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

肌肉是一种独特的组织，可以收缩，允许人类和动物的运动和必要的非自愿行动，如呼吸、心跳和消化。肌肉还可以分泌各种化学物质，称为“肌动蛋白”，与免疫系统和大脑的正常功能有关。神经元负责在大脑和肌肉组织之间传递信号，控制肌肉收缩和分泌活动。这些信号通过神经元传递，然后通过神经肌肉接头传递到肌肉。许多损伤和肌肉疾病是由于神经元和肌肉之间失去联系所致。因此，在体外(在实验室)复制连接到神经元的肌肉将使人们能够更好地理解神经肌肉连接的作用/重要性，这种理解可能会导致更好地治疗肌肉损伤和疾病。因此，迫切需要建立一种与神经元相连/神经支配的肌肉的体外生理相关模型。为此，研究人员的目标是设计和验证肌肉的收缩和分泌，以响应来自神经元的生物电信号。该项目提出，这些神经元诱导的肌肉活动取决于工程肌肉中肌肉纤维的大小、数量和排列方式。这一假说将通过在工程肌肉组织上共同培养神经形成细胞来研究。神经支配肌肉的质量将通过监测肌肉收缩和肌肉对神经冲动的反应分泌来评估。同时，研究人员将利用该研究计划培训参与生物工程相关研究的本科生和研究生。该研究计划将被纳入旨在吸引未来年轻科学家和工程师进入生物医学领域的各种外联活动。总体而言，该项目将对重建具有生物功能的肌肉组织的努力产生重大影响，并以不同的方式教育下一代生物学家和生物医学工程师。该项目专注于设计和验证运动神经元神经支配的人类骨骼肌，该肌肉收缩并继而分泌对神经递质(如谷氨酸)做出反应的肌动蛋白。实验旨在验证这样一种假设，即控制工程肌肉上乙酰胆碱受体的表达是促进神经肌肉连接形成的关键，骨骼肌母细胞在其上形成肌肉纤维的基质的拓扑结构和柔软性调节肌肉纤维的乙酰胆碱表达。这一假设将通过三个目标进行检验。第一个目标是评估成肌细胞附着底物的拓扑结构在多大程度上调节神经神经和肌肉收缩对神经冲动的反应。人骨骼肌成肌细胞将被培养在带有凹槽的基质上，凹槽(宽度为2到100微米)图案化到胶原结合的PEDGA凝胶上。研究旨在回答凹槽图案的底物在多大程度上调节肌纤维的乙酰胆碱受体的成熟和表达，调节运动神经元的排列和神经支配，并影响神经递质反应性肌肉收缩。第二个目的是研究成肌细胞黏附基质的柔软性对肌肉神经支配的影响。凝胶的弹性模数将在5到40 kpa之间变化。研究的目的是回答凝胶的柔软性和拓扑结构在多大程度上协调肌肉的成熟度和乙酰胆碱受体的表达，调节神经支配到肌肉纤维，影响神经递质响应肌肉收缩，以及神经神经支配是否与机械转导有关。第三个目标是评估神经支配的肌肉在多大程度上对神经冲动做出反应。运动神经元前体细胞将被种植在凹槽基质上形成的肌纤维上，并分化为运动神经元。重点将放在分析导致肌动蛋白分泌增加的mRNA表达和蛋白质分泌水平上。研究旨在回答神经冲动在多大程度上增加了神经肌肉的蛋白质和肌肉因子的表达，在多大程度上神经肌肉对神经冲动的反应增加了葡萄糖的摄取和脂肪的氧化，以及神经肌肉连接在多大程度上增加了神经肌肉的体积。最终结果预计将是一块神经支配的肌肉，当肌肉定期收缩时，它将积极表达和分泌肌肉因子，进而增强新陈代谢活动，增加肌肉体积。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.