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Skeletal shape depends on the transmission of contractile muscle forces from tendon to bone across the enthesis. Loss of muscle loading impairs enthesis development, yet little is known if and how the postnatal enthesis adapts to increased loading. Here, we studied adaptations in enthesis structure and function in response to increased loading, using optogenetically induced muscle contraction in young (i.e., growth) and adult (i.e., mature) mice. Daily bouts of unilateral optogenetic loading in young mice led to radial calcaneal expansion and warping. This also led to a weaker enthesis with increased collagen damage in young tendon and enthisis, with little change in adult mice. We then used RNA sequencing to identify the pathways associated with increased mechanical loading during growth. In tendon, we found enrichment of glycolysis, focal adhesion, and cell-matrix interactions. In bone, we found enrichment of inflammation and cell cycle. Together, we demonstrate the utility of optogenetic-induced muscle contraction to elicit in vivo adaptation of the enthesis.
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Overexpression of enhanced yellow fluorescent protein fused with Channelrhodopsin‐2 causes contractile dysfunction in skeletal muscle
Abstract Skeletal muscle activation using optogenetics has emerged as a promising technique for inducing noninvasive muscle contraction and assessing muscle function both in vivo and in vitro. Transgenic mice overexpressing the optogenetic fusion protein, Channelrhodopsin 2‐EYFP (ChR2‐EYFP) in skeletal muscle are widely used; however, overexpression of fluorescent proteins can negatively impact the functionality of activable tissues. In this study, we characterized the contractile properties of ChR2‐EYFP skeletal muscle and introduced the ChR2‐only mouse model that expresses light‐responsive ChR2 without the fluorescent EYFP in their skeletal muscles. We found a significant reduction in the contractile ability of ChR2‐EYFP muscles compared with ChR2‐only and WT mice, observed under both electrical and optogenetic stimulation paradigms. Bulk RNAseq identified the downregulation of genes associated with transmembrane transport and metabolism in ChR2‐EYFP muscle, while the ChR2‐only muscle did not demonstrate any notable deviations from WT muscle. The RNAseq results were further corroborated by a reduced protein‐level expression of ion channel‐related HCN2 in ChR2‐EYFP muscles and gluconeogenesis‐modulating FBP2 in both ChR2‐EYFP and ChR2‐only muscles. Overall, this study reveals an intrinsic skeletal dysfunction in the widely used ChR2‐EYFP mice model and underscores the importance of considering alternative optogenetic models, such as the ChR2‐only, for future research in skeletal muscle optogenetics.
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- Award ID(s):
- 1944448
- PAR ID:
- 10644791
- Publisher / Repository:
- DOI PREFIX: 10.1096
- Date Published:
- Journal Name:
- The FASEB Journal
- Volume:
- 38
- Issue:
- 22
- ISSN:
- 0892-6638
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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