Fig. S6.

Stimulation protocols cause mechanical fatigue (supports Figs. 2F and 6 and Table 1). The field stimulation patterns used in FRAP experiments described in the main text were tested for mechanical effects in separated experiments, with the results shown. FDB muscles dissected from adult mice (killed by protocols described in Methods) were placed in a Tyrode bath at 23 °C, suspended between an ergometer motor (Cambridge Technology Model 352) and a fixed post using 4-0 silk thread. The muscle length was adjusted to optimal by administering a series of tetanic contractions (supramaximal voltage applied via platinum wire electrodes using a 100-Hz train of 800 ms) and selecting the length and resting tension that elicited peak force. The muscle was then stimulated with pulses of 0.5-ms duration for 5 min with one of three protocols: protocol A: 40-Hz, 350-ms trains delivered once every 3 s; protocol B: 60-Hz, 350-ms trains delivered once every 3 s; and protocol C: 70-Hz, 500-ms trains delivered once every 2 s. Force signals were acquired every 10 s, starting at time = 0 min, referred to as the “Rested” condition. Following application of the fatiguing set of stimuli, the muscle was rested for 10 min before the stimulation was applied to record recovery. In FRAP experiments, which were conducted under similar conditions except for the use of thin bundles of fibers instead of whole muscles, protocol B was applied with slight adjustments (including a reduction in frequency to 50 Hz when we found evidence of action potential failure or an increase in duty cycle to up to 50% to increase the observed depletion).