Labchart pro software

After the surgery (48 h), the catheter implanted into the femoral artery was connected to a pressure transducer (DPT100, Utah Medical Products Inc., Midvale, UT, United States). PAP was recorded for 10 min before restraint stress and for 30 min during stress (detailed described below) using an amplifier connected to a digital acquisition board (ADInstruments, Dunedin-OTA, NZ) and LabChart PRO software (ADInstruments, Dunedin). The mean arterial pressure (MAP) and HR were derived from the PAP (Morais-Silva et al., 2019 (link)). The mean of the MAP and HR values each 2 min was calculated for time-course analysis.

GAS muscles were dissected from the hind limbs of euthanized mice. Stainless steel hooks were tied to the tendons of the GAS muscles using 6-0 nylon sutures and the muscles were mounted vertically between a force transducer (Model 159901, Radnoti, Monrovia, CA, USA) and an adjustable hook. The muscles were immersed in an organ bath with platinum electrodes continuously perfused with O₂/CO₂ (95/5%)-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). At the start of each experiment, muscle length was adjusted to yield the maximum force (100 V for 2 ms) using a previously described protocol with slight modification^{49 (link)}. After equilibration, GAS muscles were subjected to different stimulation frequencies to measure tetanic force (2 ms pulses at 10–200 Hz for 500 ms at 100 V with 1-min recovery intervals). Fatigue measurements of GAS muscles were evaluated through repeated bouts of stimulation, each lasting 7 min at a frequency of 1 Hz and 100 V. Data collection and analysis were performed using LabChart Pro Software (Version 8, ADInstruments, Colorado Springs, CO, USA). Muscle length and wet weight were measured at the end of each experiment.

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.