Ex vivo muscle function testing

This protocol describes the steps involved in ex-vivo muscle function testing of mouse extensor digitorum longus.

Note: these experiments require the use of an integrated muscle myograph system (e.g. DMT 820MS) with stimulating electrodes (e.g. DMT 300145) coupled to a voltage stimulator (e.g. DMT CS4) and data collection unit (e.g. ADInstruments PowerLab 8/35 and LabChart Pro software).

Solution preparation

1. The required volume of 1x modified Krebs-Henseleit (mKH) buffer was made by combining 10x concentrated stock solutions I (NaCl 1.16 M, KCl 0.046 M, KH₂PO₄ 0.0116 M, NaHCO₃ 0.253 M) and II (CaCl₂ 0.025 M, MgSO4 0.0116 M) with Milli-Q water. To avoid calcium precipitation, Milli-Q water was added to stock solution I followed by addition of stock solution II.
2. The 1x mKH buffer was gently bubbled with medical carbogen (95% O₂ and 5% CO₂) continuously for ~45 min. BSA (0.1 %), pyruvate (0.002 M), and glucose (0.011 M) were then added to the mKH buffer under constant stirring and the pH adjusted to 7.30. The mKH buffer was close-capped and stored on ice when not in use.   

Myograph preparation and muscle dissection

1. The myograph was switched on and muscle chambers heated to 30 °C. The force transducers in each chamber were calibrated according to manufacturer’s instructions and 5 ml of the 1x mKH buffer was added to relevant chambers with continuous gentle carbogen bubbling.
2. The anesthetized mouse was laid on its back on a dissecting board under a dissecting microscope, its hind leg stretched just ‘taut’ and its foot pinned, before skin from the hind leg was removed.

3. Using curved forceps, straight no. 5 forceps and dissecting scissors, the tibialis anterior was carefully excised leaving the extensor digitorum (EDL) with its tendons intact. A single strand of 5.0 braided suture was used to create a 0.4 cm diameter loop with the free ends of the suture used to tie a surgeon’s knot at the distal EDL tendomuscular junction. A stopper knot was tied distally to the surgeon’s knot. 

   This procedure was repeated at the proximal EDL tendomuscular junction with a second strand of 5.0 braided silk. 

4. The EDL tendons were cut and the muscle quickly incubated in the myograph chamber (in 5 ml 1x mKH buffer at 30 °C and bubbled with carbogen) with the suture loops attached to the respective chamber hooks.
5. The resting tension of the muscle was adjusted to ~5 mN and the muscle allowed to equilibrate for 10 min before muscle function testing.

Muscle function testing 

1. Stimulating electrodes were positioned over the mid-belly of the muscle before 2x 100 Hz tetanic stimulations (350 ms duration) were delivered, with 2 min rest in between, to stabilize the muscle. 
2. Successive single twitch stimulations were then delivered to the muscle, with 30 to 60 s rest in between, while muscle length was carefully stretched using the chamber micromanipulator. Muscle optimal length was reached when there was no further increase in twitch force (over at least 2 successive twitch responses) with increased stretch.
3. After 2 min rest, the muscle was stimulated at the following frequencies: 10, 20, 30, 40, 50, 80, 100, 150, and 200 Hz; with 2 min rest in between stimulations to avoid fatigue.
4. At the completion of function testing, muscle cross-sectional area was calculated by dividing muscle mass by the product of muscle length and muscle density (1.06 mg/mm³).