Dynamic muscle control and analysis software

At 14 days after injury, animals were anesthetized with 2% isofluorane inhalation and the plantaris and soleus on the left hindlimb were isolated for in situ isometric muscle force measurement using the ASI muscle lever system with dynamic muscle control and analysis software (Aurora Scientific, Inc.) as previously described (Saeman et al., 2015). After reaching the muscle’s optimal length (Lo), isometric twitch and tetanic function were examined with a single 200 and 150 Hz electric stimulation with impulse duration of 0.2 msec at 10 mA in the plantaris and soleus consecutively. The fatigue function of the soleus was measured at 40 Hz with impulse duration of 0.2 msec at 10 mA for a total of 240 sec (Saeman et al., 2015).

Muscle functional testing was performed in vitro using the horizontal bath of a whole-mouse test system (1300A, Aurora Scientific). Ringer’s solution (120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM KH₂PO4, 1.2 mM MgSO₄, 25 mM HEPES, 5.5 mM glucose) within the horizontal bath was bubbled with oxygen for 30 min before experimental initiation. In brief, for muscle functional testing, the EDL muscle was isolated and, using braided silk at both the proximal and distal muscle tendon junctions, secured to both a stationary lever arm hook and a force transducer (model 809c, Aurora Scientific) within the horizontal bath. In this position, the EDL muscle is aligned in parallel between two stimulation electrodes. The EDL muscle was allowed to rest for 10 min before stimulation. To determine the optimal muscle length, the EDL muscle was stimulated at different resting tensions until a maximum twitch tension was determined⁷³ . The EDL muscle rested for 2 min after this optimization. A force–frequency curve was used to determine peak tetanic force. This force determination consisted of a 1 s stimulation every 30 s beginning at 10 Hz and increasing in stimulation frequency in 10 Hz increments. All data were collected and analysed using Dynamic Muscle Control and Analysis Software (v.615A, Aurora Scientific).

Mice were anesthetized by IP injection with a mixture of 10 mg/ml dolethal (Vetoquinol, UK) and 15 μg/ml buprenodale (Dechra, UK) at five times of the bodyweight. The distal tendon of tibialis anterior (TA) muscle was dissected and attached to an isometric transducer, Dual-mode muscle lever (Aurora Scientific, Canada), through a loop made of braided silk suture (Harvard Apparatus, UK). The sciatic nerve was isolated and distally stimulated by a bipolar silver electrode using supramaximal square wave pulses at 0.1 ms duration. Data provided by the isometric transducer were recorded and analyzed using Dynamic Muscle Control and Analysis Software (Aurora Scientific, Canada). All isometric measurements were obtained at an initial length at which a maximal tension was recorded during the tetanus. Responses to tetanic stimulations at increased pulse frequencies from 10 to 180 Hz were recorded and the maximal force (mN) was determined. The specific force (mN/mm²) was subsequently calculated based on a ratio of the maximal force and the muscle cross-sectional area (CSA) that was approximated mathematically by dividing the muscle mass by the optimum fiber length and the density of mammalian muscle, as described in TREAT-NMD SOP DMD_M.2.2.005.