Engineering Neuron-Innervated Muscle with Stimulus-Responsive Contraction and Myokine Secretion

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Muscle is a unique tissue that can contract, allowing for movement and essential involuntary actions such as breathing, heart pumping, and digestion in humans and animals. Muscle can also secrete various chemicals, called "myokines," which are involved in proper function of the immune system and brain. Neurons are responsible for transmitting signals between the brain and muscle tissues that control muscle contraction and secretion activity. These signals travel through neurons and then transferred to the muscle through the neuromuscular junction. Many injuries and muscle diseases are due to the loss of connection between neurons and muscle. Therefore, reproducing muscle connected to neurons in vitro (in the lab) would enable better understanding of the role/importance of the neuromuscular junction, and this understanding could lead to better treatments for muscle injuries and diseases. Thus there is an urgent need for creating an in vitro physiologically relevant model of muscle connected with/innervated by neurons. To this end, the investigators aim to engineer and validate muscle that contracts and secretes myokines in response to bioelectrical signals from neurons. The project proposes that these neuron-induced muscular activities depend on size, number, and alignment of the muscle fibers in the engineered muscle. This hypothesis will be studied by co-culturing neuron-forming cells on engineered muscle tissue. The quality of neuron-innervated muscle will be evaluated by monitoring contractions and myokine secretion of muscles in response to a neural impulse. In parallel, the investigators will utilize the research program to train undergraduate and graduate students who are involved in bioengineering-related research. The research program will be incorporated into various outreach activities that aim to attract future young scientists and engineers to the biomedical area. Overall, the project will significantly impact efforts to recreate biologically functional muscle tissues and also educate the next generation of biologists and biomedical engineers in diverse ways.The project is focused on engineering and validating motor neuron-innervated human skeletal muscle that contracts and, in turn, secrets myokines in response to neurotransmitters (e.g., glutamate). Experiments are designed to test the hypothesis that controlling the expression of acetylcholine receptors on engineered muscle is key to enhancing formation of the neuromuscular junction and that the topology and softness of a matrix on which skeletal myoblasts form muscle fibers modulate acetylcholine expression of muscle fibers. The hypothesis will be examined via three aims. The FIRST Aim is to assess the extent that topology of myoblasts-adhered substrates modulates neural innervation and contraction of muscle in response to neural impulse. Human skeletal myoblast cells will be cultured on grooved substrates with grooves (2 to 100 micrometers in width) patterned onto a collagen conjugated PEDGA gel. Studies are designed to answer to what extent the groove-patterned substrate modulates maturity and expression of acetylcholine receptors of myofibers, modulates the alignment and innervation of motor neurons, and influences the neurotransmitter-respondent muscle contraction. The SECOND Aim is to study the effects of softness of the myoblast-adhered substrate on the neural innervation into muscle. The elastic modulus of the gel will be varied from 5 to 40 kPa. Studies are designed to answer to what extent the gel softness and topology orchestrate maturity of muscle and expression of acetylcholine receptors, modulate neural innervation into muscle fibers, influence the neurotransmitter respondent muscular contraction and if neural innervation is related to the mechanotransduction. The THIRD Aim is to evaluate the extent that neuron-innervated muscle produces myokines in response to a neural impulse. Motor neuron progenitor cells will be plated on the myofibers formed on the grooved substrates and differentiated to motor neurons. The focus will be on analyzing mRNA expression and protein secretion levels that lead to increased secretion of myokines. Studies are designed to answer to what extent the neural impulse increases protein and myokine expression by the innervated muscle, to what extent the innervated muscle increases glucose uptake and fat oxidation in response to the neural impulse and to what extent the neuromuscular junction serves to increase the volume of the innervated muscle. The end result is expected to be a neuron-innervated muscle that will actively express and secrete myokines and, in turn enhance metabolic activity and increase muscle volume over time when the muscle is regularly contracted.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.