Hypertrophic cardiomyopathy reshapes cardiomyocyte extracellular vesicle output and composition to promote inflammation and extracellular matrix remodelling

Abstract

Hypertrophic cardiomyopathy (HCM) is a prevalent inherited cardiac disease (≈1:200) marked by left-ventricular hypertrophy and most commonly driven by pathogenic variants in sarcomeric genes, including cardiac troponin I (TNNI3). In addition to cardiomyocyte dysfunction, HCM is frequently accompanied by myocardial fibrosis and immune cell infiltration, yet the pathways linking sarcomeric mutations to these non-cardiomyocyte features remain incompletely defined. Extracellular vesicles (EVs) released by cardiomyocytes provide a mechanism for intercellular signalling in HCM, but their contribution to disease progression is not well established.

Left ventricular–like cardiomyocytes (LV-CMs) derived from induced pluripotent stem cells carrying the TNNI3-R145W variant, alongside isogenic controls, were generated (N=5). Longitudinal phenotyping was performed using immunofluorescence, transcriptomics, proteomics, SarcTrack-based contractility analysis, and Seahorse metabolic profiling. EVs were isolated via size-exclusion chromatography and characterised by Nanoparticle Tracking Analysis, ExoView, electron microscopy, Western blotting, and proteomics in accordance with MISEV2023 guidance. To assess translational relevance, UK Biobank plasma proteomics (≈50,000 participants) were queried for HCM cases and integrated with cellular and EV proteomic signatures from the R145W model.

Compared with controls, R145W LV-CMs demonstrated cellular hypertrophy (P=0.0043), increased contraction amplitude (P=0.0027), and prolonged contraction duration (P=0.0001). Metabolic assessment showed elevated basal oxygen consumption (P=0.0286). Transcriptomic profiling identified 1,080 differentially expressed genes, enriched for hypertrophic extracellular matrix (ECM) remodelling and vesicle/EV-related signalling pathways. Longitudinal proteomics revealed stage-dependent perturbations, with 713 differentially expressed proteins at Day 20 and 1,080 at Day 44, repeatedly highlighting disrupted vesicle-transport programmes.

EV release was reduced two-fold in R145W LV-CMs (P<0.0001). At Day 44, EV proteomics indicated enrichment of ECM/fibrosis and metabolic cargo alongside depletion of sarcomeric proteins, consistent with altered ECM organisation and vesicle-mediated transport (P=6.3×10⁻⁵). Overlap with UK Biobank HCM plasma proteomics highlighted histidine-rich calcium-binding protein and stanniocalcin-2 (P<0.05). Functionally, early-stage R145W LV-CM EVs triggered IL-6–linked inflammatory activation in left-ventricular fibroblasts and primary monocyte-derived macrophages (P<0.05).

Collectively, TNNI3-R145W cardiomyocytes secrete fewer EVs, but their EV cargo shifts toward an early inflammatory programme and a later fibrotic signature. These EV-mediated signals promote inflammatory activation in other cardiac cell populations, supporting EVs as mechanistic biomarkers and potential therapeutic targets in HCM progression.Graphical abstract