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

FASEB J. 2024 Nov 30;38(22):e70185. doi: 10.1096/fj.202401664RR.

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.

Keywords: Channelrhodopsin‐2; function; optogenetics; skeletal muscle; structure.

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Channelrhodopsins* / genetics
  • Channelrhodopsins* / metabolism
  • Luminescent Proteins* / genetics
  • Luminescent Proteins* / metabolism
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic*
  • Muscle Contraction*
  • Muscle, Skeletal* / metabolism
  • Optogenetics* / methods

Substances

  • Luminescent Proteins
  • Channelrhodopsins
  • yellow fluorescent protein, Bacteria
  • Bacterial Proteins