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Muscle Stem Cell‐Nerve‐Vasculature Interactions Modulate Tissue Regeneration Following Critical Limb Ischemia

Critical limb ischemia, the most severe form of peripheral artery disease, is a degenerative cardiovascular disease characterized by abnormal perfusion to the limbs due to occlusions of the blood vessels. Although recent developments toward revascularization therapies have been introduced, the myopathy and dysregulation of the skeletal muscle following ischemia have not been thoroughly investigated. To elucidate the regenerative mechanism of the muscle stem cell (MuSC) and its niche components in response to ischemic insults, we explored the temporal regeneration and interactions between the vasculature, motor neurons, muscle fibers, and MuSCs up to 56 days following injury. We first used laser Doppler perfusion imaging (Fig. 1A, B, C) and immunohistochemical analysis in a surgical hindlimb ischemia model in mice to correlate blood flow to skeletal muscle regeneration. The results indicated a delayed and incomplete regenerative response compared to common chemical modes of injury. Dependent upon reperfusion to the hindlimb, the late expression of embryonic myosin heavy chain (Fig. 1D) illustrated slower regeneration kinetics while the presence of centrally nucleated myofibers with smaller mean cross‐sectional area (Fig. 1E, F) displayed incomplete regeneration 56 days after surgery. Next, we investigated ischemia‐induced changes in motor neuron innervation to demonstrate that the size of the neuromuscular junction (NMJ) was determined by the number of subsynaptic nuclei per NMJ (Fig. 1G, H, I). Further examination of the nuclei throughout isolated muscle fibers revealed an increase in myonuclear number per fiber and a concomitant decrease in myonuclear domain size (Fig. 1J, K, L) mediated by the MuSC (Fig. 1M, N, O). In parallel with the accreted myonuclei, ischemic muscle also contained significantly increased total RNA content, suggesting augmented transcriptional output of the regenerating fibers. Finally, because the vast majority of genes required for mitochondrial biogenesis are nuclear‐encoded, we noted that the increase in myonuclei also facilitated an accumulation of mitochondrial content per myonucleus (Fig. 1P, Q, R) to compensate for the mitochondrial dysfunction following injury. Overall, these results indicate that as a regenerative response to critical limb ischemia, the neurovascular network is remodeled and newly regenerated myofibers exhibit MuSC‐derived myonuclear expansion to allow enhanced transcriptional support and an increase in mitochondrial content for bioenergetic need of the energy‐demanding tissue regeneration.