Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study

Abstract

Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology.

Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies

“…Thus SSM may serve as a protective barrier to the cell, maintaining permissive oxygen levels within (66,122). Similar findings have been reported in mouse skeletal muscle using electron microscopy, suggesting physical continuities between SSM and IFM (101). Additionally, considerable evidence from network modeling studies suggests that cardiac mitochondria communicate across the cell (at distances of up to ϳ100 m), providing a platform for synchronized mitochondrial behavior (147).…”

Mitochondrial dysfunction and mitophagy defect triggered by heterozygous GBA mutations

“…The ratio of MitoRed to MitoGreen, which reports levels of polarized functional mitochondria, was significantly lower in Gba L444P/WT neurons than in controls, supporting impaired mitochondrial function (Figure 2(A)). We then assessed mitochondrial morphology by calculating the aspect ratio (length to width ratio) of individual mitochondria labeled by MitoGreen [17], which was increased in Gba L444P/WT neurons, indicating an elongated mitochondrial phenotype in the mutants (Figure 2(B)). The form factor, which reflects mitochondrial complexity and branching, remained similar between WT controls and mutant neurons.

10.1080/15548627.2018.1509818-F0002Figure 2.Mitochondrial dysfunction in Gba L444P/WT mouse hippocampal neurons (A) Representative micrographs of MitoTracker Red- and MitoTracker Green-labeled WT and Gba L444P/WT primary hippocampal neurons in primary cultures (14–15 DIV).

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“…Image stacks were acquired with a Leica Multiphoton system, projected and binarized before the analysis. Mitochondrial morphological characteristics, including mitochondrial aspect ratio (the ratio between the major and minor axes of the ellipse equivalent to the mitochondrion), degree of branching or form factor ((perimeter 2 )/(4π· surface area)) [17], were quantified using ImageJ Mito-Morphology Macro (http://imagejdocu.tudor.lu/lib/exe/fetch.php?media=plugin:morphology: mitochondrial_Morphology_macro_plug-in:mitophagy_ver _1_44_final_colocalization_f.txt).…”

Wnt-5a Ligand Modulates Mitochondrial Fission-Fusion in Rat Hippocampal Neurons

“…Z-projection was performed with those images to reconstruct 4.2-and 4.8-m thickness from the whole neuron. Sphericity of each reconstructed mitochondria was determined as described (31).…”