Characterization and temporal development of cores in a mouse model of malignant hyperthermia

Abstract

Malignant hyperthermia (MH) and central core disease are related skeletal muscle diseases often linked to mutations in the type 1 ryanodine receptor (RYR1) gene, encoding for the Ca(2+) release channel of the sarcoplasmic reticulum (SR). In humans, the Y522S RYR1 mutation is associated with malignant hyperthermia susceptibility (MHS) and the presence in skeletal muscle fibers of core regions that lack mitochondria. In heterozygous Y522S knock-in mice (RYR1(Y522S/WT)), the mutation causes SR Ca(2+) leak and MHS. Here, we identified mitochondrial-deficient core regions in skeletal muscle fibers from RYR1(Y522S/WT) knock-in mice and characterized the structural and temporal aspects involved in their formation. Mitochondrial swelling/disruption, the initial detectable structural change observed in young-adult RYR1(Y522S/WT) mice (2 months), does not occur randomly but rather is confined to discrete areas termed presumptive cores. This localized mitochondrial damage is followed by local disruption/loss of nearby SR and transverse tubules, resulting in early cores (2-4 months) and small contracture cores characterized by extreme sarcomere shortening and lack of mitochondria. At later stages (1 year), contracture cores are extended, frequent, and accompanied by areas in which contractile elements are also severely compromised (unstructured cores). Based on these observations, we propose a possible series of events leading to core formation in skeletal muscle fibers of RYR1(Y522S/WT) mice: Initial mitochondrial/SR disruption in confined areas causes significant loss of local Ca(2+) sequestration that eventually results in the formation of contractures and progressive degradation of the contractile elements.

Fig. 1.

Abnormal mitochondria are confined to discrete subcellular domains termed presumptive cores in FDB fibers from 2–4-month-old RYR1^Y522S/WTmice. (A and B) Two different areas of the same muscle fiber are shown: one with apparently normal mitochondrial disposition and morphology (A, black arrows) and another, within a presumptive core region, containing swollen and structurally disrupted mitochondria (B, white arrows). (C) Representative tomographic slice of an abnormal mitochondrion. (D–F) Respective 3D reconstructions in which shading is used to provide depth. White arrowheads in E and F point to irregularities (grooves and indentation) of the outer membrane.

Fig. 2.

Severe mitochondrial damage is associated with local disruption of adjacent SR, T-tubules, and contractile elements within early cores. (A) Large open arrows point to localized areas of damage termed early cores. Black arrows, marking Z lines, highlight local sarcomeric misalignment. The area marked by the asterisk in A is enlarged in B. (B) Early core showing severely damaged mitochondria (white arrows) and disrupted membrane systems (star). (C and D) Significant distortion and dilation of T-tubules (arrows) and misorientation of triads in two early cores. (E–H) Anti-RYR labeling (E and G) and selective T-tubule staining (F and H) are used to emphasize triad and T-tubule positioning (F, arrows). Arrows in E mark the position of Z lines. The proper positioning of triads along two transverse bands/sarcomeres (E) and of T-tubules at the A-I junction (F) are disrupted in confined regions of fibers (early cores) from RYR1^Y522S/WT mice (G and H, dashed ovals). The star in G marks the position of a nucleus.

Fig. 3.

Contracture cores are frequent at 1 year of age and are characterized by severe sarcomere shortening in areas lacking mitochondria and SR. (A–C) A large contracture core (lower part of the micrograph) in a Soleus fiber from a 1-year-old RYR1^Y522S/WT mouse (A). Contracture cores are characterized by short sarcomere length and near-complete lack of mitochondria and SR (C), whereas both mitochondria and triads are present in apparently normal adjacent areas (B). (D–F) Immunolabeling of mitochondria (E, anti-apoptosis-inducing factor) and triads (F, anti-RYR) confirms the absence of SR and mitochondria in contracture cores (arrowheads in D, phase contrast). Other examples are shown in SI Text.

Fig. 4.

Large unstructured cores define areas of the fiber with near-complete degeneration of myofibrils and intracellular organelles. Large unstructured cores (large open arrows) are common at 1 year of age in both EDL and Soleus, although somewhat more frequent in EDL. They are often in proximity to contracture cores (lower left corner of A).

Fig. 5.

Proposed model for core formation in RYR1^Y522S/WT fibers. Local mitochondrial damage within presumptive cores and concomitant disruption of the sarcotubular system in early cores occur in young (2–4 months) RYR1^Y522S/WT mice. At later stages (≈1 year), contracture cores and unstructured cores are frequently observed. The potential contributions of increased SR Ca²⁺ leak (14) and ROS/RNS stress (15) to the initial mitochondrial/SR damage and subsequent formation of contracture and unstructured cores observed in muscle from RYR1^Y522S/WT mice are shown in gray, because those steps remain to be demonstrated.