Single Muscle Fiber Physiology
Copyright – PLoS One DOI: 10.1371/journal.pone.0108601
Using the BioPen (formerly the Multifunctional pipette), compounds were delivered locally to the end or side of single adult mouse skeletal muscle ﬁbres to test whether changes in mitochondrial membrane potential were transmitted to more distant located mitochondria. Mitochondrial membrane potential was monitored with tetramethylrhodamine ethyl ester (TMRE). Cytosolic free [Ca2+] was monitored with ﬂuo-3. A pulse of carbonyl cyanide 4-(triﬂuoromethoxy) phenylhydrazone (FCCP) applied to a small area of the muscle ﬁbre (30 µm in diameter) which produced a rapid decrease in the mitochondrial TMRE signal (indicative of depolarization) to 38% of its initial value. After washout of FCCP, the TMRE signal partially recovered. At distances greater than 50 µm away from the site of FCCP application, the mitochondrial TMRE signal was unchanged. Similar results were observed when two sites along the ﬁbre were pulsed sequentially with FCCP. After a pulse of FCCP, cytosolic [Ca2+] was unchanged and ﬁbres contracted in response to electrical stimulation. These results indicate that extensive networks of interconnected mitochondria do not exist in skeletal muscle. Furthermore, the limited and reversible effects of targeted FCCP application with the BioPen highlight its advantages over bulk application of compounds to isolated cells.