Integrating snRNA-seq and gene perturbation reveals regulatory networks of intramuscular fat deposition in bovine skeletal muscle

Objective: Intramuscular fat (IMF) deposition is a key factor for meat quality traits like juiciness, tenderness, and flavor. Although fibro/adipogenic progenitor cells (FAPs) participate in adipogenesis, the roles of other stromal cells in the muscle microenvironment are unclear. This study aimed to characterize cellular heterogeneity in cattle IMF deposition and identify key cell types and molecular mechanisms regulating intramuscular adipogenesis. Methods: We performed single-nucleus RNA sequencing (snRNA-seq) on Longissimus dorsi muscle samples from three cattle breeds-Angus, Hereford, and Simmental-which exhibit divergent IMF content. Cell populations were annotated using multiple reference-based methods. We applied Scissor to identify phenotype-associated cells, CellChat to infer intercellular communication, hdWGCNA to detect co-expression modules, and scFEA to evaluate metabolic activity. Virtual gene perturbation was conducted using scTenifoldKnk, and drug-responsive cell subpopulations were assessed with scRank. Results: We identified ten distinct cell types in bovine skeletal muscle. Angus cattle, known for high marbling, had elevated proportions of adipocytes, FAPs, and vascular endothelial cells (VEndoCs), while Hereford cattle were enriched in type II fibers. VEndoCs were consistently linked to high IMF and acted as central signaling hubs, interacting extensively with adipocytes and FAPs. These cells also showed high glucose metabolic activity. Co-expression analysis within VEndoCs identified 40 candidate regulators, including nine BMP signaling pathway genes. Virtual knockdown highlighted BMPR1A as the top regulator, altering expression of 13 lipid metabolism-related genes. Neurocytes were most sensitive to resveratrol treatment, largely via ALDH2, whereas FAPs were least responsive. Conclusion: Our findings establish VEndoCs as active regulators of intramuscular adipogenesis via a BMPR1A-mediated signaling pathway. These results provide new insights into the cellular and molecular basis of marbling in cattle and highlight BMPR1A as a potential target for genetic or nutritional strategies aimed at improving meat quality.