Poster U12 - 2.5D Actuating Substrates Enable Decoupling the Mechanical and Biochemical Effects of Muscle Exercise on Motor Neurons

Introduction: Exercise has systemic beneficial effects on the body. Growing evidence has shown that muscle-secreted cytokines, often termed “myokines”, are released during exercise to upregulate a range of cell signaling pathways. Concurrently, emerging studies in mechanobiology illustrate the need to consider the impact of forces generated during muscle contraction on neighboring cells. There is thus a critical need to develop a further understanding of the underlying mechanical and biochemical mechanisms by which muscle contraction influences intercellular signaling. However, current experimental techniques make it difficult to decouple the inherent biochemical and mechanical effects of muscle contraction on other cells. We have developed a robust in vitro platform to investigate these mechanisms, with a particular focus on unraveling the impact of exercise on motor neurons.

Materials and

Methods: Harvesting secreted myokines from exercised muscle in an efficient manner requires collecting conditioned media from contractile muscle monolayers, which typically delaminate from cell culture substrates. We thus tuned the mechanical properties of a thick fibrin gel to enable stable long-term muscle monolayer culture, and used non-invasive light stimulation of optogenetic muscle to generate large volumes of exercise-secreted myokines We differentiated HBG3 HB9:GFP mouse embryonic stem cells into mature motor neuron spheroids to be seeded onto our finely tuned fibrin hydrogel and supplemented these spheroids with secreted myokines from low- and high-intensity exercise groups. In a separate experiment, we seeded motor neuron spheroids on fibrin gels containing magnetic microparticles that could be actuated via an external magnet. This method of Magnetic Matrix Actuation (MagMA) enabled us to mechanically stimulate nerves in a manner that mimicked the forces generated during muscle contraction, this simulating the physical impacts of exercise.
Motor neuron spheroids were imaged during the duration of the experiment using an EVOS fluorescent microscope. RNA sequencing was performed at the end of the experiment to analyze the transcriptomic impact of exercise stimulation. Morphological data was characterized using FIJI’s NeuronJ plugin and data was plotted and statistically examined in GraphPad Prism.

Results, Conclusions, and Discussions: After live imaging for 5 growth days, in the biochemical stimulation groups both high intensity and low intensity exercise, we saw an ~3X increase in magnitude and ~3.2X rate of total neurite length and a ~2.5X increased growth in migration area magnitude and rate when compared to control. There was a significant impact between groups for each of our three main metrics, total neurite length, max neurite length and migration area (Fig 1). For the mechanically stimulated group, we saw a similar effect size with total neurite length being increased by ~2.7X in magnitude and rate when compared to the magnetic control (Fig 2).
Despite morphological similarities between biochemically and mechanically stimulated neurons, RNA sequencing analysis revealed different transcriptomic signatures in response to both modes of signaling, with biochemical stimulation (Fig 3 & 4) have a significantly greater impact on cell signaling related to axon growth and development, neuron projection guidance, and neuron-neuron and neuron-muscle synapse maturation.
Our study robustly validates a previously hypothesized role for muscle exercise in regulating motor neuron growth and maturation through both mechanical and biochemical signaling. In future work, we aim to study how muscle exercise can be used to tune peripheral nerve remodeling in pathological conditions, such as nerve injury.

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