Matrix Topography Regulates Synaptic Transmission at the Neuromuscular Junction

Abstract

Recreation of a muscle that can be controlled by the nervous system would provide a major breakthrough for treatments of injury and diseases. However, the underlying basis of how neuron-muscle interfaces are formed is still not understood sufficiently. Here, it is hypothesized that substrate topography regulates neural innervation and synaptic transmission by mediating the cross-talk between neurons and muscles. This hypothesis is examined by differentiating neural stem cells on the myotubes, formed on the substrate with controlled groove width. The substrate with the groove width of 1600 nm, a similar size to the myofibril diameter, serves to produce larger and aligned myotubes than the flat substrate. The myotubes formed on the grooved substrate display increases in the acetylcholine receptor expression. Reciprocally, motor neuron progenitor cells differentiated from neural stem cells innervate the larger and aligned myotubes more actively than randomly oriented myotubes. As a consequence, mature and aligned myotubes respond to glutamate (i.e., an excitatory neurotransmitter) and curare (i.e., a neuromuscular antagonist) more rapidly and homogeneously than randomly oriented myotubes. The results of this study will be broadly useful for improving the quality of engineered muscle used in a series of applications including drug screening, regeneration therapies, and biological machinery assembly.

Keywords: acetylcholine receptors; motor neurons; myotubes; neural innervation; neuromuscular junctions.

Figure 1

Schematic description of engineering the neuromuscular junction through the sequential coculture of skeletal myoblasts and NSCs on the matrigel‐coated PUA substrates. The substrates were engineered to present grooves with 200, 800, and 1600 nm width.

Figure 2

Fabrication procedure and analysis of the grooved PUA substrates. A) Schematic description of the fabrication procedure of the grooved substrates. B) Scanning electron microscope images of the flat PUA substrate, PUA substrate with 200 nm groove width (w) and PUA substrate with 1600 nm groove width (w). Images in the first and second rows represent the top view and the side view of the substrates, respectively.

Figure 3

Immunofluorescence staining of F‐actin (green), vinculin (red), and nucleus (blue) of A) primary myoblasts and B) C2C12 myoblasts. The images on the second row of A are magnified views of the area boxed in images. Images were captured 3 d after culture. C) The vinculin expression level quantified with the immunofluorescence images. Each condition was normalized to the vinculin expression value of primary myoblasts adhered to the flat substrate. * represents the statistical significance of the difference of the values between conditions noted in brackets (n = 4, *p < 0.05).

Figure 4

Angular orientation analysis of the skeletal myoblasts. A,B) The primary myoblasts and C,D) C2C12 myoblasts were cultured for 7 d on the flat substrate, a grooved pattern with 200 nm width, and grooved pattern with 1600 nm width. A,C) Morphology images of the primary myoblasts and C2C12 myoblasts, respectively. B,D) Representative histograms of the orientation of primary myoblasts and C2C12 myoblasts. The average value of goodness of fit (R ²) is indicated as µ, and the standard deviation is indicated as σ (n = 4).

Figure 5

Analysis of the myogenic differentiation of skeletal myoblasts. A,B) Immunofluorescence images of the MHC (red), F‐actin (green), and nucleus (blue) in the differentiated A) primary myoblasts and B) C2C12 myoblasts taken after 10 d of culture in myogenic differentiation medium. C,D) Immunofluorescence images of the sarcomeric‐actinin (red), F‐actin (green), and nucleus (blue) in the differentiated C) primary myoblasts and D) C2C12 myoblasts taken after 10 d of culture in myogenic differentiation medium. E–H) Morphometric analysis of the differentiated skeletal myoblasts based on the immunofluorescence images. The E) myotube width, F) myotube lengths, G) MHC‐positive area, and H) fusion index were quantitatively examined. In each plot, * and ** represent the statistical significance of the difference of the values between conditions noted in brackets (n = 4, *p < 0.01, **p < 0.05).

Figure 6

Analysis of the differentiation and alignment of NSCs cultured on the myotubes. Immunofluorescence images of the differentiated NSCs on the myotubes formed with A,B) primary myoblasts and C,D) C2C12 myoblasts. NSCs were stained positively for islet‐1 (Isl‐1, green in panels (A,C), neurogenin‐2 (NGN, green in panels (B,D), and nucleus (blue) after 5 d of culture in the neural differentiation medium. Representative histogram showing the angular orientation of the differentiated NSCs on myotubes formed with E) primary myoblasts and F) C2C12 myoblasts. The average value of goodness of fit (R ²) is indicated as µ, and the standard deviation is indicated as σ (n = 4).

Figure 7

Immunocytochemistry of the neuron‐innervated myotubes. Images were captured after the coculture of primary myoblasts and C2C12 myoblasts with NSCs for 7 d. A,C) Myotubes and neurons stained for MHC (red), NF (green), AchR (orange), and nucleus (blue). B,D) Myotubes and neurons stained for SNP (green), AchRs (orange), and nucleus (blue). B) The motor neuron progenitor cells were additionally labeled with MAP2 (red). E,G) Quantified acetylcholine receptor expression levels (n = 4, *p < 0.01, **p < 0.05). The relative acetylcholine receptor expression level was calculated by counting the number of pixels stained positively for acetylcholine receptors in each image and normalizing it to the number obtained with the flat substrate condition. F,H) Average percentage of area where neurofilaments and acetylcholine receptors are colocalized in the myotubes (n = 4, *p < 0.01, **p < 0.05). Images of panels (A,B) and graphs of panels (C,D) are the results for primary myoblasts‐derived myotubes. Images of panels (C,D) and graphs of panels (G,H) are the results for C2C12 myoblasts‐derived myotubes.

Figure 8

Functionality analysis of the neuron‐innervated myotubes. A) Schematic description of the increased contraction of neuron‐innervated muscle by glutamate and the inhibited contraction by curare. B) Triggered contraction of the primary myoblast‐derived myotubes with the addition of glutamate. C) Inhibited contraction of the primary myoblast‐derived myotubes upon exposure to curare. D) Triggered contraction of the C2C12 myoblast‐derived myotubes with the addition of glutamate. E) Inhibited contraction of the C2C12 myoblast‐derived myotubes upon exposure to curare. In panels (B,D) arrows indicate the time point when glutamate was added. In panels (C,E) arrows indicate the time point when curare was added.