3 in 1 animal system

For the measurements of diaphragm contractile force, we adapted a previously described protocol [55 (link)]. We dissected ~4 mm midcostal diaphragm strips and placed these in the organ bath of the 3-in-1 animal system (Aurora Scientific Inc., Aurora, ON, Canada) filled with mammalian Ringer solution containing 120.5 mM NaCl, 20.4 mM NaHCO₃, 10 mM dextrose, 4.8 mM KCl, 1.6 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM NaH₂PO₄, and 1.0 mM pyruvate. Upon dissection and force measurement, the solution was perfused continuously with 95% O₂–5% CO₂ and maintained at 25 °C. Diaphragm strips were then set to optimal length, maximum force (F₀) was meassured by stimulating for 1000 ms at 120 Hz. Specific force (sFo) was calculated by normalizing maximum force with the division of the diaphragm strip mass (mg) by the product of diaphragm strip length (mm) and 1.06 mg/mm³ as the density of skeletal muscle. The force frequency was determined by stimulating the diaphragm strip with 1000 ms trains of 0.5 ms stimuli at 10, 25, 50, 75, 100, 125, 140, and 150 Hz.

Measurements of TA muscle force were performed using a 3-in-1 animal system (Aurora Scientific). Mice were maintained anesthetized with isoflurane TA muscle was exposed, the knee stabilized with a knee pin and clamp, and the TA distal tendon was cut and connected to the force transducer with a silk loop. Electrodes were positioned under the TA. The optimal length (L₀) was determined using a series of rectangular unipolar pulses of 0.2 ms at different muscle base tensions to stimulate the TA muscles. Then, the maximal tetanic force (F₀) was recorded at 150 Hz using the same rectangular unipolar pulses of 0.2 ms for 300 ms. The cross-sectional area (CSA) and the specific force (sF₀) were calculated after the measurement of muscle optimal length and weight for each muscle^{65 (link)}.