ABSTRACT: Purpose: Extraocular muscles (EOMs) display unique physiological and pathological features distinct from limb skeletal muscles. To elucidate the molecular and cellular basis of these differences, we performed a comparative single-nucleus transcriptomic analysis of macaque EOM and quadriceps femoris muscle (QFM). Methods: EOM and QFM tissues from Macaca fascicularis were subjected to single-nucleus RNA sequencing (snRNA-seq). Unsupervised clustering, cell-type annotation, differential gene expression, pathway enrichment, and intercellular communication analyses were performed to compare cellular composition, gene expression, and signaling patterns between the two muscles. Results: We constructed a high-resolution single-nucleus transcriptomic atlas of EOM, identifying major cell types including multiple myofiber subtypes, fibro-adipogenic progenitors (FAPs), Schwann cells, astrocytes, and immune cells. Compared to QFM, EOM exhibited a higher proportion of fast-twitch fibers (2A/2X), FAPs, and neural-associated cells. EOM myonuclei showed enriched expression of genes related to synaptic transmission and axon development, while QFM fibers expressed genes involved in oxidative phosphorylation and glycolysis. EOM uniquely expressed diverse myosin isoforms, including MYH13 and MYH4, supporting its specialized contractile properties. Disease-associated gene profiling revealed that EOM had lower expression of limb muscle disease genes but was susceptible to mitochondrial dysfunction. Cell-cell communication analysis showed that EOM relied more on neuromuscular and regenerative signaling (e.g., WNT, nWNT), while QFM favored immune and metabolic pathways. Conclusions: Our study reveals the unique cellular composition, gene expression patterns, and intercellular signaling features of EOM compared to QFM. These findings provide a comprehensive molecular framework for understanding the specialized function and disease susceptibility of EOM.
