Decoupling the Biochemical and Mechanical Effects of Muscle Exercise on Motor Neurons with 2.5D Actuating Matrices [230828Ram_RNA-seq]

Muscles' function as an endocrine organ is still poorly understood, but an emerging body of literature has provided evidence that myokines may aid in the development of surrounding tissues. Another outcome of muscle contraction in vivo is the localized mechanical forces produced. Current experimental techniques are not equipped to isolate and study the two effects independently. We have developed a system to culture contractile 2D optogenetic muscle long-term to provide a source for myokine secretion. We supplement our neuron monoculture with myokine secreted media and saw a 3X increase in total neurite length magnitude and a 3.2X increase in the rate of growth. As for mechanical stimulation, we optimize our Magnetic Matrix Actuation (MagMA) to provide localized stressors on the growing neurites. The mechanical stimulation regime results in a ~2.7X increase in both magnitude and rate of total neurite length. We validate our morphological findings with bulk RNA-sequencing analysis, the results show a large number of differentially expressed genes between the biochemical stimulation group and control. On the other hand, there was minimal difference between the mechanical stimulation group and control. Our study mechanistically unravels how repeated muscle contraction, or exercise, regulates motor neuron growth and maturation. Overall design: HBG3 mESC neuron monoculture spheroids on day 8 of differentiation plated onto five different conditions. MC1-4 are magnetic controls, they contain the magnetically controlled silicone array but was never dynamically actuated. M1-4 consisted of are magnetically actuated samples. As for the biochemical stimulation portion, samples S1-6 were supplemented with myokine-infused media from optically stimulated muscle for 30 minutes at 1 Hz. The NE1-6 samples were infused with media that sat on the optogenetic muscle monolayer for 30 minutes, but it was not stimulated. C1-6 are the biological controls.