Characterizing Murine Skeletal Muscle Fatigue

Intact TA muscles were dissected from the hindlimb of euthanized mice and mounted vertically between a force transducer (Model FT03, Glass Instruments, USA) in an organ bath with platinum electrodes and continuous perfusion with 95% O₂ + 5% CO₂-saturated Krebs-Ringer solution (118 mM NaCl, 4.75 mM KCl, 24.8 mM NaHCO₃, 1.18 mM KH₂PO₄, 2.5 mM CaCl₂∙2H₂0, 1.18 mM MgSO₄, and 10 mM glucose). Optimal muscle stretch was determined by applying a single twitch at supramaximal voltage (100 V for 1 ms) using a previously described protocol with slight modification^{43 (link),44 (link)}, and set at the length that generated maximal twitch force. After 10 min of equilibration, TA muscles were subjected to different force frequencies (tetani with increasing stimulation frequencies at 30–200 Hz every 500 ms with 2-min recovery intervals). The fatigue properties of TA muscles were assessed through repeated stimulation for 10 min at frequency of 1 Hz and 100 V. All experiments were performed at 25 °C. Data acquisition and analysis were performed using LabChart Pro Software (Version 8; AD instruments, Pty Ltd.). Muscle length, diameter, and wet weight were measured at the end of each experiment.