(119d) Secretome of Engineered Neuron-Innervated Muscle

Skeletal muscles can secrete cytokines and small proteins, so-called myokines, in response to the contraction. Myokines act to retain the homeostasis of muscle and other organs through autocrine, paracrine, or endocrine effects. Efforts have been made to create in vitro platforms to recapitulate skeletal muscle physiology and use it to study myokine secretion mechanisms. Most approaches expose an engineered muscle sheet or 3D muscle strips to electrical pulse stimulation. However, these studies miss a crucial element that regulates muscle contraction physiologically, the neuromuscular junction (NMJ).

The NMJ is a chemical synapse at the junction of a motor neuron and a muscle fiber. At the junction site, neurons transmit a stimulatory or inhibitory signal to the muscle by releasing neural transmitters and, in turn, controlling the muscular contraction. Recently, neuromuscular system engineered in vitro has gained attention because of their potential as neuromuscular disorder drug screening platforms and autonomous bio-actuators. However, these platforms have not been used to address the extent to which neural innervation affects myokine secretion.

In this work, we reproduced the neuron-innervated muscles to address the extent to which neural innervation affects myokine secretion and subsequently modulates the physiological activities of muscle and other organs. The muscles were cultured on a grooved substrate with a myofibril-like pattern and co-cultured with neural stem cell-derived motor neurons to recreate the high functional NMJs. We examined the contraction capability of resulting neuron-innervated muscles with the addition of excitatory neurotransmitters compared to neuron-free muscles. Then, we assessed the change in myokine gene expression and secretion between neuron-innervated muscles and neuron-free muscles. We found that the substrate with grooved pattern served to enhance the differentiation and alignment of myotubes, resulting better neural innervation. The neuron-innervated muscles contracted more actively in responded to glutamate. In addition, the neuron-innervation upregulated the myokine gene expression and increased the myokine secretion of the muscles. The results of this study will be useful for better understanding the crosstalk between muscles and neurons and provide a potential treatment for neural disorders.