A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction

Abstract

Background: In many neurodegenerative and muscular disorders, and loss of innervation in sarcopenia, improper reinnervation of muscle and dysfunction of the motor unit (MU) are key pathogenic features. In vivo studies of MUs are constrained due to difficulties isolating and extracting functional MUs, so there is a need for a simplified and reproducible system of engineered in vitro MUs.

Objective: to develop and characterise a functional MU model in vitro, permitting the analysis of MU development and function.

Methods: an immortalised human myoblast cell line was co-cultured with rat embryo spinal cord explants in a serum-free/growth fact media. MUs developed and the morphology of their components (neuromuscular junction (NMJ), myotubes and motor neurons) were characterised using immunocytochemistry, phase contrast and confocal microscopy. The function of the MU was evaluated through live observations and videography of spontaneous myotube contractions after challenge with cholinergic antagonists and glutamatergic agonists.

Results: blocking acetylcholine receptors with α-bungarotoxin resulted in complete, cessation of myotube contractions, which was reversible with tubocurarine. Furthermore, myotube activity was significantly higher with the application of L-glutamic acid. All these observations indicate the formed MU are functional.

Conclusion: a functional nerve-muscle co-culture model was established that has potential for drug screening and pathophysiological studies of neuromuscular interactions.

Keywords: human myoblast; motor neuron (MN) co-culture; motor unit (MU); myotube; neuromuscular junction (NMJ).

Figure 1

Immortalised human skeletal muscle cells co-cultured with rat embryo spinal cord explants. (a) Phase contrast image of explant (orange star) sprouting neurites (shown in enlarged inset) after 24 h over undifferentiated myocytes. Image captured at 10x magnification. Scale bar: 100 µm. (b) Multinucleated myotube formation (indicated with yellow lines) after 48 h with continued expansion of neural projections (shown in enlarged inset) emanating from the spinal cord explant (orange star). Image captured at 10x magnification. Scale bar: 100 µm. (c) Maintained neurite growth (shown in inset) and continued myotube formation (yellow lines) at 72 h. Image captured at 10x magnification. Scale bar: 100 µm. (d) Neuronal axons (pink arrows) form a visible link (circled in red) with a myotube (green arrow). Image captured at 40x magnification. Scale bar: 25 µm.

Figure 2

Cholinergic motor neurons co-localise with myotubes at Day 14. Panel a is a representative image of co-culture stained for vesicular acetylcholine transporter (VaChT) (red), myosin heavy chain (MHC) (green) and DAPI (blue). Scale bar = 7.5 µm.

Figure 3

Interaction between neuronal axons and non-myelinating Schwann cells at Day 14. Image is representative of neuronal cells in the co-culture stained for β-III-Tubulin (green), Schwann cells stained for glial fibrillary acidic protein (GFAP) (red) and DAPI (blue). Enlarged Inset shows cellular interaction. Scale bar = 10 µm.

Figure 4

Characterisation of neuromuscular junction formation at Day 14. Representative image of co-culture stained for β-III-tubulin (green), alpha-bungarotoxin (α-BTX) (red) and DAPI (blue). Scale bar = 5 µm. White arrows indicate the NMJ formation and the multiple innervation.

Figure 5

Characterisation of presynaptic neuromuscular junction activity at Day 14. Representative image of co-culture stained for neurofilament heavy (NFH) (green), synaptotagmin (Syt1) (red) and DAPI (blue). Scale bar = 7.5 µm.

Figure 6

Characterisation of postsynaptic neuromuscular junction formation at Day 14. (a) shows co-culture stained for alpha-bungarotoxin (α-BTX) (red), MuSK (magenta) and DAPI (blue). (b) is representative of co-culture stained for α-BTX (red), Rapsyn (green) and DAPI (blue). (c) reveals interaction and detailed conformation of postsynaptic proteins MuSK (magenta) and Rapsyn (green) at the AChR stained with α-BTX (red) and DAPI (blue). Scale bar = 25 µm.

Figure 7

Functional assessment of NMJ formation via the effects of α-bungarotoxin, tubocurarine and L-glutamic acid on myotube contraction frequency at day 14. (a) shows the effect of α-bungarotoxin on myotube contraction frequency compared to controls. (b) demonstrates tubocurarine effects on myotube contraction frequency compared to controls. (c) displays the impact of L-glutamic acid on myotube contraction frequency compared to controls. Data are means ± SD, n = 12, each time point analysed with unpaired T-test, ** p < 0.01, **** p < 0.0001. Time points: (1) -30 s, (2) 0 s, (3) 1 min, (4) 2 min, (5) 5 min, (6) 10 min, (7) 30 min, (8) 1 h, (9) 1 h 1 min (washout), (10) 1 h 30 min, (11) 24 h.