Abstract
BackgroundThe 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.MethodsWe 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.ResultsThe 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.ConclusionsTaken 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.
Highlights
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
Mice carrying the Human α-skeletal actin (HSA)-Cre79 and H2B/GPI alleles produced by breeding, referred to with the acronym “RG” in accordance with the original manuscript characterizing this reporter construct [21], have skeletal muscle fibers in which the myonuclei are labeled with mCherry, and green fluorescent protein (GFP) is inserted into the sarcolemma
Myonuclei-specific transgenic fluorescent labeling in chemically fixed muscle preparations The HSA-Cre79 transgene used in the RG mice is highly specific for myofibers in skeletal muscle tissue, based off our own observations presented in this paper and experiments performed by the group that developed the transgenic line [19, 25]
Summary
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. Muscle fiber damage followed by regeneration of the myofiber results in the appearance of “central nuclei, ” or myonuclei in the approximate center of the myofiber, rather than the periphery [3]. These central nuclei appear in skeletal muscle fibers after deliberate muscle damage induced by a variety of insults, including myotoxic drug exposure, muscle transection, laser ablation, and eccentric contractions [4,5,6,7,8,9,10]. Central nuclei have long been accepted as a morphological indicator for muscle health, for myofibers having undergone a degeneration/regeneration event. A better understanding of myonuclear disposition and dynamics may be informative about the aging, disease, and injury of skeletal muscle tissue
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