Striated muscle is a highly organized structure composed of well-defined anatomical domains with integrated but distinct assignments. So far, the lack of a direct correlation between tissue architecture and gene expression has limited our understanding of how each unit responds to physio-pathologic contexts. Here, we show how the combined use of spatially resolved transcriptomics and immunofluorescence can bridge this gap by enabling the unbiased identification of such domains and the characterization of their response to external perturbations. Using a spatiotemporal analysis, we follow changes in the transcriptome of specific domains in muscle in a model of denervation. Furthermore, our approach enables us to identify the spatial distribution and nerve dependence of atrophic signaling pathway and polyamine metabolism to glycolytic fibers. Indeed, we demonstrate that perturbations of polyamine pathway can affect muscle function. Our dataset serves as a resource for future studies of the mechanisms underlying skeletal muscle homeostasis and innervation. Display omitted •Characterization of functional cluster in adult muscle using spatial transcriptomics•Identification of differentially activated pathway during denervation•Polyamine pathway is spatially restricted and innervation responsive Using spatially resolved transcriptomics, D’Ercole et al. have analyzed the different morphofunctional domains in muscle and their response to reversible nerve damage, highlighting the polyamine pathway as a potential contributor to the atrophic response.