Download Supplementary methods Twelve plantaris muscles from 10 bullfrogs

Supplementary methods
Twelve plantaris muscles from 10 bullfrogs (Lithobates catesbeianus) were used (Supplementary
table 1). Individuals were housed in small groups in vivaria with wet and dry regions, and fed crickets
biweekly. Room temperature was maintained at 20 °C. All animal housing and procedures were
approved by the UC Irvine Institutional Animal Care and Use Committee. Frogs were killed by
double pithing, the skin stripped from the hindlimbs, and the legs removed at the hip. The plantaris
longus muscles and the sciatic nerves were carefully dissected out in oxygenated anuran Ringer’s
solution (concentrations in g l-1, 6.78 NaCl, 0.09 KCl, 0.11 CaCl2, 0.23 NaHCO3, 2.0 dextrose; pH7.4)
leaving the origin of the muscle connected to the knee joint and the distal tendon largely intact.
Sonomicrometry transducers (1 mm diameter; Sonometrics Inc., London, Ontario, Canada) were
implanted along a fascicle and secured using 5-0 silk. The knee was clamped to a stereotaxic frame
and the distal tendon was secured as close to the end of the muscle as possible in a custom built
clamp. The distal clamp was connected to the lever arm of an ergometer (310 B-LR, Aurora Scientific
Inc., Ontario, Canada) using steel cable (10 cm). Care was taken to minimize the compliance of the
setup. A nerve cuff containing stimulus and ground silver wire electrodes was placed around the
sciatic nerve. The preparation was immersed in oxygenated Ringer’s solution at room temperature.
Single or trains of square wave stimuli were delivered to the nerve (Grass S48 stimulator, Grass
medical instruments, Quincy, MA, USA)
and force, muscle length and fibre length logged
simultaneously at 5000 Hz (National Instruments AD board (NI USB-6212); Igor Pro 6.31,
Wavemetrics Inc., Lake Oswego, OR, USA). In all contractions, muscle length was held constant;
however, fibres shortened as series elastic elements stretched during force production. Muscle
performance was monitored throughout the experiment by performing regular supra-maximal tetanic
contractions. The experiment was terminated once force fell below 80 % of its original value. Peak
isometric stress was calculated as peak isometric force in the TetMax condition divided by muscle cross
sectional area and corrected for pennation angle (Supplementary table 1).
Supplemental Table 1. The basic morphological and
physiological properties of the animals and muscle used
Frog mass (g)
96.4±7.5 (n=10)
Muscle mass (g)
1.5±0.13 (n=12)
Resting muscle length (mm)
37.0±0.96 (n=12)
Resting fibre length (mm)
14.5±1.08 (n=12)
Peak isometric stress (N cm-1)
20.7±2.0 (n=12)
Supplemental Table 1. Stimulation parameters for the
various stimulation conditions
Tetmax
Tetsub
Twitch
Pulse duration (ms)
Train duration (ms)
Pulse frequency (Hz)
Pulse amplitude (V)
0.2
400
100
3-5
0.2
400
100
0.3-0.9
0.2
N/A
N/A
3-5