Exclusive vital labeling of myonuclei for studying myonuclear arrangement in mouse skeletal muscle tissue

Abstract

Background: The arrangement of myonuclei in skeletal muscle tissue has long been used as a biomarker for muscle health, but there is a dearth of in vivo exploration of potential effects of myonuclear organization on the function and regeneration of skeletal muscle because traditional nuclear stains are performed on postmortem tissue. Therefore, we sought a transgenic method to produce a selective and persistent myonuclear label in whole muscles of living mice. Methods: We bred together a mouse line with skeletal muscle fiber-selective expression of Cre recombinase and a second mouse line with a Cre-inducible fluorescently tagged histone protein to generate a mouse line that produces a myonuclear label suitable for vital imaging and histology of fixed tissue. We tested the effectiveness of this vital label in three conditions known to generate abnormal myonuclear positioning. First, we injured myofibers of young mice with cardiotoxin. Second, this nuclear label was bred into a murine model of Duchenne muscular dystrophy. Finally, we examined old mice from this line that have undergone the natural aging process. Welch's t test was used to compare wild type and transgenic mice. Results: The resulting mouse line transgenically produces a vital red fluorescent label of myonuclei, which facilitates their in vivo imaging in skeletal muscle tissue. Transgenic fluorescent labeling of myonuclei has no significant effect on skeletal muscle function, as determined by twitch and tetanic force recordings. In each muscle examined, including those under damaged, dystrophic, and aged conditions, the labeled myonuclei exhibit morphology consistent with established literature, and reveal a specialized arrangement of subsynaptic myonuclei at the neuromuscular junction. Conclusions: Taken together, our results demonstrate that this mouse line provides a versatile tool to selectively visualize myonuclei within both living and fixed preparations of healthy, injured, diseased, and aged muscles.

Bioengineering a Miniaturized In Vitro 3D Myotube Contraction Monitoring Chip For Modelization of Muscular Dystrophies

“…The overall volume of 2D monolayer nuclei was found to be more important than the one of 3D myotube, confirming the relative morphological aberrance caused by 2D culture and proving the need for three-dimensional systems of culture. The shape of 3D myotube nuclei is closer to the in vivo conformation of myonuclei (Bruusgaard et al, 2010)(Hastings et al, 2020), which makes our culture chip a relevant tool for the analysis of nuclei-linked pathology, such as laminopathies.…”

Lifetime analysis of mdx skeletal muscle reveals a progressive pathology that leads to myofiber loss

“…We found that endplate fragmentation is dramatically elevated throughout the lifespan of mdx mice and increases in severity through the first year of life. Previous reports have induced endplate fragmentation by injection of myotoxic drugs, physically damaging myofibers and by eccentric contraction protocols 37,51,52 . These results indicate that fragmentation can be the result of full myofiber replacement or from local damage and repair.…”

Synergistically Acting on Myostatin and Agrin Pathways Increases Neuromuscular Junction Stability and Endurance in Old Mice

“…The arrangement of myonuclei in skeletal muscle tissue has long been used as a biomarker for muscle health [ 50 ]. ActR-Fc-nLG3 has no major effect on muscle fibers, as confirmed by the significant lower number of peripheral nuclei per fiber than the other groups.…”