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.

Amyotrophic lateral sclerosis (ALS) is a fatal and heterogeneous neurodegenerative syndrome characterized by loss of upper and lower motor neurons and their tracts, leading to spasticity, muscle weakness, muscle atrophy, paresis of skeletal muscles, and respiratory failure.^1,2 Symptom onset commonly starts in an upper or lower limb, termed spinal-onset ALS, or in the head and neck region, presenting with dysarthria or dysphagia, termed bulbar-onset ALS.^1,2 The median survival time from symptom onset to death is approximately 2 to 4 years, with a shorter disease duration for bulbar-onset ALS patients.^3–6 Numerous genes have been associated with ALS; the two most common are mutations in the superoxide dismutase 1 gene (SOD1) and a hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 gene (C9orf72HRE).^7,8 

Patients with ALS typically show few to no clinical symptoms from the extraocular muscles (EOMs), even late in the course of the disease.^9–11 The EOMs have also previously been shown to respond differently to both aging and muscular dystrophies compared to limb muscles.¹² The EOMs differ significantly from other striated muscles^13,14 and have a unique and complex myosin heavy chain (MyHC) composition, which includes the concurrent presence of several MyHC isoforms along the length of the same myofiber,^15,16 rare MyHC isoforms such as MyHC extraocular muscle (MyHCeom), MyHC slow-tonic (MyHCsto), and isoforms typically associated with muscle development (e.g., MyHC embryonic).¹⁶ Despite the complexity of the EOMs, three major myofiber groups can be identified in the human EOMs on the basis of their MyHC composition: (1) myofibers containing MyHCI and/or MyHCsto, (2) myofibers containing MyHCIIa, and (3) myofibers lacking these MyHC isoforms but containing MyHCeom.¹⁶ We have previously shown that the EOMs are remarkably spared in terminal stages of ALS, showing only mild myofiber hypertrophy, atrophy and increased connective tissue.¹⁷ We have also shown that the distal axonal loss seen in limb muscles of both the SOD1 G93A transgenic mouse model and terminal ALS donors is not seen in the EOMs, where instead the neuromuscular junctions (NMJs) are well preserved.^18,19 However, the proportion of myofibers that are double-labeled for MyHCI and MyHCsto is significantly decreased in both the global layer (GL) and orbital layer (OL) of ALS donors, suggesting a selective denervation of EOMs in ALS.²⁰ Myofibers containing both MyHCI and MyHCsto are functionally distinct, with slow tonic contractions and specialized motor neurons,²¹ but they represent only about 15% of the myofibers in human EOMs.^16,20 The present study therefore sought to investigate the impact of ALS on the two remaining major groups of myofibers in human EOMs containing MyHCIIa or MyHCeom and to assess possible differences in myofiber type composition between spinal-onset and bulbar-onset ALS donors. 

Materials and Methods

Muscle Samples

Medial recti muscles were collected postmortem from human control donors (n = 6) with no previous history of neurologic disorder and from terminal spinal-onset ALS (n = 9) and bulbar-onset ALS (n = 8) donors. The ALS diagnosis was based on the European Federation of Neurological Societies guidelines for clinical management of ALS.²² The study was approved by the Regional Medical Ethical Review Board in Umeå. Informed consent was obtained in accordance with the stipulations of the Swedish law for ethical approval and the Swedish autopsy law, and the study adhered to the tenets of the Declaration of Helsinki. The medial recti muscles were collected, frozen, and processed for sectioning as previously described.²⁰ A Leica CM3050 cryostat microtome (Leica Biosystems, Nussloch, Germany) set at −23°C was used to cut 5- to 6-µm serial whole muscle cross-sections or longitudinal sections in the mid-portion of each EOM. The muscle sections were collected on SuperFrost Plus adhesion slides (Thermo Fisher Scientific, Waltham, MA, USA) and kept at −20°C until processing for indirect immunofluorescence. 

Antibodies and Immunofluorescence

Antibodies used in the present study are presented in Supplementary Table S1. Mouse monoclonal antibodies A4.74 against MyHCIIa, BA-D5 against MyHCI, and 4A6 against MyHCeom (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) were used to classify different myofiber types. Rabbit polyclonal primary antibody against laminin was used to label myofiber contours (Z 0097; Dako Denmark A/S, Glostrup, Denmark). NMJs were identified by rhodamine-conjugated α-bungarotoxin (α-BTx) labeling (Molecular Probes, Eugene, OR, USA) and mouse monoclonal antibody against acetylcholine receptor (AChR) γ-subunit (AChRγ) (GTX74890; Gentex, Landskrona, Sweden), both of which bind to postsynaptic AChR on the plasma membrane of myofibers. Antibodies against neurofilament protein (a general marker for nerve fibers) were used to detect nerve fibers at motor endplates: rabbit polyclonal antibody against neurofilament 200 kDa (NF-H, N4142; Sigma-Aldrich, St. Louis, MO, USA), sheep polyclonal antibody against neurofilament 150 kDa (NF-M, AF3029; R&D Systems, Abingdon, UK), and mouse monoclonal antibody against neurofilament 70 kDa (NF-L, M0762; Dako Denmark A/S). In addition, synaptophysin (SY38; Boehringer Mannheim, Indianapolis, IN, USA) was also used as a marker for vesicles in nerve terminals at presynaptic regions. 

The glass slides were processed for double, triple, or sequential immunolabeling as previously described.²³ In brief, the glass slides were air-dried at room temperature for 20 minutes and rinsed for 3 × 5 minutes in 0.01-M PBS (Merck KGaA, Darmstadt, Germany) containing 0.05% Tween 20 (Sigma-Aldrich). The slides were blocked with 5% normal serum in PBS containing 0.1% bovine serum albumin for 15 minutes at room temperature. The glass slides were then incubated with the first antibody or mixtures of two antibodies in a moist chamber at 4°C overnight. The following day, the slides were rinsed and blocked with normal serum at room temperature as above, followed by the application of the appropriate secondary antibody or mixtures of two antibodies, and the glass slides were incubated at 37°C for 30 minutes. Afterward, they were rinsed and blocked with normal serum as previously, and then the second or third primary antibody was applied for 1 hour at 37°C, followed by a similar rinsing and blocking procedure. The appropriate secondary antibody was applied and incubated at 37°C for 30 minutes. Finally, the slides were rinsed in PBS and the cover glasses were mounted with VECTASHIELD Mounting Medium (H-1000; Vector Laboratories, Burlingame, CA, USA). 

A multiple and sequential immunolabeling procedure was performed in order to evaluate the innervation pattern in different myofiber types following microscopic evaluation and photographing. For example, a glass slide that had been immunolabeled with antibodies against AChRγ (donkey anti-mouse Alexa Fluor 488), NF-M (donkey anti-sheep Rhodamine Red-X), and MyHCI (goat anti-mouse Alexa Fluor 647), followed by evaluation and photographing, was further incubated with the antibody against MyHCIIa (goat anti-mouse Alexa Fluor 647). Thus, we could examine the section, photograph it again, and classify the myofiber types. Control sections were treated as above, except that the primary antibodies were omitted. No labeling was observed in the control sections. 

Identification of Myofiber Types

Myofibers labeled with the antibody A4.74 against MyHCIIa were classified as myofibers containing MyHCIIa. Myofibers labeled with the antibody BA-D5 against MyHCI were classified as myofibers containing MyHCI. We confirmed that myofibers that were not labeled by either of these two antibodies were labeled with the antibody 4A6 against MyHCeom and could therefore be classified as myofibers containing MyHCeom, as previously described.¹⁶ 

Microscopy and Quantifications

During immunolabeling, microscopy, and quantifications, the clinical and genetic information about the donors was blinded to the investigator to ensure unbiased quantification. All of the glass slides were examined and photographed using a Leica DM 6000 B microscope with the software Leica Application Suite Advanced Fluorescence 2.5.0.6735 (Leica Microsystems, Wetzlar, Germany). Individual images covering the whole cross-section of each EOM were captured with a resolution of 1392 × 1040 pixels using 20× objective and digitally stitched by the software into a composite image of the whole cross-sectioned EOM. The border between the GL and OL was delineated as previously described.²⁰ At least 10 of the individual images were randomly selected from the entire cross-section of each EOM. All of the randomly selected images were analyzed, and the myofibers were quantified manually by using Adobe Photoshop CS6 Extended version 13.0.6 × 64 (Adobe Systems, San Jose, CA, USA). A counting frame was used to ensure unbiased quantification of myofibers of variable size.²⁴ Each myofiber type was classified and quantified in each image, and the proportion of myofibers with each labeling pattern was calculated. 

The total cross-sectional area of the GL and OL in the whole cross-sectioned EOMs as well as each analyzed captured image was calculated by using the marking tool provided in Adobe Photoshop CS6 Extended, excluding areas with large rifts or separations. The indicated number of pixels was converted to the estimated cross-sectional area (mm²) using the pixel ratio specified for the camera and the objective. The myofiber density in the GL and OL was estimated by adding the total number of myofibers and dividing it with the total cross-sectional area of the image analyzed. The total myofiber count for each cross-sectioned EOM was estimated by multiplying the myofiber density with the estimated total area of the GL and OL. 

Statistical Analysis

The statistical analysis was performed using IBM SPSS Statistics version 26 (IBM, Armonk, NY, USA). The proportions of myofiber types, myofiber density, cross-sectional area, and the total number of myofibers are presented as median and lower quartile (Q1) to upper quartile (Q3). The group comparisons were carried out using the independent samples t-test for two groups or one-way ANOVA with planned comparison contrast tests for three groups. A bias-corrected and accelerated 95% confidence interval (CI) bootstrap set at 3000 sample runs was used when performing the group comparisons. The Welch test was used when performing the group comparisons when homogeneity of variance could not be assumed. The a priori hypotheses for the group comparisons were as follows: (1) the EOMs in spinal-onset ALS and bulbar-onset ALS donors differ in myofiber composition compared to the control donors, and (2) the EOMs in spinal-onset ALS and bulbar-onset ALS donors differ in myofiber composition when compared to each other. Bivariate correlation tests were performed using Pearson's correlation coefficient (r) when a linear trend could be observed and Spearman's rank-order correlation coefficient (ρ) when a linear trend could not be observed. Receiver operating characteristic (ROC) analysis was performed, reporting the area under the ROC curve (AUC) and 95% CI for AUC. Youden's index highest value was used to determine the optimal cut-off for the myofiber type proportion, sensitivity, specificity, positive likelihood ratio (LR+), and negative likelihood ratio (LR−). Findings were considered statistically significant for P < 0.05. 

Results

Clinical and genotype data for the ALS donors are presented in Table 1. A heterogeneity in the genotype of ALS donors was observed, including mutation in SOD1, C9orf72HRE, mutation in the vesicle-associated membrane protein-associated protein B gene (VAPB), and mutation in the kinesin family member 5A gene (KIF5A) (Table 1). Group comparisons for the different genotypes were not performed due to the heterogeneity of genotypes identified in the ALS donors in relation to the sample size of the study population. The control donors included five males and one female, and the mean age at death for the control donors was 66.2 years (range, 42–82). The three major myofiber types containing MyHCI or MyHCIIa or lacking both of these isoforms but containing MyHCeom were present in both the GL (Figs. 1, 2) and OL (Fig. 1) of the EOM specimens in all donors studied. The proportions of the different myofiber types are presented for the GL and OL of control donors, spinal-onset ALS donors, and bulbar-onset ALS donors in Figure 3. Furthermore, the myofiber proportions in the GL and OL of control donors, all ALS donors as a combined group, spinal-onset ALS donors, and bulbar-onset ALS donors are presented in Table 2. A total of 69,331 myofibers were quantified in the EOM samples of the donors; 21,737 myofibers in control donors (GL: 13,388, OL: 8349), 24,983 myofibers in spinal-onset ALS donors (GL: 17,383, OL: 7600), and 22,611 myofibers in bulbar-onset ALS donors (GL: 16,230, OL: 6381). In general, a heterogeneous labeling pattern for myofibers labeled for MyHCIIa in the GL was observed. For quantitative consistency, all fibers labeled with the MyHCIIa antibody were treated as MyHCIIa-positive fibers, regardless of the labeling intensity. Occasionally, myofibers displaying double-labeling for MyHCI and MyHCIIa were present in some control donors and ALS donors, but these were exceedingly rare (not shown). 

Table 1.

 

Clinical and Genotype Data for the Spinal-Onset and Bulbar-Onset ALS Donors

 

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.

Table 2.

 

Proportion of the Different Myofiber Types in the GL and OL of EOMs

 

Myofiber Types in Control EOMs

In the EOM samples of control donors, the majority of the myofibers were labeled with the antibody against MyHCIIa (GL: 59.59%; 47.34%–78.24%; OL: 82.13%, 72.94%–82.93%). Myofibers containing MyHCIIa showed a very homogeneous and strong labeling intensity in the OL where myofibers were also notably smaller compared to those in the GL (Fig. 1). Labeling intensity of myofibers containing MyHCIIa in the GL was more heterogeneous, with some fibers having a strong labeling intensity and others having a moderate labeling intensity (Fig. 1). A smaller proportion of myofibers were labeled with the antibody against MyHCI in the EOM samples of all control donors (GL: 14.87%, 13.58%–17.48%; OL: 15.06%, 14.07%–16.41%), and rather evenly distributed across the whole EOM cross-section with a strong labeling intensity in both the GL and OL (Fig. 1). The myofibers containing MyHCeom accounted for the remainder of the myofibers (GL: 25.98%, 6.03%–34.93%; OL: 2.40%, 0.83%–12.55%). 

Myofiber Types in ALS EOMs

In most of the EOM specimens from the ALS donors, the labeling intensity of MyHCIIa was more heterogeneous in the GL compared to the control donors (Fig. 1). The labeling intensity of myofibers containing MyHCIIa was generally more homogeneous in the OL compared to the GL; however, notable variations in labeling intensity in the OL were present in both spinal-onset ALS and bulbar-onset ALS donors (Fig. 1). Discrete variations in labeling intensity of myofibers containing MyHCI were observed in some of the EOM specimens from ALS donors. 

There were no significant differences in the proportion of myofibers containing MyHCIIa, MyHCeom, or MyHCI in the GL or OL between males and females in the ALS donors (P > 0.05). There were no significant correlations between age at death and the proportion of myofibers containing MyHCIIa, MyHCeom, or MyHCI in the GL or OL in the ALS donors (P > 0.05). In the GL, there was a significant decrease in the proportion of myofibers containing MyHCIIa in both spinal-onset ALS and bulbar-onset ALS donors compared to control donors (P < 0.05 and P < 0.01, respectively) (Fig. 3) and a significant increase in the proportion of myofibers containing MyHCeom in both spinal-onset ALS and bulbar-onset ALS donors compared to control donors (P < 0.05 and P < 0.001, respectively) (Fig. 3). Furthermore, there was a significant increase in the proportion of myofibers containing MyHCeom in the bulbar-onset ALS donors when compared to the spinal-onset ALS donors (P < 0.05) (Fig. 3). Although the proportion of myofibers containing MyHCIIa was notably lower in the GL of bulbar-onset ALS donors compared to spinal-onset ALS donors, this difference did not reach statistical significance (P > 0.05) (Fig. 3). The proportion of myofibers containing MyHCIIa was notably lower and the proportion of myofibers containing MyHCeom was notably higher in the OL of bulbar-onset ALS donors; however, no significant differences in the proportion of these myofiber types in the OL were detected when comparing the three groups (P > 0.05) (Fig. 3). No significant differences in the proportion of myofibers containing MyHCI were detected in the GL or the OL of spinal-onset ALS donors, bulbar-onset ALS donors, and control donors (P > 0.05) (Fig. 3). Including control donors, spinal-onset ALS donors, and bulbar-onset ALS donors in the correlation analyzes, there were significant negative correlations between the proportion of myofibers containing MyHCIIa and the proportion of myofibers containing MyHCeom in both the GL (r = −0.989, P < 0.0001) (Fig. 4) and OL (r = −0.984, P < 0.0001) (Fig. 4). 

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).

Table 3 presents the estimates for the GL and OL of the myofiber density, cross-sectional area, total number of myofibers, and the proportion of the EOMs constituting the GL and OL in control donors, all ALS donors as a merged group, spinal-onset ALS donors, and bulbar-onset ALS donors. The median cross-sectional areas of the EOMs were 29.7 mm² (24.1–42.8 mm²) in control donors, 43.6 mm² (33.9–55.0 mm²) in the ALS donors as a merged group, 41.6 mm² (31.9–45.1 mm²) in the spinal-onset ALS donors, and 50.0 mm² (35.5–67.9 mm²) in the bulbar-onset ALS donors. The median total number of myofibers in the EOMs was 18902 (15,472–26,926) myofibers in the control donors, 21,480 (15,808–28,293) myofibers in all ALS donors as a combined group, 20,036 (15,642–28,887) myofibers in spinal-onset ALS donors, and 25,281 (16,653–28,108) myofibers in the bulbar-onset ALS donors. The whole area of the cross-sectioned EOMs, the total number of myofibers, and the relative proportions of the GL and OL did not differ significantly among control donors, spinal-onset ALS donors, and bulbar-onset ALS donors (P > 0.05). However, when comparing the cross-sectional area of the GL and OL separately, the GL was significantly larger in the bulbar-onset ALS donors compared to control donors (P < 0.05), whereas no significant differences in the area of the GL were observed when comparing spinal-onset ALS donors to control donors or bulbar-onset ALS donors (P > 0.05). No significant differences in the cross-sectional area in the OL were detected when comparing control donors, spinal-onset ALS donors, and bulbar-onset ALS donors to each other (P > 0.05). Furthermore, the myofiber density was significantly lower in the GL in the bulbar-onset ALS donors compared to control donors (P < 0.01), whereas no significant differences were observed when comparing the myofiber density in the GL in spinal-onset ALS donors to control donors or bulbar-onset ALS donors (P > 0.05). The myofiber density in the OL was significantly lower in the bulbar-onset ALS donors compared to the spinal-onset ALS donors (P < 0.05), whereas no significant differences in the myofiber density in the OL were detected when comparing control donors to spinal-onset ALS donors or bulbar-onset ALS donors (P > 0.05). In the GL of the ALS donors, the myofiber density was significantly positively correlated with the proportion of myofibers containing MyHCIIa (r = 0.551, P = 0.022) and significantly negatively correlated with the proportion of myofibers containing MyHCeom (r = −0.496, P = 0.043), whereas the proportion of myofibers containing MyHCI did not show a significant correlation with the myofiber density (P > 0.05). No significant correlations were detected between the myofiber density and the proportion of myofibers containing MyHCIIa, MyHCeom, or MyHCI in the OL of ALS donors (P > 0.05). 

Table 3.

 

Estimates of the Myofiber Density, Cross-Sectional Area, Total Number of Myofibers, and Proportions of the GL and OL in the EOMs

 

In the spinal-onset ALS donors, disease duration was significantly correlated with the proportion of myofibers containing MyHCIIa in the GL (ρ = 0.700, P = 0.036) (Fig. 4) and the proportion of myofibers containing MyHCeom in the OL (ρ = 0.733, P = 0.025) (Fig. 4), whereas none of the remainder myofiber proportions in the GL or the OL showed any significant correlation with the disease duration (P > 0.05). There were no significant correlations between disease duration and the myofiber proportions in the GL or the OL of bulbar-onset ALS donors (P > 0.05). ROC analysis of the proportion of myofibers containing MyHCIIa was performed for control donors versus all ALS donors (Fig. 5), yielding an AUC = 0.863 (95% CI, 0.705–1.000; P = 0.010), where Youden's index highest value of 0.706 yielded a MyHCIIa cut-off of 44.98% for differentiating control donors and ALS donors (sensitivity, 100%; specificity, 70.6%; LR+, 3.4; LR−, 0.0). 

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.

Neurofilaments and Synaptophysin at Motor Endplates

We used general markers of neurofilaments and synaptophysin, which are crucial proteins at the nerve terminal or NMJs, in order to further understand the significant changes in the proportion of myofibers containing MyHCIIa or MyHCeom in the GL of ALS donors. Immunolabeling with antibodies against neurofilaments (NF-H, NF-M, and NF-L) and synaptophysin were present in the vast majority of NMJs or motor endplates examined in the EOMs of control donors in the present study, and the immunoreactivity was similar in both spinal-onset ALS and bulbar-onset ALS donors as compared to control donors (Fig. 6). However, the absence of neurofilament immunolabeling was occasionally observed in the NMJs detected by labeling with α-BTx or AChRγ or at the nerve terminal identified by labeling with synaptophysin (not shown). 

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.

When investigating the immunoreactivity with antibodies against neurofilament and synaptophysin in different myofiber types in the GL of ALS donors, we found that myofibers labeled with α-BTx in which neurofilaments and synaptophysin immunoreactivity were present mostly contained MyHCeom. In contrast, only sporadic myofibers containing MyHCIIa were observed displaying NMJs with α-BTx in the GL of ALS donors. The myofibers containing MyHCIIa displayed immunolabeling with neurofilaments (Fig. 6), and synaptophysin (not shown) was present in the presynaptic region at the nerve terminal in these myofibers containing MyHCIIa. A subgroup of myofibers containing MyHCeom in longitudinal sections of bulbar-onset ALS donors showed multiterminal en plaque motor endplates identified by α-BTx labeling (Fig. 7). Both neurofilament and synaptophysin were present at the multiterminal motor endplates in the myofibers containing MyHCeom (Fig. 7). 

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.

Discussion

We here report changes in the myofiber composition and density in the GL of the mid-portion of the medial recti muscles of ALS donors with different sites of symptom onset: (1) The proportion of myofibers containing MyHCIIa was significantly decreased and the proportion of myofibers containing MyHCeom was significantly increased in the GL of spinal-onset ALS and bulbar-onset ALS donors compared to control donors. (2) There was an increase in the cross-sectional area and a decrease in myofiber density in the GL of bulbar-onset ALS donors compared to control donors. (3) The proportion of myofibers containing MyHCeom was significantly increased in the GL of bulbar-onset ALS donors compared to spinal-onset ALS donors. (4) The myofiber density in the GL of ALS donors was positively correlated with the proportion of myofibers containing MyHCIIa and negatively correlated with the proportion of myofibers containing MyHCeom. 

Our findings further emphasize the importance of investigating pathological changes in ALS patients by stratifying them according to their site of symptom onset. It has previously been reported that bulbar-onset ALS patients have worse prognosis compared to spinal-onset ALS patients.^3,4,6 In a large study population, we could show that bulbar-onset ALS patients had both significantly higher plasma neurofilament light-chain concentrations and significantly shorter disease duration compared to the spinal-onset ALS patients, suggesting that the bulbar-onset ALS patients have a more aggressive neurodegenerative process.⁶ Our present results are in agreement with the higher plasma neurofilament light-chain levels and worse survival after symptom onset in bulbar-onset ALS patients.⁶ We could also show that the proportion of myofibers containing MyHCIIa in the GL of spinal-onset ALS donors was significantly correlated with the disease duration, supporting that more pronounced changes in myofiber composition were observed in spinal-onset ALS donors with a more aggressive disease. 

Oculomotor functions such as saccade velocity and smooth pursuit movements have been shown to be affected in ALS patients and more so in bulbar-onset ALS patients than in spinal-onset ALS patients.^25,26 The slowing of saccade velocity in bulbar-onset ALS patients previously reported was interpreted as possibly derived from pathological changes at the brainstem level,²⁵ but our findings of a major myofiber type shift and decrease in myofiber density in bulbar-onset ALS donors suggest that changes at the muscle level also could play a role in the reported changes in velocities. The more pronounced myofiber-type shift with a significantly higher proportion of myofibers containing MyHCeom in the GL of bulbar-onset ALS donors compared to spinal-onset ALS donors in our study is in agreement with previous reports suggesting more advanced oculomotor dysfunction in bulbar-onset ALS patients compared to spinal-onset ALS patients.^25,26 

The present study further elucidates differences between the myofibers in the GL and the OL. A previous study from our laboratory reported minor morphological changes in EOMs where mild changes in myofiber size and increase in connective tissue could be observed in ALS donors compared to control donors.¹⁷ The proportion of the myofibers containing MyHCeom in the GL was significantly increased in bulbar-onset ALS donors compared to both control donors and spinal-onset ALS donors. Notably, despite changes in the myofiber type proportion in ALS donors, the total number of myofibers in the EOMs was preserved, which strongly suggests that myofibers that contained MyHCIIa had shifted into MyHCeom-containing myofibers and not simply degenerated. This points toward a broader remodeling of EOM function in ALS that goes beyond just a shift in myofiber type. More elaborate studies are needed to reveal whether these changes are compensatory mechanisms that help in the preservation of basic eye motility in ALS patients or a more ominous sign that, given a sufficiently extended lifespan in patients with invasive ventilation, even the EOMs will become clinically involved. Interestingly, we could not detect any significant differences in the proportion of myofibers containing MyHCIIa or MyHCeom in the OL of spinal-onset ALS and bulbar-onset ALS donors compared to control donors, which might suggest a higher resilience to the pathological processes in ALS in the myofibers of the OL compared to the GL. It has previously been reported that myofibers in the OL are exceptionally fatigue resistant, having a high mitochondrial and oxidative enzyme content^13,27 and a higher vascularization.²⁸ The differences in vascularization might potentially be a contributing factor for a more robust resilience to pathological changes in the OL in ALS donors. 

The heterogeneous and somewhat weaker labeling intensity observed in myofibers containing MyHCIIa in both spinal-onset ALS and bulbar-onset ALS donors was most prominent in the GL, whereas the labeling intensity in the GL and OL of the control donors and the OL of ALS donor was more homogeneous and stronger. The differences in labeling patterns in the GL of ALS donors might reflect an increased variability in the total content of different MyHC isoforms, which could suggest an ongoing myofiber type shift, as ALS seems to lead to a gradual shift from MyHCIIa to MyHCeom. The present data on the EOMs of the control donors showed similar patterns of distribution in myofiber composition as previously reported,¹⁶ although our previous study examined other EOMs than the medial recti muscles. 

A study from our laboratory reported that myofibers containing MyHCeom have a novel type of multiple motor endplates mostly found in the GL but also present in the OL.²³ It is of particular interest to further study the myofibers containing MyHCeom and their motor endplates, as the myofiber type shift observed in the GL of medial recti muscles in the present study might reflect adaptive changes in the oculomotor nuclei and the myofiber innervation. It is possible that myofibers innervated by motor neurons conferring a MyHCIIa-dominant composition are denervated and reinnervated by motor neurons conferring a more MyHCeom-dominant composition. It will be particularly interesting to study palisade endings, which are complex axonal constructs at the end of the multiply innervated myofibers whose function is incompletely understood but which have been shown to confer a large capacity for vesicular trafficking of, among others, growth factors.²⁹ A previous study involving the oculomotor nuclei indicated differences in expression in approximately 1700 genes and suggests a lower vulnerability to excitotoxicity compared to spinal motor neurons.³⁰ Our results prompt further studies into the association between changes in the myofiber composition of EOMs and changes in the corresponding motor neuron populations at the brainstem level. Further inquiry might also reveal what changes in the EOMs are purely pathological consequences and which ones represent adaptive changes selective to the EOMs. This could in turn give insights into protective mechanisms that could be harnessed in the development of new treatment strategies for ALS. 

Acknowledgments

The authors are indebted to the donors who made this study possible. The authors thank Mona Lindström and Anna-Karin Olofsson for excellent technical assistance, and Farhan Shah and Erik Flinth for valuable advice. 

Supported by research grants from the Swedish Research Council, the Västerbotten County Council (Central ALF and Spjutspetsmedel), the Ulla-Carin Lindquist Foundation, the Neuroförbundet Association, Stiftelsen Kronprinsessan Margaretas Arbetsnämnd för synskadade (KMA), Ögonfonden, the Swedish Brain Foundation, and the Knut and Alice Wallenberg Foundation. 

Disclosure: A. Behzadi, Pharma Industry Publishing AB (F); A.E. Tjust, None; J.-X. Liu, None; P.M. Andersen, Biogen (C, F), Orphazyme (C, F), Roche (C, F), Regeneron (C, F), Avrion (C, F), uniQure (C, F), AL-S Pharma (C, F), Sanofi (C, F), Amylyx (C, F), Alexion (C, F), Orion Pharma (C, F), Eli Lilly (C, F), PTC Pharmaceuticals (C, F); T. Brännström, None; F. Pedrosa Domellöf, None