1264 - Integrated Spatial Transcriptomics and Single-Nucleus RNA-Seq of FFPE Human Myocardium After Cardiac SBRT for Refractory Ventricular Tachycardia
Presenter(s)
U. Lawrynowicz1, M. Bienkowski2, A. Misztak1, K. Chojnowska1, M. Jaskiewicz1,3, M. Jakalski1,4, U. Juhas5, M. Miszczyk6,7, S. Blamek8, M. Sajdok9, J. Nozynski10, K. S. Golba11, T. Latusek12, T. Jadczyk9, J. Bednarek9, M. Cybulska13,14, R. Kurzelowski9, W. Wojakowski11, J. Mieczkowski1, and B. Tomasik15,16; 13P-Medicine Laboratory, Medical University of Gdansk, Gdansk, Poland, 2Department of Pathomorphology, Medical University of Gdansk, Gdansk, Poland, 3Laboratory of Photobiology and Molecular Diagnostics, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland, 4Applied Genomics and Bioinformatics Centre, Faculty of Biology, University of Gdansk, Gdansk, Poland, 5Division of Bioenergetics and Physiology of Exercise, Medical University of Gdansk, Gdansk, Poland, 6Department of Urology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria, 7Collegium Medicum - Faculty of Medicine, WSB University, Dabrowa Górnicza, Poland, 8Department of Radiotherapy, Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice Branch., Gliwice, Poland, 9Medical University of Silesia in Katowice, Katowice, Poland, 10Silesian Centre for Heart Diseases, Zabrze, Poland, 11Upper Silesian Heart Center, Katowice, Poland, 12Maria Sklodowska-Curie National Research Institute of Oncology, Gliwice, Slaskie, Poland, 13Department of Electrocardiology, Upper Silesian Heart Center, Medical University of Silesia, Katowice, Poland, 14Department of Electrocardiology and Heart Failure, Medical University of Silesia, Katowice, Poland, 15Department of Oncology and Radiotherapy, Medical University of Gdansk, Gdansk, Poland, 16Centre for Experimental Cardiooncology, Medical University of Gdansk, Gdansk, Poland
Purpose/Objective(s): Cardiac stereotactic body radiotherapy (SBRT) has emerged as a treatment option for patients with refractory ventricular tachycardia (VT), achieving durable anti-arrhythmic control without uniform transmural fibrosis and thereby suggesting non-fibrotic mechanisms of myocardial remodeling. We hypothesized that radiation induces spatially localized, cell-type-specific transcriptional programs in human myocardium. We mapped these changes using spatial transcriptomics in an intra-patient model, integrating an internal FFPE-derived single-nucleus RNA sequencing (snRNA-seq) reference to enhance resolution.
Materials/Methods: FFPE myocardial sections from a human explanted heart obtained one year after cardiac SBRT for refractory ventricular tachycardia were profiled using a probe-based spatial whole-transcriptome assay. We compared high-dose (25 Gy, 8272 spots) and low-dose (1-3 Gy, 9261 spots) regions. Spots were initially annotated using published human heart references, then refined by integrating internally generated FFPE snRNA-seq from non-irradiated myocardium (24547 isolated nuclei, FACS-enriched, QC passed). Differential expression and pathway enrichment were performed within cardiomyocyte-, fibroblast-, endothelial-, and immune-enriched regions; inflammatory foci were analyzed separately from adjacent myocardium.
Results: Reliance on external atlas-based annotation provided limited cell-type resolution. Internal snRNA-seq integration substantially improved spot-level classification and cell-type assignment confidence, increasing mean cell-type probability scores by 0.23 (from 0.278-0.313 to 0.478-0.559). In cardiomyocyte-enriched regions, high-dose samples showed differential expression of structural and contractile genes (TNNC1, MYL2, ACTN2, ANKRD1) and ion-handling/conduction genes (KCNIP2, FGF12, TECRL), suggesting parallel structural and electrical remodeling. Extracellular matrix genes (COL1A1, COL1A2, COL3A1, FN1) were downregulated in high-dose regions, arguing against a dominant pro-fibrotic response. Signals in fibroblast-enriched areas were largely attributable to cardiomyocyte admixture. Spatially defined inflammatory foci displayed distinct transcriptional signatures relative to neighboring non-inflamed tissue, consistent with localized remodeling.
Conclusion: We demonstrate that single-nucleus transcriptomics from FFPE human heart tissue is feasible and enhances cell-type resolution in spatial transcriptomics. This unique intra-patient spatial analysis of explanted human myocardium one year after SBRT reveals spatially restricted, cardiomyocyte-enriched transcriptional remodeling without evidence of a dominant pro-fibrotic program. These findings link molecular changes with clinical response and support the concept that structural and electrical remodeling, rather than uniform fibrosis, may contribute to the therapeutic effects of cardiac SBRT.