Three dimensional visualisation and quantitative analysis of mitochondrial networks in human skeletal muscle

Vincent, A.E.1, White, K.2, Davey, T.2, Philips, J.1, Hall, M.G.3, Ng, Y.S.1, Falkous, G.1, Holden, T.1, Taylor, R.W.1, Turnbull, D.M.1, Picard, M4

1Wellcome Centre for Mitochondrial Research, Newcastle University; Newcastle, UK
2Electron Microscopy Research Services, Newcastle University; Newcastle, UK
3Institute of Child Health, University College London, London, UK
4Division of Behavioral Medicine, Dept. of Psychiatry, Dept. of Neurology, Columbia University, New York, USA

Mitochondrial morphology is linked to mitochondrial function, but the three-dimensional organization of the mitochondrial network in human skeletal muscle (SKM) has not been defined. Here we used serial block face scanning electron microscopy (SBF-SEM) and IMOD analysis to visualise and quantify mitochondria. Mitochondrial morphology was quantified in SKM biopsies from controls (n=8) and mitochondrial disease patients with single, large scale mtDNA deletions (n=2), m.8344A>G (n=3) or m.3243A>G (n=1). Branching was assessed in transverse and longitudinal planes, and a 3D mitochondrial shape descriptor, the Mitochondrial complexity index (MCI), developed. All methods were validated in mouse muscle.
Mitochondria morphology is highly variable in controls. MCI was variable within SKM fibres (Coefficient of Variance (C.V.) (11.7-170.6%), between SKM fibres (C.V. = 51.6-137.1%), as well as between patients (C.V. = 53.8%). This was also true of mitochondrial volume. In m.8344A>G patients, increasing mtDNA heteroplasmy was associated with reduced MCI, consistent with fragmentation. 3D reconstructions and high-resolution EM revealed mitochondrial membrane protrusions not previously observed in humans. These mitochondrial “nanotunnels” are more frequent in patients than controls. When investigating mitochondrial networks between groups, computer-based classifier algorithms identified the proportion of very small mitochondria as most discriminant parameter between paients and controls. Assessment of mitochondrial branching in the longitudinal and transverse orientations of SKM fibres demonstrated mitochondrial anisotropy.
Because connected mitochondria exchange mtDNA and other molecules, network organisation and branching directionality has implications for understanding the spread and segregation of mtDNA mutations. These findings also help to account for segmental respiratory chain deficiency.