Effects of in vivo injury on the neuromuscular junction in healthy and dystrophic muscles

Abstract

The most common and severe form of muscular dystrophy is Duchenne muscular dystrophy (DMD), a disorder caused by the absence of dystrophin, a structural protein found on the cytoplasmic surface of the sarcolemma of striated muscle fibres. Considerable attention has been dedicated to studying myofibre damage and muscle plasticity, but there is little information to determine if damage from contraction-induced injury occurs at or near the nerve terminal axon. We used α-bungarotoxin to compare neuromuscular junction (NMJ) morphology in healthy (wild-type, WT) and dystrophic (mdx) mouse quadriceps muscles and evaluated transcript levels of the post-synaptic muscle-specific kinase signalling complex. Our focus was to study changes in NMJs after injury induced with an established in vivo animal injury model. Neuromuscular transmission, electromyography (EMG), and NMJ morphology were assessed 24 h after injury. In non-injured muscle, muscle-specific kinase expression was significantly decreased in mdx compared to WT. Injury resulted in a significant loss of maximal torque in WT (39 ± 6%) and mdx (76 ± 8%) quadriceps, but significant changes in NMJ morphology, neuromuscular transmission and EMG data were found only in mdx following injury. Compared with WT mice, motor end-plates of mdx mice demonstrated less continuous morphology, more disperse acetylcholine receptor aggregates and increased number of individual acetylcholine receptor clusters, an effect that was exacerbated following injury. Neuromuscular transmission failure increased and the EMG measures decreased after injury in mdx mice only. The data show that eccentric contraction-induced injury causes morphological and functional changes to the NMJs in mdx skeletal muscle, which may play a role in excitation-contraction coupling failure and progression of the dystrophic process.

Figure 1. Structure of the neuromuscular junction (NMJ)

A, flat plane projection of a Z-stacked confocal image of a non-injured wild-type (WT) NMJ in the quadriceps muscle. The terminal neuron is labelled with antibodies against neurofilament (red) and the motor end-plate is stained with α-bungarotoxin (green). Scale bar, 10 μm. B, electron microscopy image showing ultrastructure of neuromuscular synapse. Scale bar, 500 nm. C, structure of the MuSK signalling complex is shown in the schematic. Activation of this complex by neuronal Agrin drives AChR clustering. D, constituents of the MuSK complex were examined by qRT-PCR. Interestingly, MuSK was the only constituent that showed significant differences between WT and mdx. All data are presented as mean ± SD.*P < 0.05. mdx, mice lacking dystrophin; WT, wild-type.

Figure 2. Apparatus used to induce injury

A, to produce the injury, the femur was stabilized and the ankle attached to a motor-driven lever arm. The femoral nerve was used to stimulate the quadriceps supramaximally (causing knee extension, blue arrow) while the lever arm forced the knee joint into flexion (blue arrow). To complete one repetition, the quadriceps was stimulated for 200 ms to induce a peak isometric contraction before lengthening by the lever arm. B, representative trace recordings of torque for the first and final repetition, from wild-type (WT) mice and mice lacking dystrophin (mdx). C, lengthening contractions resulted in a significant loss of maximal isometric torque for both WT and mdx mice; however, mdx mice showed a significantly greater loss in torque (76 ± 8%) compared to WT (39 ± 6%). All data are presented as mean ± SD, P < 0.05. * indicates statistical significance from respective non-injured quadriceps; †indicates statistical significance from injured WT quadriceps.

Figure 3. Representative images of injured and non-injured neuromuscular junctions (NMJs)

A total of 140 NMJs from quadriceps whole-mount preparations were stained with α-bungarotoxin, Z-stacked and analysed. Shown are representative confocal images of NMJs in normal (WT) and dystrophic (mdx) quadriceps before and after lengthening contractions: A, WT; B, WT-injured; C, mdx; D, mdx-injured. Similar to the force data, WT mice showed little to no change in NMJ morphology after injury. Non-injured mdx muscle showed a non-continuous, punctate pattern in NMJ morphology compared to non-injured and injured WT muscles; however, mdx motor end-plates showed significantly exacerbated morphological changes after injury, in particular, less dense aggregates, increased discontinuity and greater number of separate ACh clusters within total end-plate area (see Results). Scale bar, 10 μm. All data were measured from processed binary images generated in Image J (NIH) software. Pixel positions of skeletonized images were used in quantifying discontinuity and branching patterns using a binary connectivity plugin.

Figure 4. Neuromuscular transmission failure

A, representative data from contractile assay to assess NMJ transmission failure. Nerve stimulation: the femoral nerve was stimulated every second for 2 min with a 330 ms duration maximum tetanic contraction. Muscle stimulation: with electrodes placed directly over the muscle, a maximum tetanic contraction was superimposed on to the protocol every 15 s. B, the relative contribution of neuromuscular transmission failure (NTF) to muscle fatigue was estimated as: (NF − MF)/(1 − MF), where NF is a percentage decrement in force during repetitive nerve stimulation and MF is the percentage force decrement during direct muscle stimulation. WT-injured quadriceps (8 ± 4) showed no difference in NTF compared to controls (WT non-injured, 8 ± 2). Mdx non-injured quadriceps tended toward a slightly higher NTF (11 ± 0.2) from that of WT; however, after injury, mdx quadriceps showed a significant increase in NTF (26 ± 6). All data are presented as mean ± SD, P < 0.05.

Figure 5. Effects of injury on electromyography (EMG)

A, representative EMG curves for tetanic contraction in wild-type (WT) and dystrophic (mdx) mice, before and after injury. B, percentage change in EMG characteristics after injury. Data are derived from mouse quadriceps muscles. PA, peak amplitude; RMS, root mean square. *Indicates significance compared to WT. All data are presented as mean ± SEM, P < 0.05.