PSV-5 Single-nucleus analysis of intramuscular fat reveals predominance of lipid associated macrophages in beef steers with high marbling.

Abstract Intramuscular adipose tissue (IMAT; marbling) deposition is the deciding factor of beef quality grade in the U.S. Cellular mechanisms mediating IMAT growth remain little understood. We aimed to define IMAT cellular transcriptional heterogeneity in beef steers with High (HM) and Lower (LM) marbling scores. IMAT was dissected from Longissimus dorsi muscle (9-11th rib) from eight half-sibling finished Angus steers. IMAT nuclei were isolated, then sequenced via single-nuclei RNA sequencing with 10x Genomics in an Illumina NovaSeq-6000. Analysis was conducted using R-Studio. Marbling score (P= 0.0018) and percent muscle fat (P=0.0003) were higher in HM (798.5 ± 31 and 21±2.17%, respectively) vs. LM (629.0 ± 7 and 7.0±0.08%), which graded prime and choice, respectively. No differences were observed on backfat thickness and carcass KPH% between groups. Serum free fatty acids were higher in HM vs. LM (0.27 ±.008 vs. 0.19 ±.016 mEq/dL; P= 0.0023). The cellular composition of HM vs. LM IMAT was, respectively: 29% vs. 8% mature adipocytes (AD), 12% vs. 9% adipose progenitor cells (APC), 10% vs. 24% endothelial cells (EC), 6% vs.10% pericytes and smooth muscle cells (PESMC), 19% vs. 37% muscle cells (MUSC), and 24% vs. 12% immune cells (IMC), mainly represented by macrophages (Figure 1A-B). The higher abundance of ASPC and AD in HM vs. LM suggests a higher adipogenic and lipogenic propensity in HM, befitting increased IMAT deposition. High frequency of MUSC expressing high MYOG and MYOD1 were likely due to remaining muscle attached to IMAT after its dissection. Lipid associated macrophages (LAMs) were notably more abundant in HM (80.8% of IMC) compared to LM (26.5% of IMC) (Figure 1B). LAMs play a central role in tissue remodeling and inflammation in adipose tissue. We identified two LAM subtypes (Figure 1C): LAM1-TREM2+ (Triggering receptor expressed on myeloid cells 2), which play a proinflammatory role, scavenging excess lipid; and LAM2- LPL+ (Lipoprotein lipase), which exhibit anti-inflammatory properties by performing lipid homeostasis. The increased abundance LAMs in HM vs. LM may suggest a response to higher amounts of ‘ectopic’ IMAT associated with a chronic positive energy balance during the finishing period, which triggers inflammatory responses and require lipid homeostasis. Functional transcriptional analysis (Gene ontology terms) of LAMs compared to the other IMAT cells suggest LAMs play a complex role in regulating lipid accumulation, adipocyte proliferation and differentiation, and inflammation in conditions where excess or higher marbling is present (Figure 1D). Overall, our results highlight the predominance of specific LAM populations in IMAT of HM steers and imply higher marbling deposition induces an increased LAM migration into IMAT, which’s specific roles are yet to be defined.