Usage of a Localised Microflow Device to Show that Mitochondrial Networks Are Not Extensive in Skeletal Muscle Fibres

In cells, such as neurones and immune cells, mitochondria can form dynamic and extensive networks that change over the minute timescale. In contrast, mitochondria in adult mammalian skeletal muscle fibres show little motility over several hours. Here, we use a novel three channelled microflow device, the multifunctional pipette, to test whether mitochondria in mouse skeletal muscle connect to each other. The central channel in the pipette delivers compounds to a restricted region of the sarcolemma, typically 30 µm in diameter. Two channels on either side of the central channel use suction to create a hydrodynamically confined flow zone and remove compounds completely from the bulk solution to internal waste compartments. 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 [Ca(2+)] was monitored with fluo-3. A pulse of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 100 µM) applied to a small area of the muscle fibre (30 µm in diameter) 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 [Ca(2+)] was unchanged and fibres contracted in response to electrical stimulation. In conclusion, our 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 multifunctional pipette highlight its advantages over bulk application of compounds to isolated cells.