Usage of a localised microflow device to show that mitochondrial networks are not extensive in skeletal muscle fibres.

Joseph Bruton,Gavin D M Jeffries,Håkan Westerblad,Agustin Guerrero-Hernandez

doi:10.1371/journal.pone.0108601

Joseph Bruton, Gavin D M Jeffries + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0108601

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Abstract

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 [Ca2+] 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 [Ca2+] 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.

Highlights

In mammalian cells, mitochondria exist in a variety of forms from the almost universal picture of an ovoid structure not more than one mm long seen in cells ranging in size from hepatocytes to neurones to the long thread-like branching structures attaining a length of 50 mm or longer found in human fibroblasts [1,2]
Even when relatively large areas of the fibre were exposed to the FCCP, mitochondrial depolarization was confined to those mitochondria underneath and very close to the site of FCCP application
Mitochondrial depolarisation was not detected in mitochondria that lay as little as 50 mm away from the border of the FCCP application

Summary

Introduction

Mitochondria exist in a variety of forms from the almost universal picture of an ovoid structure not more than one mm long seen in cells ranging in size from hepatocytes to neurones to the long thread-like branching structures attaining a length of 50 mm or longer found in human fibroblasts [1,2]. Mitochondria in organisms as diverse as fungi and mice can adapt quickly to metabolic disturbances within the cell. Dynamic formation of mitochondria to mitochondria connections has been demonstrated in cells as diverse as cortical neurones [1] and fibroblasts [2]. These connections can form and break quite readily and allow diffusion of fluorescent labels between distant mitochondrial areas which suggest that the mitochondrial matrix has a uniform internal ionic and protein solution [8,9,10,11,12]

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