Integrating single-nucleus RNA sequencing and gene perturbation reveals regulatory networks of intramuscular fat deposition in bovine skeletal muscle.

Abstract

Objective: Intramuscular fat (IMF) is key for meat quality. Although fibro/adipogenic progenitor cells (FAPs) participate in adipogenesis, roles of other stromal cells remain unclear. This study characterized cellular heterogeneity in cattle IMF deposition and identified essential cell types and mechanisms regulating intramuscular adipogenesis.

Methods: We performed single-nucleus RNA sequencing on longissimus dorsi muscle samples from three cattle breeds (Angus, Hereford, and Simmental) with 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, high-dimensional weighted gene co-expression network analysis to detect co-expression modules, and single-cell flux estimation analysis to evaluate metabolic activity. Virtual gene perturbation was evaluated using scTenifoldKnk, and drug-responsive cell subpopulations were assessed with scRank. Additional in vitro validation experiments were conducted to confirm the regulatory effect of BMPR1A.

Results: Ten cell types were identified. Angus (high marbling) showed elevated proportions of adipocytes, FAPs, and vascular endothelial cells (VEndoCs), while Hereford were enriched in type II fibers. VEndoCs were consistently linked to high IMF, acting as central signaling hubs that interact extensively with adipocytes and FAPs, and exhibited high glucose metabolic activity. Co-expression analysis within VEndoCs revealed 40 candidate regulators including nine BMP signaling pathway genes. Virtual knockdown identified BMPR1A as top regulator, altering expression of 13 lipid metabolism-related genes. In vitro validation confirmed that BMPR1A inhibition downregulated PPARγ mRNA, validating its positive regulatory role in intramuscular adipogenesis. Neurocytes showed highest sensitivity to resveratrol (via ALDH2), whereas FAPs were least responsive.

Conclusion: Our findings indicate VEndoCs actively regulate intramuscular adipogenesis via a BMPR1A-mediated signaling pathway, supported by in vitro validation. These results provide insights into the cellular and molecular basis of marbling in cattle, highlighting BMPR1A as a potential target for genetic or nutritional strategies to improve meat quality.