Abstract 134: A Single-cell Atlas Of Human Skeletal Muscle Tissue In Chronic Limb-threatening Ischemia

Introduction: Chronic limb-threatening ischemia (CLTI) represents end-stage peripheral arterial disease (PAD) and is a significant cause of limb amputation and mortality. Evidence supports the importance of skeletal muscle responses in the pathobiology of CLTI. Skeletal muscle regeneration depends on the coordinated activity of muscle stem cells (MuSCs) and an array of non-myogenic cells, including immune cells and fibroadipogenic progenitors (FAPs). The mechanisms underlying failed muscle regeneration in the CLTI limb are unknown. Single-cell RNA sequencing (scRNA-seq) facilitates the deconvolution of multicellular tissues in homeostatic and pathologic conditions. Herein, we profiled the transcriptomes of 135,203 cells from lower extremity skeletal muscle of PAD patients to construct a cellular atlas of CLTI. Methods: We recruited 12 PAD patients undergoing major limb amputation for CLTI. We obtained proximal (non-ischemic) and distal (ischemic) muscle samples from each patient and used scRNA-seq to profile the transcriptomes of 1,700 – 20,000 cells per sample. Results: We observed 10 distinct cellular populations in the CLTI limb. The predominant cell types were MuSCs, FAPs, and immune cells. There was significant transcriptional heterogeneity in each population; we defined a range of MuSC, FAP, and macrophage sub-populations and identified specific FAPs and macrophages associated with tissue ischemia. Using CellChat analysis, we found differential cell-cell signaling probabilities in ischemic vs. non-ischemic muscle. In ischemic muscle, FAPs are the primary source of outgoing signals, while macrophages are the primary receiver. Conclusions: Our single-cell atlas highlights the transcriptional heterogeneity of cellular responses in the CLTI limb. This atlas will serve as a resource for future studies of the molecular underpinnings of CLTI, advance our understanding of its pathogenesis, and support the development of novel therapies.