Volumetric muscle loss (VML) produces chronic functional deficit extending beyond the injury, even in muscle regions that retain innervation. Neuromuscular junction (NMJ) associated gene regulation depends on sustained trophic and electrical input, and disruptions to these signals in other contexts lead to reduced NMJ transcriptional activity. Since VML alters both neural and mechanical environments in the remaining muscle, we hypothesize that VML may impair the ability of surviving fibers to maintain NMJ associated transcriptional responses over time. Understanding how NMJ associated gene expression and nuclear structure in the muscle remaining responds in these surviving regions is needed to identify mechanisms that limit recovery. Our objective was to characterize the temporal changes in NMJ associated gene expression and subsynaptic nuclear architecture in surviving muscle after VML. We hypothesized that VML would induce an early compensatory increase in NMJ associated transcripts that would not be sustained, and that subsynaptic nuclei would undergo progressive structural remodeling consistent with reduced transcriptional responsiveness. Adult male and female mice (n=3 per sex per timepoint) underwent VML of the posterior compartment (~20% excised) or served time 0 controls. Bulk muscle was collected at 0, 7, 14, 48 and 112 days post-injury (dpi) for qPCR analysis of transcripts. VML induced a temporal pattern of transcriptional dysregulation. NMJ related and denervation associated transcripts increased (Etv4, Pdzrn4, Musk, Myog, Runx1) at early timepoints (7, 14 dpi), consistent with an attempted compensatory reactivation in surviving fibers (main effect of time for 4/6 genes, p≤0.0002; Musk p=0.724). By 48 dpi and persisting at 112 dpi, these transcripts declined toward uninjured levels, indicating a failure to sustain synaptic gene expression at the NMJ rather than true recovery. In a separate subset of mice NMJ subsynaptic nuclei were evaluated at 48 and 112 dpi using 3D confocal imaging, nuclei were isolated computationally using a custom Nikon NIS-Elements GA3 segmentation pipeline, and nuclear structural features were analyzed using R-based clustering and effect size statistics. Nuclear structural analysis demonstrated minimal remodeling at 48 dpi; however, by 112 dpi subsynaptic nuclei showed substantial increases in volume (g=0.98), sphericity (g=0.88), chromatin intensity (g=0.63), and dense foci content (g=0.31), reflecting predominantly medium to large structural effect. Despite progressive structural remodeling, the mismatch between the bulk transcription and nuclei structure suggests that critical changes are occurring within specific nuclear populations. These findings reveal an early, but unsustained, attempt at NMJ associated transcriptional rescue followed by late-stage structural remodeling of NMJ subsynaptic nuclei, consistent with a persistent low plasticity NMJ nuclear state that may limit long term neuromuscular recovery. The dissociation between bulk transcript normalization and nuclear pathology provides a strong rationale for applying single-nucleus RNA sequencing to resolve the transcriptional state of NMJ subsynaptic nuclei and identify regulatory pathways that may limit their ability to sustain NMJ associated gene expression after traumatic muscle loss. Ongoing work is examining whether this low plasticity state shares features with neuromuscular aging. Funding: R01-AR078903 (JAC and SMG), K02-AG081488 (SMG) This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.
Ng et al. (Fri,) studied this question.