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Mechanically patterned neuromuscular junctions-in-a-dish have improved functional maturation.

ABSTRACT: Motor neuron (MN) diseases are progressive disorders resulting from degeneration of neuromuscular junctions (NMJs), which form the connection between MNs and muscle fibers. NMJ-in-a-dish models have been developed to examine human MN-associated dysfunction with disease; however such coculture models have randomly oriented myotubes with immature synapses that contract asynchronously. Mechanically patterned (MP) extracellular matrix with alternating soft and stiff stripes improves current NMJ-in-a-dish models by inducing both mouse and human myoblast durotaxis to stripes where they aligned, differentiated, and fused into patterned myotubes. Compared to conventional culture on rigid substrates or unpatterned hydrogels, MP substrates supported increased differentiation and fusion, significantly larger acetylcholine (ACh) receptor clusters, and increased expression of MuSK and Lrp4, two cell surface receptors required for NMJ formation. Robust contractions were observed when mouse myotubes were stimulated by ACh, with twitch duration and frequency most closely resembling those for mature muscle on MP substrates. Fused myotubes, when cocultured with MNs, were able to form even larger NMJs. Thus MP matrices produce more functionally active NMJs-in-a-dish, which could be used to elucidate disease pathology and facilitate drug discovery.

SUBMITTER: Happe CL

PROVIDER: S-EPMC5541845 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Mechanically patterned neuromuscular junctions-in-a-dish have improved functional maturation.

Happe Cassandra L CL Tenerelli Kevin P KP Gromova Anastasia K AK Kolb Frederic F Engler Adam J AJ

Molecular biology of the cell 20170511 14

Motor neuron (MN) diseases are progressive disorders resulting from degeneration of neuromuscular junctions (NMJs), which form the connection between MNs and muscle fibers. NMJ-in-a-dish models have been developed to examine human MN-associated dysfunction with disease; however such coculture models have randomly oriented myotubes with immature synapses that contract asynchronously. Mechanically patterned (MP) extracellular matrix with alternating soft and stiff stripes improves current NMJ-in-a ...[more]

PMID: 28495800

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Project description:Desmin, the major intermediate filament (IF) protein in muscle cells, interlinks neighboring myofibrils and connects the whole myofibrillar apparatus to myonuclei, mitochondria, and the sarcolemma. However, desmin is also known to be enriched at postsynaptic membranes of neuromuscular junctions (NMJs). The pivotal role of the desmin IF cytoskeletal network is underscored by the fact that over 100 mutations of the human DES gene cause hereditary and sporadic myopathies and cardiomyopathies. A subgroup of human desminopathies comprises autosomal recessive cases resulting in complete abolition of desmin protein. In these patients, who display a more severe phenotype than the autosomal dominant cases, it has been reported that some individuals also suffer from a myasthenic syndrome in addition to the classical occurrence of myopathy and cardiomyopathy. Since further studies on the NMJ pathology is hampered by the lack of available human striated muscle biopsy specimens, we exploited homozygous desmin knock-out mice which closely mirror the striated muscle pathology of human patients lacking desmin protein. Here, we report on the impact of the lack of desmin on the structure and function of NMJs and on the transcription of genes coding for postsynaptic proteins. Desmin knock-out mice display a fragmentation of NMJs in soleus, but not in extensor digitorum longus muscle. Moreover, soleus muscle fibers show larger NMJs. Further, transcription levels of acetylcholine receptor (AChR) genes are increased in muscles from desmin knock-out mice, especially of the AChR subunit, which is known as a marker of muscle fiber regeneration. Electrophysiological recordings depicted a pathological decrement of nerve-dependent endplate potentials and a faster rise time of the nerve-independent miniature endplate potentials. The latter is indicating an enhanced opening time of the AChR channels. Our study highlights the essential role of desmin for the structural and functional integrity of mammalian neuromuscular junctions.

Project description:Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.

Project description:Desmin, the major intermediate filament (IF) protein in muscle cells, interlinks neighboring myofibrils and connects the whole myofibrillar apparatus to myonuclei, mitochondria, and the sarcolemma. However, desmin is also known to be enriched at postsynaptic membranes of neuromuscular junctions (NMJs). The pivotal role of the desmin IF cytoskeletal network is underscored by the fact that over 120 mutations of the human DES gene cause hereditary and sporadic myopathies and cardiomyopathies. A subgroup of human desminopathies comprises autosomal recessive cases resulting in the complete abolition of desmin protein. In these patients, who display a more severe phenotype than the autosomal dominant cases, it has been reported that some individuals also suffer from a myasthenic syndrome in addition to the classical occurrence of myopathy and cardiomyopathy. Since further studies on the NMJ pathology are hampered by the lack of available human striated muscle biopsy specimens, we exploited homozygous desmin knock-out mice which closely mirror the striated muscle pathology of human patients lacking desmin protein. Here, we report on the impact of the lack of desmin on the structure and function of NMJs and the transcription of genes coding for postsynaptic proteins. Desmin knock-out mice display a fragmentation of NMJs in soleus, but not in the extensor digitorum longus muscle. Moreover, soleus muscle fibers show larger NMJs. Further, transcription levels of acetylcholine receptor (AChR) genes are increased in muscles from desmin knock-out mice, especially of the AChRγ subunit, which is known as a marker of muscle fiber regeneration. Electrophysiological recordings depicted a pathological decrement of nerve-dependent endplate potentials and an increased rise time of the nerve-independent miniature endplate potentials. The latter appears related to the fragmentation of NMJs in desmin knockout mice. Our study highlights the essential role of desmin for the structural and functional integrity of mammalian NMJs.

Dataset Information

Mechanically patterned neuromuscular junctions-in-a-dish have improved functional maturation.

Publications

Mechanically patterned neuromuscular junctions-in-a-dish have improved functional maturation.

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