Adult mouse muscle satellite cells (MuSCs) are quiescent in uninjured muscles. Upon muscle injury, MuSCs exit quiescence, reenter the cell cycle to proliferate and self-renew, and then differentiate and fuse to drive muscle regeneration. However, it remains poorly understood how MuSCs transition from quiescence to the cycling state. Here, we report that Pax3 and Pax7 binding protein 1 (Paxbp1) controls a key checkpoint during this critical transition. Deletion of Paxbp1 in adult MuSCs prevented them from reentering the cell cycle upon injury, resulting in a total regeneration failure. Mechanistically, we found an abnormal elevation of reactive oxygen species (ROS) in Paxbp1-null MuSCs, which induced p53 activation and impaired mTORC1 signaling, leading to defective cell growth, apoptosis, and failure in S-phase reentry. Deliberate ROS reduction partially rescued the cell-cycle reentry defect in mutant MuSCs. Our study reveals that Paxbp1 regulates a late cell-growth checkpoint essential for quiescent MuSCs to reenter the cell cycle upon activation.

Skeletal muscle regeneration requires the highly coordinated cooperation of muscle satellite cells (MuSCs) with other cellular components. However, due to technical limitations, it remains unclear how MuSCs dynamically interact with non-myogenic cells, especially myeloid cells, in live animals. In this work, we developed a dual-laser multimodal nonlinear optical microscope platform to serve as an effective tool for studying the real-time interaction between MuSCs and non-myogenic cells during the early phase of muscle regeneration. Increased cell volume and mitochondrial mass, cell density, and myotube formation are indicative of MuSCs activation/growth, proliferation, and differentiation, respectively. Using 3D time-lapse imaging on live reporter mice containing both red fluorescence protein (RFP)-labeled macrophages and yellow fluorescence protein (YFP)-labeled MuSCs, and taking advantages of the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH), we monitored the real-time spatiotemporal interaction between RFP+ macrophages/RFP- non-myogenic cells and YFP+ muscle stem/progenitor cells during the activation and the proliferation stages of regeneration. Our results indicated that their cell-cell contact was transient in nature. By inhibiting macrophage infiltration, we further showed that direct cell-cell contact between macrophages and MuSCs was not required for early activation of MuSCs before the proliferation stage. However, decreased macrophage infiltration impeded the proliferation and differentiation of MuSCs and also led to intramuscular fibrosis. Besides, neutrophil depletion in the CCR2 deficient mice did not delay the initial growth of MuSCs. These findings provide a new perspective on the role of myeloid cells during muscle regeneration.

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