The effect of plasma from muscle-specific tyrosine kinase myasthenia patients on regenerating endplates

Abstract

Muscle-specific tyrosine kinase (MuSK) is essential for clustering of acetylcholine receptors (AChRs) at embryogenesis and likely also important for maintaining synaptic structure in adult muscle. In 5 to 7% of myasthenia gravis (MG) cases, the patients' blood contains antibodies to MuSK. To investigate the effect of MuSK-MG antibody on synapse regeneration, notexin was used to induce damage to the flexor digitorum brevis muscle. We administered aliquots of MuSK-MG patients' plasma to the flexor digitorum brevis twice daily for a period up to 21 days, and muscles were investigated ex vivo in contraction experiments. AChR levels were measured with (125)I-alpha-bungarotoxin, and endplates were studied with quantitative immunohistochemistry. In normal muscles and in 14-day regenerated muscles, MuSK plasma caused impairment of nerve stimulus-induced contraction in the presence of 0.35 and 0.5 mmol/L Ca(2+) with or without 100 to 400 nmol/L tubocurarine. Endplate size was decreased in regenerated muscles relative to controls; however, we did not observe such differences in muscle not treated with notexin. MuSK plasma had no effect on the amount and turnover rate of AChRs. Our results suggest that anti-MuSK antibodies influence the activity of MuSK molecules without reducing their number, thereby diminishing the size of the endplate and affecting the functioning of AChRs.

Figure 1

Effect of calcium and MuSK-MG plasma treatment (patient 1) on the contraction of regenerating FDB muscle (14 to 16 days after notexin). Plasma injections were started 1 day after notexin. Nerve stimulation with single pulses in muscles treated with patient’s plasma (squares) and control plasma (triangles). Direct stimulation of the same muscles with single pulses (open squares and triangles for patient and control, respectively). Means ± SEM of values from six to eight muscles. *P < 0.005; **P < 0.05 (Student’s t-test).

Figure 2

Effect of anti-MuSK⁺ plasma (patient 1) and tubocurarine on the contraction of normal (upper panel) and notexin-treated (14 days, lower panel) FDB muscles in the presence of 0.5 mmol/L (upper panel) or 0.35 mmol/L Ca²⁺ (lower panel). Nerve stimulation at 40 Hz. Ordinate, the contractions of muscles are expressed as a fraction of the contractions caused by direct stimulation of the muscle. Data derived from mice treated with control plasma (black bars) and patient 1 plasma (white bars). Means ± SEM of three muscles. *P < 0.05 (ANOVA two-way test).

Figure 3

Loss of bound ¹²⁵I-α-BuTX from notexin-treated FDB muscle treated with plasma from patient 1 (triangles) and control plasma (squares). Injections of ¹²⁵I-α-BuTX were given (at t = 0 in the figure) either at day 7 or at day 13 (open symbols) after notexin-treatment. The continuous lines were exponentially fitted to the data points, with the values of half-life time as indicated in the figure. Values at t = 0 were normalized to 2500 cpm. Means ± SEM of four to eight muscles.

Figure 4

Immunohistochemical analysis of the NMJ using fluorescent microscopy. Muscles were analyzed after 7 days’ treatment with patient’s or control plasma. AChR (postsynaptic membrane) is stained in red, VAChT (nerve bouton) is stained in green; merge on the right. Scale bar is 10 μm. A: Normal endplate in a muscle treated with control serum. B: Endplate in a muscle treated with patient 1 serum appears normal. C: In a muscle treated with control serum, new endplates are formed 7 days after notexin treatment. These endplates are enlarged compared with normal adult endplates. D: Endplate in a muscle treated with patient 1 serum 7 days after notexin treatment. The enlargement of newly formed endplates is not present in this condition. E: Measurement of fluorescence intensities of AChR relative to VAChT staining in an average of 105 endplates per muscle. The average AChR/VAChT staining was significantly decreased at endplates of patient serum treated animals compared with control serum treated muscles in the group of 7 days after notexin. F: Surface area of photographed endplates of identically treated muscles. The average surface area was significantly decreased at endplates of patient serum treated animals compared with control serum treated muscles in 7 days after notexin treatment. Means ± SEM of four to six muscles. The level of statistical significance (unpaired t-test) is as indicated in the figure.