Critical Limb Ischemia Induces Remodeling of Skeletal Muscle Motor Unit, Myonuclear-, and Mitochondrial-Domains

Abstract

Critical limb ischemia, the most severe form of peripheral artery disease, leads to extensive damage and alterations to skeletal muscle homeostasis. Although recent research has investigated the tissue-specific responses to ischemia, the role of the muscle stem cell in the regeneration of its niche components within skeletal muscle has been limited. To elucidate the regenerative mechanism of the muscle stem cell in response to ischemic insults, we explored cellular interactions between the vasculature, neural network, and muscle fiber within the muscle stem cell niche. Using a surgical murine hindlimb ischemia model, we first discovered a significant increase in subsynaptic nuclei and remodeling of the neuromuscular junction following ischemia-induced denervation. In addition, ischemic injury causes significant alterations to the myofiber through a muscle stem cell-mediated accumulation of total myonuclei and a concomitant decrease in myonuclear domain size, possibly to enhance the transcriptional and translation output and restore muscle mass. Results also revealed an accumulation of total mitochondrial content per myonucleus in ischemic myofibers to compensate for impaired mitochondrial function and high turnover rate. Taken together, the findings from this study suggest that the muscle stem cell plays a role in motor neuron reinnervation, myonuclear accretion, and mitochondrial biogenesis for skeletal muscle regeneration following ischemic injury.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Characterization of CLI mouse model and skeletal muscle regeneration. (a) LDPI of ventral mouse hindlimbs 1 hour, 3 days, 7 days, 14 days, 28 days, and 56 days following CLI. Control leg on left, ischemic leg on right. Scale bar represents blood flow perfusion by color. (b) H&E staining of TA cross-sections in control, and 3 days, 7 days, 14 days, 28 days, and 56 days following CLI. (c) Immunohistochemistry of TA cross-sections in control, and 3 days, 7 days, 14 days, 28 days, and 56 days following CLI. Dystrophin pseudo-colored in red, eMHC in green, nuclei in blue. Scale bars on cross-sections represent 50 µm. (d) Total number of eMHC+ fibers within a 0.33 mm2 field of view for control, 7 days, 14 days, and 56 days following CLI. (e) Percentage of centrally nucleated fibers in control, 14 days, 28 days, and 56 days following surgery. (f) Mean cross-sectional fiber area of 4 random 0.33 mm2 fields of view of the TA using dystrophin in control, 14 days, 28 days, and 56 days following CLI. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to control for all figures. (g) Mean cross-sectional area of centrally nucleated myofibers of the TA. n = 3, ****p < 0.0001 compared to control. ##p < 0.01, ###p < 0.001 compared to day 56.

Figure 2

Remodeling of the motor unit. (a) NMJ in EDL 1 hour (day 0), 3 days, 7 days, 14 days, 28 days, and 56 days following CLI. Nuclei pseudo-colored in blue, α-bungarotoxin (for acetylcholine receptor-α subunit) in red, Thy1 (for motor neuron axon terminal) in green. Maximum intensity projection was performed on images from confocal microscopy. (b) NMJ on single myofibers from TA from 1 hour (day 0), 3 days, 7 days, 14 days, 28 days, and 56 days following CLI. Nuclei pseudo-colored in blue, acetylcholine receptor-α subunit in red, actin in green. (c) Representative images of normal, partially denervated, and completely denervated NMJ. Acetylcholine receptor-α subunit (α-bungarotoxin) pseudo-colored in red, Thy1 in green. (d) Percentages of normal, partially denervated, and completely denervated NMJ in EDL (at least 20 NMJs per sample) 1 hour (day 0), 3 days, 7 days, 14 days, 28 days, and 56 days following CLI. (e) Number of subsynaptic myonuclei within each NMJ (at least 15 NMJ’s per sample) and their associated NMJ areas of single myofibers from the TA. All scale bars represent 50 µm. n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to day 0 for all figures.

Figure 3

Changes in myonuclear domain following CLI. (a) Z-stack confocal imaging of single myofibers from TA of control, 3 days, 7 days, 14 days, 28 days, and 56 days following CLI. Nuclei pseudo-colored in light blue, actin in red. (b) Number of myonuclei per 500 µm of myofiber (at least 20 fibers per sample) at various timepoints (n = 6). (c) Myonuclear domain of at least 20 single myofibers per sample over 500 µm at various timepoints, calculated as myofiber volume divided by number of myonuclei. Myofiber volume was approximated as the volume of a cylinder using the average radius along a 500 µm length of myofiber (n = 6). (d) Total isolated RNA levels in gastrocnemius homogenate normalized to muscle mass. (e) Z-stack confocal imaging of single myofibers from TA of Pax7-TdTomato mice control, 7 days, 14 days, and 28 days following CLI. Nuclei pseudo-colored in blue, Pax7 in red, actin in green. All scale bars represent 100 µm. Maximum intensity projection performed on all z-stack images (n = 3). (f) MuSC frequency of at least 20 single myofibers per sample at various timepoints reported as percentage of Pax7+ cells out of total myonuclei (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to control for all figures. (g) Myofiber volume, myonuclei number, myonuclear domain, and MuSC frequency as a ratio of ischemic to control myofiber. Dashed line represents a ratio of 1.

Figure 4

Changes in mitochondrial domain following CLI. (a) Confocal imaging of single myofibers from TA of mitoDendra2 mice control, 7 days, 14 days, and 28 days following CLI using constant gain and laser power. Nuclei pseudo-colored in blue, mitochondria in white. Red arrowheads indicate areas of high mitochondrial density. (b) Confocal imaging of single myofibers from TA of mtKeima mice in GFP channel to delineate healthy mitochondria. (c) Confocal imaging of single myofibers from TA of mtKeima mice in dsRed channel to delineate Mitophagy. (d) Merged images of mtKeima myofibers in green (b) and red (c) channels to portray relative levels of healthy to autophagic mitochondria. Scale bars represent 50 µm. (e) Mitochondrial domain of at least 20 single myofibers per sample at various timepoints calculated as relative integrated fluorescent density of total mitochondria in mitoDendra2 mice divided by the number of myonuclei (n = 3). (f) Relative mtDNA copy number quantified as expression of mitochondrial-encoded mt-Co1 normalized by expression of nuclear-encoded Sdha (n = 3). (g) Relative mitochondrial content of healthy and autophagic mitochondria of at least 20 myofibers per sample in mtKeima mice calculated as mean fluorescent intensity of each fiber (n = 3). (h) Red to green ratio of myofibers in mtKeima mice to represent mitochondrial autophagy to healthy mitochondria (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared to control for all figures.

Figure 5

Altered mitochondrial membrane protein expression after CLI. (a) Quantified Western blot analyses 7, 14, and 28 days following CLI for the long isoform of OPA1 protein expression (n = 3). (b) Quantified Western blot analyses 7, 14, and 28 days following CLI for the short isoform of OPA1 protein expression (n = 3). (c) Relative gene expression of Opa17, 14, and 28 days following critical limb ischemia (n = 3). (d) Quantified Western blot analyses 7, 14, and 28 days following CLI for prohibitin 2 protein expression (n = 3). (e) Relative gene expression of prohibitin 2 7, 14, and 28 days following critical limb ischemia (n = 3). (f) Relative gene expression of prohibitin 17, 14, and 28 days following critical limb ischemia (n = 3). *p < 0.05, **p < 0.01 compared to control for all figures. Black bars represent control and gray bars represent hindlimb ischemia for all figures.

Figure 6

Impaired mitochondrial function following CLI. (a) Basal (state 1 respiration) oxygen consumption rate and mitochondrial hydrogen peroxide (H2O2) production from hindlimb skeletal muscles 7 days following CLI using Oroboros Oxygraph-2k (n = 3). (b) Quantified Western blot analyses 7, 14, and 28 days following CLI for mitochondrial ETC complex I (NDUFB8-subunit), complex II (SDHB-subunit), complex III (UQCRC2-subunit), complex IV (MTCO1-subunit), and complex V (ATP5A-subunit) relative protein expression (n = 3). (c) Quantified Western blot analyses 14 days following CLI for SOD2 (n = 3), SOD1 (n = 6), and myoglobin (n = 6) protein expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared to control for all figures.