PROJECT 1: ABSTRACT/SUMMARY Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a heterogeneous debilitating disorder characterized by severe disabling fatigue, especially post-exertion, in the absence of alternative diagnosis. The etiology of ME/CFS remains unknown and underexplored and is likely multifaceted. Given the prevalence of muscle fatigue symptoms, the pathophysiology of skeletal muscle has received attention in ME/CFS studies. Skeletal muscles of ME/CFS individuals do not exhibit overt changes in muscle fiber architecture, quality, or type, though studies have identified altered muscle metabolism, peripheral vascular dysfunction, and local and systemic inflammation as potential mediators of CE/MFS. This project will address the premise that skeletal muscle dysfunction in ME/CFS arises from a multifaceted and heterogeneous dysregulation of myogenic, vascular endothelial, and immune cell identities and interactions in skeletal muscles. Myogenic cells and resident immune and vascular endothelial cells are in close proximity and have varied interactions through cell-cell signaling pathways and coupled metabolic programs that are critical for muscle health and disease. Here, we will collect a new cohort of ME/CFS and control skeletal muscle biopsies to investigate the interactions between myogenic, endothelial, and muscle-resident immune cells using innovative single-cell technologies. First, we will collect a new cohort of skeletal muscle biopsies and blood samples from ME/CFS patients (n = 40) and healthy controls (n = 20), along with accompanying survey and physiological data (grip strength and orthostatic vascular function). Building on our prior track record of generating a multi-donor scRNAseq atlas in human skeletal muscle, we will analyze these new biopsies by single-nucleus paired RNA/ATAC-sequencing (snRNA/ATACseq) and a novel method of spatial total RNA-sequencing (STRS). snRNA/ATACseq will reveal changes in cellular identities and compositions, as well as alterations in metabolic and transcriptional programs through various bioinformatic analyses. STRS will reveal changes in the spatial mapping of muscle cell types and their noncoding (e.g., regulatory miRNA and lncRNA) RNA profiles. Together these data will be integrated to identify changes in cell-cell communication (ligand-receptor) pathways involving myogenic, endothelial, and immune cells and their spatial organization between control and ME/CFS muscles. Various metrics from these single-cell datasets will be tested through association studies as possible biomarkers that may explain the heterogeneous manifestations of ME/CFS in this patient cohort. Third, hypotheses related to signaling interactions driving endothelial dysfunction in ME/CFS skeletal muscles will be evaluated using cell culture models involving skeletal muscle- specific human endothelial cells exposed to plasma and monocytes isolated from ME/CFS or control samples. Together, these aims may shed light on the cellular and molecular etiologies of ME/CFS in human skeletal muscles.