Norbert Hübner

Hypertrophic cardiomyopathy (HCM) is marked by asymmetric cardiac wall thickening, hypercontractility, diastolic dysfunction, and fibrosis. Pathogenic sarcomere gene variants cause HCM, but comparable abnormalities occur in patients with unexplained disease, albeit with fewer adverse events. To investigate stage- and genotype-specific disease mechanisms, we performed single-nucleus RNA sequencing of cardiac tissues from 47 patients with HCM, spanning obstructive HCM with preserved systolic function and end-stage HCM, and compared them with nonfailing donor and dilated cardiomyopathy hearts. We identified transcriptional programs associated with cardiomyocyte hypertrophy, fibrosis, and vascular remodeling. Pathogenic variant–positive early-stage HCM samples showed reduced cardiomyocyte abundance and expansion of a proarrhythmogenic cardiomyocyte state. We identified proline-rich 16 ( PRR16 ) as a cardiomyocyte growth–associated gene in HCM and validated its increased expression by RNA in situ hybridization and in a human induced pluripotent stem cell–derived cardiomyocyte HCM model. In HCM samples, fibroblast compositional shifts were associated with profibrotic activation and adverse extracellular matrix remodeling, accompanied by reduced collagen IV ( COL4A1/COL4A2 ) expression and ultrastructural basement membrane abnormalities. HCM samples also exhibited extensive vascular alterations, including shifts in endothelial cell subpopulations, reduced pericyte abundance suggestive of microvascular dysfunction, and increased lymphangiogenic vascular endothelial growth factor C signaling. Unsupervised and supervised machine learning approaches distinguished HCM from dilated cardiomyopathy and accurately predicted genotype status in early-stage HCM from cell type–resolved transcriptional profiles, revealing widespread genotype-driven remodeling. Together, our findings uncover multicellular, genotype-associated remodeling programs in HCM, providing insight into mechanisms underlying arrhythmia, fibrosis, microvascular dysfunction, and heart failure progression.

Supplementary Table 2: Anti-mouse antibodies for flow cytometry

Supplementary Table 6: Microscope configurations for live cell imaging.

Figure S1. Retroviral CCR7 Overexpression Elevates CCR7 in Both CAR-T Cell Subsets Despite Baseline Differences.

Figure S9. Transcriptional profiling of CXCR5-CAR.CCR7 T cells shows no adverse effect on oncogene or tumor suppressor gene expression.

Live-cell imaging of a CCR7-engineered mouse anti-CXCR5 CAR-T cell (cyan) engaging and killing a CXCR5-expressing NIH3T3 tumor cell (red). Sustained immune synapse formation is accompanied by repeated intracellular Ca²⁺ influx events (green), in the target cell, consistent with CAR-T cell–mediated cytotoxic granule release and target cell death. Close-Up of a single interaction, magnified from Video 2.

Supplementary Table 7: Summary of used package versions.

Figure S4. Exemplary Dot Plots from Mouse In Vitro Killing Assay.

Figure S5. Assessing CAR-T Cytolytic Activity and Surface Expression In Vitro.

Supplementary Table 4: Anti-human antibodies for microscopy.