Forelimb grip strength was measured on a weekly basis, by means of a grip strength meter (Columbus Instruments, USA), according to a standard protocol [33 (link),34 (link),35 (link),36 (link),37 (link),40 (link),41 (link)]. Maximal force, absolute (expressed in kg force, KGF) and normalized to body mass (in KGF/kg), obtained from five repeated measurements per mouse, was used for data analysis [33 (link),34 (link),35 (link),36 (link),37 (link),40 (link),41 (link)]. At the beginning (T0) and the end (T4) of the training phase, all mice underwent an acute exercise resistance test on a treadmill at incremental speed to assess in vivo fatigability. Each mouse was let run until exhaustion, i.e., inability to re-start the running after a 20 s pause, and the total distance run (in m) up to that time was calculated and used for data analysis [33 (link),34 (link),35 (link),36 (link),37 (link),40 (link),41 (link)].
In vivo isometric torque produced by hind limb plantar flexor muscles (gastrocnemius, soleus, and other minor muscles) was measured in anesthetized mice at T4 by using the 1300A 3-in-1 Whole Animal Muscle Test System (Aurora Scientific Inc., Aurora, ON, Canada). Inhalation anesthesia (≈3% isoflurane in an induction chamber, then ≈2% isoflurane via nose cone for maintenance, both with 1.5 L/min O2) was delivered by using an anesthetic vaporizer (Harvard Apparatus Fluovac and Datex Ohmeda Isotec 4, Holliston, MA, USA) with an oxygen concentrator (LFY-1-5A, Longfei Group Co., Wenzhou, China; distributed by 2Biological Instruments, Besozzo, VA, Italy). The animal was positioned on a thermostatically controlled plate (36 °C); the right foot was placed on a pedal connected to a servomotor, forming a 90° angle with the secured hind limb. Contractions were elicited with the best stimulus intensity at increasing frequencies (200 ms trains at 1, 10, 30, 50, 80, 100, 120, 150, 180, and 200 Hz), via percutaneous electrical stimulation of the sciatic nerve through a pair of needle electrodes connected to a stimulator. Torque values were calculated with the Dynamic Muscle Analysis software (ASI DMAv5.201) and normalized to mouse body mass (N*mm3/kg). Normalized values were used to construct torque–frequency curves [36 (link),41 (link)].
In vivo isometric torque produced by hind limb plantar flexor muscles (gastrocnemius, soleus, and other minor muscles) was measured in anesthetized mice at T4 by using the 1300A 3-in-1 Whole Animal Muscle Test System (Aurora Scientific Inc., Aurora, ON, Canada). Inhalation anesthesia (≈3% isoflurane in an induction chamber, then ≈2% isoflurane via nose cone for maintenance, both with 1.5 L/min O2) was delivered by using an anesthetic vaporizer (Harvard Apparatus Fluovac and Datex Ohmeda Isotec 4, Holliston, MA, USA) with an oxygen concentrator (LFY-1-5A, Longfei Group Co., Wenzhou, China; distributed by 2Biological Instruments, Besozzo, VA, Italy). The animal was positioned on a thermostatically controlled plate (36 °C); the right foot was placed on a pedal connected to a servomotor, forming a 90° angle with the secured hind limb. Contractions were elicited with the best stimulus intensity at increasing frequencies (200 ms trains at 1, 10, 30, 50, 80, 100, 120, 150, 180, and 200 Hz), via percutaneous electrical stimulation of the sciatic nerve through a pair of needle electrodes connected to a stimulator. Torque values were calculated with the Dynamic Muscle Analysis software (ASI DMAv5.201) and normalized to mouse body mass (N*mm3/kg). Normalized values were used to construct torque–frequency curves [36 (link),41 (link)].
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