Overexpression of enhanced yellow fluorescent protein fused with Channelrhodopsin-2 causes contractile dysfunction in skeletal muscle

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, Channelphodopsin2-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 to ChR2-only and WT mice, observed under both electrical and optogenetic stimulation paradigms. Bulk RNAseq identified 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.