PSVII-24 - Single-nuclei transcriptome of subcutaneous and intramuscular adipose tissues in beef cattle reveals depot-specific cellular heterogeneity

Abstract: The deposition of intramuscular adipose tissue (IM; marbling) is the primary determination of beef quality grade in the U.S. While marbling has improved, carcass fatness has increased. The linear relationship between IM content and the deposition of subcutaneous adipose tissue (e.g. backfat; BF) poses a great challenge to the beef industry, as large amounts of ‘‘waste fat” are generated for every percent of IM. Our objective was to define IM and BF-specific transcriptional heterogeneity at the single-cell level in beef cattle. Paired IM and BF samples from the longissimus muscle (9-11^th ribs) were collected from two beef steers at harvest and immediately flash frozen in liquid nitrogen. Nuclei was isolated by dounce homogenization. Single-nuclei RNA sequencing (snRNAseq) was performed using 10x Genomics and Cell Ranger pipelines and analyzed using Seurat and R Studio. Across BF and IM, we identified 13 cell populations among 5,500 sequenced nuclei, including major adipose tissue (AT) cell types, such as mature adipocytes (AD), adipose progenitor cells (APC), lymphatic (LEC) and vascular (VEC) endothelial cells (EC), macrophages (MAC), and pericytes and smooth muscle cells (PE/SM). Cell types were identified by signature genes e.g., PDGRFA or PPARG for APC; ADIPOQ, LEP and/or LPIN for AD; VWF and PECAM1 for EC endothelial; MSR1 for MAC; and NOTCH3 and ACTA2 for PE/SM, respectively. Almost 50% of nuclei across IM and BF expressed mature adipocyte signature genes being the most frequent cell type, followed by APCs (20%) and ECs (16%). We identified four subtypes of AD in both BF and IM, and a BF-specific AD subpopulation expressing LPIN+ and high levels of VEGFA, GPAM and PPARGC1A. There was an IM-enriched subpopulation expressing PPARG and high KRT19, a marker of mesenchymal cells, which was practically missing in BF (0.2% of total nuclei vs. 7% in IM). These findings suggest depot-specific APC and AD subpopulations that may arise form distinct APC. Across BF and IM, we identified three APC subtypes, two of which expressing the fibro-adipogenic (FAP) marker PDGFRA, and one expressing high levels of PPARG and FASN, master regulators of adipogenesis. Both FAP subpopulations expressed high levels of extracellular matrix and fibrogenic genes, such as LUM, FBN1, COL1A1 and COL6A1, however, these genes were higher in IM vs. BF, suggesting a pro-fibrogenic profile of IM. BF had increased proportion of VEC (7% vs. 1%) and half of LEC (14% vs 7%) compared to IM, indicating high AT vascularization. Unprecedently, we demonstrate depot-specific transcriptional heterogeneity at a single-cell level in BF and IM of beef cattle. Defining the molecular and cellular mechanisms regulating differential AT growth will aid in the development of approaches to enhance marbling while limiting fatness.