The Spectrum of Mitochondrial Ultrastructural Defects in Mitochondrial Myopathy

Abstract

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling, and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease.

Figure 1. Normal mitochondrial ultrastructure.

(A) Transmission electron micrograph of normal mitochondria in human skeletal muscle showing typical tubular cristae, and crista junctions. (B) Schematic representation of crista junctions, associated structures, and parameters measured in this study. (C) Three-dimensional schematic of normal tubular cristae structure. The outer mitochondrial membrane (OMM, red), and three functionally distinct regions of the inner mitochondrial membrane (IMM): inner boundary membrane (blue), crista junction (orange), and crista membrane (green). (D) Same image as in A with pseudocolored adjacent but distinct mitochondria and outlined crisate membranes undergoing trans-mitochondrial cristae coordination (see ref. and text for discussion). (E) Unprocessed and (F) pseudocolored transmission electron micrographs of normal human skeletal muscle illustrating mitochondria with normal shapes and sizes, with electron-dense curvilinear cristae, with some exhibiting trans-mitochondrial cristae coordination.

Figure 2. Paracrystalline inclusions (PCIs).

(A) Type I PCIs occupying most of a mitochondrion’s volume in skeletal muscle with a single mtDNA deletion (patient 1). (B) Linear Type I PCI in mitochondrion from a case of m.8344A>G (patient 5). (C) Disruption of IMM and OMM (arrowhead) by a rigid type II PCI, and (D) other examples of type II PCIs from a case of m.8344A>G (patient 4). (E) Subsarcolemmal region of a muscle fiber showing the extracellular space (EC, yellow) and sarcolemma, profile of a nucleus (N, blue), myofibrils (MF), mitochondria with PCI (red) and normal mitochondria (green), with (F) pseudocolored mask (patient 1). Note the greater abundance of PCI-containing mitochondria in the perinuclear SS compartment compared to the intermyofibrillar compartment, consistent with the distinctive requirement for de novo protein synthesis for PCI formation.

Figure 3. Linearization and geometrical cristae features.

(A,B) Enlarged mitochondria containing linearized cristae juxtaposed at nearly identical angles, and (C) linearized cristae forming various geometrical shapes, and (D) linearized IMM segments (arrows) joined by curved segments from a case of m.8344A>G (patient 4). (E) SBF-SEM image with example of linearized cristae and geometrical shapes and (F) three-dimensional reconstruction of mitochondrion in E (patient 4).

Figure 4. Concentric “onion-shaped” cristae.

(A) Multiple overlaid layers of double OMM/IMM membranes forming two major compartments in a case of the m.8344A>G (patient 4), and (B) two-dimensional reconstruction of membrane structures. (C) Example of concentric cristae compartments (patient 4) imaged with SBF-SEM, with (D) corresponding three-dimensional reconstruction.

Figure 5. Compartmentalization.

(A) Example of membrane-bound sub-mitochondrial compartments located centrally and (B) peripherally in contact with the inner boundary membrane, from cases of m.8344A>G mutation (patient 5) and single mtDNA deletion (patient 3), respectively. (C) Electron-dense round compartments distributed in the mitochondrial matrix in a case of single mtDNA deletion (patient 3). (D) Compartment bound by linearized electron-dense membranes in a case of m.8344A>G (patient 4). (E) Cross-sectional image of a mitochondrion with two distinct compartments of different electron density, and (F) three-dimensional reconstruction from SBF-SEM. (G,H) Examples of OMM protrusion and distension consistent with the release of mitochondrial components in the cytoplasm in a case of m.8344A>G mutation (patient 4). Pseudocolored areas indicate the major compartment bound by the OMM (green), and sub-compartments (red and blue).

Figure 6. Nanotunelling and hyperbranching.

(A,B) Examples of mitochondrial nanotunnels composed of both OMM and IMM, but devoid of cristae in a case of m.3243A>G (patient 7). (C) Variation in nanotunnel diameter, with each datapoint representing the average of the smallest and largest diameters measured for individual nanotunnels across all subjects (n = 26). (D) Three-dimensional reconstruction of three mitochondria connected via nanotunnels from a case of m.3242A>G (patient 7). (E,F) Examples of highly branched mitochondria (hyperfusion) from cases of m.3243A>G (patient 7) (G) Donut-shaped mitochondrion denoting self-fusion from a case of single mtDNA deletion (patient 2). (H) Three-dimensional reconstruction of a donut-shaped mitochondrion from a case of m.8344A>G (patient 6).