Skeletal Muscle Nuclei in Mice are not Post-mitotic

Abstract

The skeletal muscle research field generally accepts that nuclei in skeletal muscle fibers (ie, myonuclei) are post-mitotic and unable to proliferate. Because our deuterium oxide (D₂O) labeling studies showed DNA synthesis in skeletal muscle tissue, we hypothesized that resident myonuclei can replicate in vivo. To test this hypothesis, we used a mouse model that temporally labeled myonuclei with GFP followed by D₂O labeling during normal cage activity, functional overload, and with satellite cell ablation. During normal cage activity, we observed deuterium enrichment into myonuclear DNA in 7 out of 7 plantaris (PLA), 6 out of 6 tibialis anterior (TA), 5 out of 7 gastrocnemius (GAST), and 7 out of 7 quadriceps (QUAD). The average fractional synthesis rates (FSR) of DNA in myonuclei were: 0.0202 ± 0.0093 in PLA, 0.0239 ± 0.0040 in TA, 0.0076 ± 0. 0058 in GAST, and 0.0138 ± 0.0039 in QUAD, while there was no replication in myonuclei from EDL. These FSR values were largely reproduced in the overload and satellite cell ablation conditions, although there were higher synthesis rates in the overloaded PLA muscle. We further provided evidence that myonuclear replication is through endoreplication, which results in polyploidy. These novel findings contradict the dogma that skeletal muscle nuclei are post-mitotic and open potential avenues to harness the intrinsic replicative ability of myonuclei for muscle maintenance and growth.

Keywords: DNA synthesis; Growth; Muscle; Stable isotope; myonuclei.

Graphical Abstract

Figure 1.

Overview of the experimental design. (A) To identify the source of measured DNA synthesis in skeletal muscle, we used Pax7-DTA mice. Animals underwent TAM or vehicle treatment to induce conditional ablation of SCs. After washout, mice were assigned into sedentary or exercise group (wheel running) for 6 wk. Mice were labeled with the stable isotope D₂O for the entire time of the intervention. (B) To test if skeletal muscle resident myonuclei possess the ability to replicate in vivo, we used the HSA-GFP mouse model, which uses doxycycline (DOX) treatment for the temporal labeling (Tet-ON) of resident myonuclei with GFP. (C) To test if myonuclear replication can be stimulated by mechanical overload, DOX-treated HSA-GFP mice were subjected to SA surgery. (D) To test if SCs depletion stimulates myonuclei to replicate, we used the Pax7-DTA; HSA-GFP mouse model, which uses TAM treatment to deplete SCs and DOX treatment to label resident myonuclei with GFP. In myonuclei replication experiments after removing DOX, and a washout period, we provided D2O for 6–10 wk to measure DNA synthesis in sorted GFP+ (resident myonuclei) and GFP- (nonmyonuclei) fractions by GC-QQQ.

Figure 2.

Impact of SCs ablation on Fractional Synthesis Rate (FSR) of DNA in PLA (A, B) and soleus (C, D). FSR (%/day) was determined in the PLA and soleus muscles of young (7-mo) and old (25-mo) control and exercised mice. A two-way ANOVA (SC presence by exercise) determined a significant effect of exercise, which increased the FSR of DNA. However, there were no main effects of SC ablation. Teal boxes = control (+SC); purple boxes = ablated (-SC). Values expressed as mean ± SEM.

Figure 3.

Representative microscopic images of the validation of the HSA-GFP skeletal muscle after DOX treatment. (A) Longitudinal sections of TA muscle from the HSA-GFP mouse. DAPI = total nuclei, GFP = myonuclei, PAX7 = SCs. The GFP signal from myonuclei does not overlap with the PAX7 signal from SCs. Scale bar = 100 μm. (B) Single-fiber images of EDL muscle from the HSA-GFP mouse with GFP-labeled myonuclei. Scale bar = 80 μm.

Figure 4.

Fluorescence activated cell sorting (FACS) gating strategy for myonuclei and nonmyonuclei sorting from skeletal muscle homogenates. Upper panel = gating strategy for muscle homogenates from noninduced HSA-GFP control mice. Lower panel = gating strategy for muscle homogenates from DOX-treaded HSA-GFP mice. (A, D) Scatterplot of forward scatter (area) by side scatter (area) of muscle homogenate. (B, E) Scatterplot of PI (PI, height) by forward scatter (area). Gate R2 indicates the total nuclear population. (C, F) Scatterplot of PI (PI, height) by GFP (height). Gate R3 indicates myonuclei (PI+/GFP+); gate R4 indicates nonmyonuclei (PI+/GFP-). (G) Representative images of pre-sorted crude nuclear fraction; sorted myonuclear fraction (PI+/GFP+) and sorted nonmyonuclear fraction (PI+/GFP-). Scale bar = 80 μm.

Figure 5.

DNA proliferation rates in sorted myonuclear and nonmyonuclear fractions. (A–C) Nuclei sorting yields from skeletal muscles homogenates. (D–F) DNA fractional synthesis rates (FSR, %/day) in myonuclei and nonmyonuclei fractions. (G–I) Percentage of nuclei that contain new DNA within the labeling time in myonuclei and nonmyonuclei fractions. Red boxes = myonuclei fraction; blue boxes = nonmyonuclei fraction.

Figure 6.

Representative analysis of ploidy levels in sorted myonuclei from PLA muscles. Cell flow cytometry of hepatocytes (A), myonuclei isolated from PLA skeletal muscles from animals kept in the normal cage conditions (B), mice after functional overload (C), and mice after SCs ablation (D). The peaks corresponding to diploid nuclei are labeled 2 N, triploid nuclei as 3 N, tetraploid nuclei as 4 N, and hexaploid nuclei as 6 N.