Single-cell and Spatial Transcriptomic Profiling of the Type 2 Diabetic Coronary Microcirculation

Coronary microvascular disease (CMD) is an early complication of type 2 diabetes (T2D) that manifests due to endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction. Coronary resistance microvessels (CRMs) from T2D mice and pigs are less stiff compared to normal, a finding that is dictated by less stiff coronary VSMCs, not ECs. These data suggest that cell- and vascular layer-specific mechanisms are responsible for CMD. To interrogate these mechanisms, we hypothesized that single-cell fixed RNA profiling (scRNA-seq) and spatial transcriptomic (ST) analysis of the CRM layers can identify new pathways to target in CMD. 16-week-old control Db/db (n=4) and T2D db/db (n=3) mouse hearts were fixed and embedded. Scrolls for scRNA-seq were cut and cells isolated from tissue sections using the Chromium fixed RNA profiling protocol from 10x Genomics. Sections were also placed onto Visium reaction slides (10X Genomics), and libraries were generated using the 10X Visium Spatial Gene Expression for FFPE kit. Data were analyzed with Seurat. scRNA-seq findings revealed an upregulation of genes associated with adipogenesis, fatty acid metabolism, and oxidative phosphorylation in many of the T2D cell clusters, and numerous genes associated with the succinate dehydrogenase enzyme that plays a critical role in mitochondrial function were differentially regulated in several of the cell clusters. In ECs, VSMCs, cardiomyocyte clusters, fibroblasts, and macrophages, the upregulation of adipogenesis is directed by Angplt4 and Ephx2, whereas Hmgcs2 and Acot2 are the key players in the upregulation of fatty acid metabolism, and Pdk4 and Ech1 are the pivotal contributors to oxidative phosphorylation upregulation. Not surprisingly, many of the upregulated pathways from the scRNA-seq experiments were also upregulated in diabetic CRM-enriched regions of the ST data, specifically oxidative phosphorylation, fatty acid metabolism, and adipogenesis. Interestingly, scRNA-seq data revealed that apoptosis and reactive oxygen species (ROS) pathways were upregulated in VSMCs but not in ECs nor fibroblasts. Cat and Mgst1 are the main contributors to ROS upregulation and Cdkn1a and Txnip to apoptosis upregulation. These data collectively support the idea that fatty acid metabolism and oxidative phosphorylation are dysregulated in CRM cells in diabetic mouse hearts, and VSMCs are distinctly upregulating ROS and apoptosis pathways. These data are intriguing and support the well-documented concept that heart failure in T2DM patients is led by cardiac metabolic inflexibility, from reduced mitochondrial function, increased reliance on fatty acid oxidation, and impaired glucose utilization resulting in oxidative stress and lipotoxicity. Continuing analysis will identify specific molecular targets involved in the pathophysiology of CMD with the goal of identifying novel therapeutic targets. R01 HL165124 and S10 OD023438 and internal NCH funds to AJT. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.