Transcriptomics and Functionality of Optogenetic Skeletal Muscle

Optogenetic muscle stimulation is a novel and reproducible technique that uses light and genetic modification to elicit muscle contractions. This stimulation technique relies on light-sensitive opsins, such as Channelrhodopsin-2 (ChR2), which, when exposed to blue light (455 nm), opens to allow cation flow, depolarizing muscle fibers and inducing contraction. Until now, studies have demonstrated the utility of this technique with transgenic mouse models expressing ChR2-enhanced yellow fluorescent protein (EYFP) fusion in their skeletal muscle, for activating skeletal muscle both in vivo and ex vivo. However, while the mechanism of skeletal muscle optogenetics is well understood, the alterations in musculoskeletal tissues, i.e., muscle, tendon, and bone induced by optogenetic loading, as well as the effects of ChR2-EYFP fusion protein on muscle functionality, remain unknown. Therefore, the primary goals of this dissertation have been to identify the effects of repeated optogenetic stimulation on the transcriptional responses of skeletal muscle and its associated tendon and bone in transgenic mouse models, and to understand the influence of optogenetic fusion protein on skeletal muscle's structure, function, and transcriptomic responses. In my first aim (chapter 2), I investigated how repeated loading influences the transcriptional response of tendon and bone, following optogenetic skeletal muscle activations in field-standard transgenic mouse models. Using next-generation sequencing, I determined the molecular mechanisms governing the adaptation of young tendon and bone to mechanical loading. The findings of this study showed that repeated loading of young tendon and bone led to differential responses of genes associated with mechanosensitive processes. In my second aim (chapter 3), I investigated the role of ChR2-EYFP fusion protein on skeletal muscle structure, function, and transcriptomic response at baseline and following repeated bouts of optogenetic stimulation. By measuring muscle contraction force using both optogenetic and gold-standard electrical simulation techniques, I demonstrated that the presence of EYFP with ChR2 caused 30-40% force reduction in fast-twitch skeletal muscles and reduced muscle fiber size, which improved upon EYFP removal. Moreover, ChR2-EYFP altered the transcriptomic profile of skeletal muscle compared to wildtype muscles and had a less pronounced transcriptional response to repeated muscle stimulation compared to ChR2-only muscle. In my third and final aim (chapter 4), I designed an in vitro microtissue culture platform to grow muscle tissues expressing ChR2 using flexible polydimethylsiloxane (PDMS). To achieve this, I first experimentally determined the Young’s moduli of PDMS (0.68 and 1.68 MPa, for monomer to curing agent ratios of 15:1 and 10:1, respectively) and then utilized finite element modeling to determine the mechanical properties of mini to microscale culture device designs. In summary, my dissertation adopts interdisciplinary approaches to explore the functionality and applications of skeletal muscle optogenetics. The research presented in this dissertation advances the field of musculoskeletal bioengineering by elucidating the role of repeated optogenetic-induced loading on the transcriptional responses of musculoskeletal tissues in transgenic mouse models, and by providing a comprehensive understanding of how optogenetic fusion proteins influence the physiology and transcriptomics of skeletal muscle. My studies pave the way for new research directions, encompassing both musculoskeletal tissue adaptation and the exploration of cellular-level mechanisms of optogenetics.