Myofiber Type Shift in Extraocular Muscles in Amyotrophic Lateral Sclerosis

Abstract

Purpose: To investigate changes in myofiber composition in the global layer (GL) and orbital layer (OL) of extraocular muscles (EOMs) from terminal amyotrophic lateral sclerosis (ALS) donors.

Methods: Medial recti muscles collected postmortem from spinal-onset ALS, bulbar-onset ALS, and healthy control donors were processed for immunofluorescence with antibodies against myosin heavy chain (MyHC) IIa, MyHCI, MyHCeom, laminin, neurofilaments, synaptophysin, acetylcholine receptor γ-subunit, and α-bungarotoxin.

Results: The proportion of myofibers containing MyHCIIa was significantly smaller and MyHCeom was significantly larger in the GL of spinal-onset ALS and bulbar-onset ALS donors compared to control donors. Changes in the GL were more prominent in the bulbar-onset ALS donors, with a significantly larger proportion of myofibers containing MyHCeom being present compared to spinal-onset ALS donors. There were no significant differences in the myofiber composition in the OL. In the spinal-onset ALS donors, the proportions of myofibers containing MyHCIIa in the GL and MyHCeom in the OL were significantly correlated with the disease duration. Neurofilament and synaptophysin were present at motor endplates of myofibers containing MyHCeom in ALS donors.

Conclusions: The EOMs of terminal ALS donors displayed changes in the fast-type myofiber composition in the GL, with a more pronounced alteration in bulbar-onset ALS donors. Our results align with the worse prognosis and subclinical changes in eye movement function previously observed in bulbar-onset ALS patients and suggest that the myofibers in the OL might be more resistant to the pathological process in ALS.

Figure 1.

Cross-sections of the OL and GL in the mid-portion of medial recti muscles from a control donor (OL, a1–a3; GL, b1–b3), a spinal-onset ALS donor (OL, c1–c3; GL, d1–d3), and a bulbar-onset ALS donor (OL, e1–e3; GL, f1–f3), triple-labeled for MyHCIIa (long arrows, green myofibers), MyHCI (arrowheads, red myofibers), and laminin (gray), which labels the basal lamina, thereby delineating the myofiber contours as well as making nerve fascicles and capillaries visible. A proportion of the myofibers was not labeled with either antibody and was inferred to contain MyHCeom (short arrows). The labeling of myofibers containing MyHCIIa was more homogeneous and stronger in the GL of the control donors (b1, b3) compared to the GL in both spinal-onset ALS donors (d1, d3) and bulbar-onset ALS donors (f1, f3). The labeling intensity of myofibers containing MyHCIIa was less heterogeneous in the OL of control donors (a1, a3) than both spinal-onset ALS donors (c1, c3) and bulbar-onset donors (e1, e3). Myofibers that were not labeled with antibodies against either MyHCIIa or MyHCI were more frequent in the GL of the spinal-onset ALS donors (d3) and even more abundant in the bulbar-onset ALS donors (f3) compared to the myofibers in the GL of control donors (b3). Scale bar: 50 µm.

Figure 2.

Identification of myofibers containing MyHCeom in cross-sections of the GL from a control donor (a1–a4), a spinal-onset ALS donor (b1–b4), and a bulbar-onset ALS donor (c1–c4). Triple immunolabeling with antibodies against MyHCeom (a1, b1, c1; green myofibers), MyHCI (a2, b2, c2; red myofibers), and MyHCIIa (a3, b3, c3; gray myofibers) is shown. Merged images are presented in a4, b4, and c4. The short arrows denote examples of myofibers containing MyHCIIa, and the arrowheads denote examples of myofibers containing MyHCI. Note that the myofibers unlabeled with antibodies against MyHCI and MyHCIIa were labeled with the anti-MyHCeom antibody (long arrows), confirming that they were correctly classified as myofibers containing MyHCeom. Scale bar: 50 µm.

Figure 3.

Box plots showing the proportions of myofibers containing MyHCIIa, MyHCeom, or MyHCI in the GL and OL of medial recti muscles in control donors (n = 6), spinal-onset ALS donors (n = 9), and bulbar-onset ALS donors (n = 8). (a) Proportion of myofibers containing MyHCIIa in the GL. (b) Proportion of myofibers containing MyHCeom in the GL. (c) Proportion of myofibers containing MyHCI in the GL. (d) Proportion of myofibers containing MyHCIIa in the OL. (e) Proportion of myofibers containing MyHCeom in the OL. (f) Proportion of myofibers containing MyHCI in the OL. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4.

Scatterplots showing a significant negative correlation of the proportion of the myofibers containing MyHCIIa or MyHCeom in the GL (a) and OL (b) of control donors (n = 6), spinal-onset ALS donors (n = 9), and bulbar-onset ALS donors (n = 8) combined. In the spinal-onset ALS donors, disease duration was significantly correlated with the proportion of myofibers containing MyHCIIa in the GL (c) and MyHCeom in the OL (d).

Figure 5.

ROC analysis of the proportion of myofibers containing MyHCIIa in the GL of control donors (n = 6) versus all ALS donors (n = 17), yielding a significant AUC = 0.863 (95% CI, 0.705–1.000; P = 0.010). Youden's index highest value of 0.706 yielded a MyHCIIa cut-off of 44.98% with a LR+ of 3.4 for differentiating control donors and ALS donors.

Figure 6.

Immunolabeling with antibodies against neurofilaments (NF-M or NF-H, left column) at motor endplates identified with AChRγ, α-BTx at postsynaptic regions, or synaptophysin (SYN, middle column) at presynaptic nerve terminals in myofibers containing MyHCeom (long arrows) in the GL of EOMs in cross-sections from a control donor (a1–a3), a spinal-onset ALS donor (b1–b3), and a bulbar-onset ALS donor (c1–c3). Note that NMJs were mostly observed in myofibers containing MyHCeom, and immunolabeling of NF-M or NF-H was present at their motor endplates. NF-M was also present at NMJs in myofibers containing MyHCIIa (d1–d3, short arrows) in longitudinal section from a bulbar-onset ALS donor. Scale bar: 50 µm.

Figure 7.

Longitudinal consecutive sections (a1–a5 and b1–b3) of EOMs in the GL from a bulbar-onset ALS donor showing immunoreactivity with antibodies against synaptophysin (SYN, green, arrows in a1 and a4) and neurofilament (NF-H, red, arrows in a3 and a4) at multiterminal endplates in one myofiber containing MyHCeom (*). NMJs were identified by α-BTx (red, arrows in a2). Sequential immunolabeling with different combinations of antibodies was performed to correlate the presence of synaptic proteins at NMJs with the myofiber types. The myofiber unlabeled with antibodies against MyHCIIa (gray in a4) and MyHCI (gray in b1) but labeled with antibody against MyHCeom (green in a5 and green in b3, respectively) was identified as a myofiber containing MyHCeom (*). Scale bar: 50 µm.