characteristic (ROC) curves, we found that differences in normalized fiber length (114 ± 13%, MHC I; 124 ± 17%, MHC IIA, P < 0.01) could be used to predict fiber type with excellent reliability (area under the curve = 0.72). We extended these analyses to include older adults (2 females, 1 male) to demonstrate the durability of this approach in fibers with likely different morphology and mechanical characteristics. We report that MHC isoform expression in human skeletal muscle fibers can be predicted at the time of dissection, regardless of origin.NEW & NOTEWORTHY A priori estimation of myosin heavy chain (MHC) isoform in individual muscle fibers may bias the relative abundance of fiber types in subsequent assessment. Until now, no standardized assessment approach has been proposed to characterize fibers at the time of dissection. We demonstrate an approach based on normalized fiber length that may dramatically bias a sample toward slow twitch (MHC I) or fast twitch (not MHC I) fiber populations.

Fatiguing exercise reduces cellular passive Young's modulus in human vastus lateralis muscle.

Previous studies demonstrated that acute fatiguing exercise transiently reduces whole-muscle stiffness, which might contribute to increased risk of injury and impaired contractile performance. We sought to elucidate potential intracellular mechanisms underlying these reductions. To that end, the cellular passive Young's modulus was measured in muscle fibres from healthy, young males and females. Eight volunteers (four male and four female) completed unilateral, repeated maximal voluntary knee extensions until task failure, immediately followed by bilateral percutaneous needle muscle biopsy of the post-fatigued followed by the non-fatigued control vastus lateralis. Muscle samples were processed for mechanical assessment and separately for imaging and phosphoproteomics. Fibres were passively (pCa 8.0) stretched incrementally to 156% of initial sarcomere length to assess Young's modulus, calculated as the slope of the resulting stress-strain curve at short (sarcomere length = 2.4-3.0 µm) and long (sarcomere length = 3.2-3.8 µm) lengths. Titin phosphorylation was assessed by liquid chromatography followed by high-resolution mass spectrometry. The passive modulus was significantly reduced in post-fatigued versus control fibres from male, but not female, participants. Post-fatigued samples showed altered phosphorylation of five serine residues (four located within the elastic region of titin) but did not exhibit altered active tension or sarcomere ultrastructure. Collectively, these results suggest that acute fatigue is sufficient to alter phosphorylation of skeletal titin in multiple locations. We also found reductions in the passive modulus, consistent with prior reports in the literature investigating striated muscle stiffness. These results provide mechanistic insight contributing to the understanding of dynamic regulation of whole-muscle tissue mechanics in vivo. HIGHLIGHTS: What is the central question of this study? Previous studies have shown that skeletal muscle stiffness is reduced following a single bout of fatiguing exercise in whole muscle, but it is not known whether these changes manifest at the cellular level, and their potential mechanisms remain unexplored. What is the main finding and its importance? Fatiguing exercise reduces cellular stiffness in skeletal muscle from males but not females, suggesting that fatigue alters tissue compliance in a sex-dependent manner. The phosphorylation status of titin, a potential mediator of skeletal muscle cellular stiffness, is modified by fatiguing exercise. Previous studies have shown that passive skeletal muscle stiffness is reduced following a single bout of fatiguing exercise. Lower muscle passive stiffness following fatiguing exercise might increase risk for soft-tissue injury; however, the underlying mechanisms of this change are unclear. Our findings show that fatiguing exercise reduces the passive Young's modulus in skeletal muscle cells from males but not females, suggesting that intracellular proteins contribute to reduced muscle stiffness following repeated loading to task failure in a sex-dependent manner. The phosphorylation status of the intracellular protein titin is modified by fatiguing exercise in a way that might contribute to altered muscle stiffness after fatiguing exercise. These results provide important mechanistic insight that might help to explain why biological sex impacts the risk for soft-tissue injury with repeated or high-intensity mechanical loading in athletes and the risk of falls in older adults.