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.

Fig. 1.

Subsarcolemmal and intermyofibrillar mitochondrial morphology in intact mouse soleus muscle fibers. Scanning electron microscopy (EM) of a freeze-fractured muscle sample (A) and transmission EM in the longitudinal plane (B), showing a myofiber's outer surface lined with collagen fibers forming the extracellular matrix. A breach within the plasma membrane exposes globular subsarcolemmal (SS) mitochondria in A, which can be observed in B between the plasma membrane and myofibrils. Scanning EM of a freeze-fractured myofiber (C) and transmission EM in the transverse plane (D) exposing elongated intermyofibrillar (IMF) mitochondria clustering around Z-lines between sarcomeres. Insets represent lower magnification micrographs with regions magnified in the main panels. PM, plasma membrane; Myofibr, myofibrils; S, sarcomeres; Cap, capillary. Scanning electron microscopy (SEM) images were captured at magnifications of ×10,300 (A) and ×1,140 (A, inset), and ×5,800 (B) and ×1,310 (C, inset).

Fig. 2.

Interactions among intermyofibrillar mitochondrial membranes. Transmission EM of myofibers in the longitudinal plane showing distinct mitochondria on either sides of the Z-line (A) or continuous mitochondria spanning the Z-lines (B and C; arrowheads). C and D: mitochondrial contact sites characterized by electron densities localized at the outer mitochondrial membranes. C' and D': higher magnifications demonstrating electron-dense outer membranes of adjacent mitochondria that become indistinguishable at the contact sites. The arrowhead in C' indicates separation of the outer and inner mitochondrial membranes suggestive of forces acting upon the outer membrane. E: an irregularly shaped mitochondrion with distinct matrix spaces bound by (E') distinct inner mitochondrial membranes (arrows), but surrounded by a common outer mitochondrial membrane (arrowhead). OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; MA, mitochondrial matrix.

Fig. 3.

Electron dense mitochondrial contact sites among SS and IMF mitochondria. Transmission EM showing representative electron dense mitochondrial contact sites among subsarcolemmal (SS; A) and intermyofibrillar (IMF; B) mitochondria. Arrowheads indicate electron densities associated with membrane juxtaposition; the arrow indicates a restricted but continuous neck between two mitochondrial units.

Fig. 4.

Physical interactions between SS and IMF mitochondria. Transmission electron micrograph of myofibers in the transverse plane. A: SS and IMF mitochondria are distinct organelles. B and C: some SS and IMF mitochondria form continuous organelles (arrowheads) that coexist in both subcellular compartments. SS, subsarcolemmal; IMF, intermyofibrillar; PM, plasma membrane (sarcolemma); Myofibr, Myofibrils; Cap, capillary.

Fig. 5.

Three-dimensional visualization of intermyofibrillar mitochondrial morphology in muscle fibers. A: schematic representation of a myofiber sectioned in the two major planes. A combination of transmission EM in the longitudinal (B) and transverse planes (C) reveals the complexity of intermyofibrillar (IMF) mitochondria within the natural organization of sarcomeres. The Z-line is not visible in C and highly branched mitochondria are apparent due to the almost perfect transverse orientation of the muscle fiber. D: scanning EM micrograph showing how the freeze-fracture process tends to rupture sarcomeres at the I band near the Z-line, where thick filaments are absent and most IMF mitochondria are located. Myofibers are composed of multiple sarcomeres arranged in series, where each sarcomeric plane is populated by reticular mitochondria along its transverse axis (see also Fig. 1C). Insets show B: IMF mitochondria in a zone of high mitochondrial density, note also sarcomeric shortening due to myofiber contraction. C: a minimally inclined transverse section showing an expanded Z-line (dark line) and mitochondria on either sides; D: lower magnification of the main image showing the whole-width myofiber after freeze-fracture. SEM images were captured at magnifications of ×5,800 (D), ×2,900 (D, inset). S, sarcomere; M, mitochondria.

Fig. 6.

Quantification of form factor and aspect ratio among SS and IMF mitochondria. Mitochondrial form factor and aspect ratio were quantified by manually tracing SS (A) and IMF (B) mitochondria, and plotted against each another. Electron micrographs numbered 1–4 correspond to individual mitochondria plotted above. Inset in A shows mean values and 95% confidence internal (CI) for form factor and aspect ratio among both SS and IMF subpopulations. *Denotes statistical significance (P < 0.01) compared to SS. n = 505 SS and 563 IMF mitochondria.

Fig. 7.

Analysis of morphological parameters in SS and IMF mitochondria. A: multiple shape descriptors were determined for manually traced SS and IMF mitochondria. Bars represent the range of values that contain 50% of all mitochondria (interquartile range) for each parameter. B–H: frequency distributions (%total mitochondria) were plotted for each shape descriptor with 20 bins of equal sizes. n = 505 SS and 563 IMF mitochondria.