{"doi":"10.1002/ctm2.979","title":"Cardiac troponin T in extracellular vesicles as a novel biomarker in human cardiovascular disease","abstract":"Dear Editor, Soluble cardiac troponin T (cTnT), an indicator of myocardial injury and stress, is used in decision management for patients with cardiovascular disease (CVD). As highly sensitive assays can detect elevated concentrations of cTnT even in healthy individuals (e.g. outside of myocardial necrosis, electrocardiographic changes or angina) and cannot distinguish among disease conditions,1, 2 a comprehensive understanding of the cTnT-secretome is an unmet need. Within the secretome, cTnT is not only present as a soluble factor but may also be contained within extracellular vesicles (EVs).3 EVs are nanoscale particles secreted by all cells, the cargoes of which can reflect the molecular composition of the cells of origin4 and indicate disease or injury.5 As EVs are easily sampled from plasma,6 they are being developed as a ‘liquid’ biopsy reflecting the disease state from the tissue of origin. Here, we advanced a fluorescence-based super-resolution microscopy technique, quantitative single-molecule localization microscopy (qSMLM), to robustly characterize cTnT-positive EVs. Importantly, we provide the first report of cTnT-secretome across a spectrum of CVDs. EVs were purified from induced pluripotent stem cell–derived cardiomyocyte cell media (CCM), representing a source of cardiomyocyte-derived EVs (Figure S1), and patient plasma (Figures S2): healthy subjects (n = 5), patients with heart failure (HF; n = 5), hypertrophic cardiomyopathy (n = 3), type 1 myocardial infarction (MI-TI, n = 5) or type 2 myocardial infarction (MI-TII; n = 5) and chronic kidney disease (CKD; n = 5). In all cases (Figures S1 and S2), EVs had intact morphology and contained canonical EV markers (tetraspanins CD9, CD63, CD81; luminal marker TSG101) with low amounts of soluble proteins. According to dot blots (Figure S2C), the CD81 content of patient EVs was highly variable, whereas combined tetraspanins had more uniform expression. Table S1 shows patient characteristics. Control patients were younger and more likely to be female. Patients with MI and CKD had a higher incidence of diabetes and CAD, whereas HF patients had lower mean left ventricular ejection fraction and higher NT-proBNP. To enable molecular quantification of EVs using qSMLM, the following five steps were performed (see the Supplemental Methods): (1) covalent labelling of membrane proteins with fluorescent dye CF568 to detect EV membranes; (2) affinity labelling of cTnT with a specific AF647-tagged antibody (in mild permeabilizing conditions) to detect cTnT-EV cargo; (3) affinity labelling of EVs with unmodified tetraspanin antibodies to isolate tetraspanin-enriched EVs onto coverslips; (4) two-colour imaging to detect EV membrane and cTnT; and (5) data analysis7 with robust molecular counting (Figure S3)8 to quantify images. We first validated the assay and assessed CD81-enriched CCM-EVs (Figure S4). Overall, ∼14% of EVs contained cTnT; on average, these EVs had a diameter of 118 nm with two detected molecules of cTnT per EV (Figure S4D,E). Next, we affinity isolated tetraspanin-enriched plasma EVs (using a combination of antibodies against the canonical EV markers CD81, CD63 and CD9 for affinity pull-down). EV membrane and cTnT were fluorescently labelled and detected using qSMLM (Figure 1A). Characterizations of EVs for individual subjects and controls are provided in Figures S5 and S6. The percentage of cTnT-positive EVs did not vary significantly across CVDs (Figure 1B). Although the overall size distribution of plasma EVs was similar across all CVDs (Figure 1C, left), cTnT-positive EVs were on average significantly larger (Figure 1C, right) with a typically narrower range of sizes (Figure 1D). This was in agreement with CCM-EVs (Figure S4D,E): cTnT-positive EVs had a larger average diameter with a narrower range of sizes. Importantly, cTnT-positive EVs in MI-TI, MI-TII and CKD (but not in HF) were smaller than those in healthy donors; the difference was significant both when ","journal":"Clinical and Translational Medicine","year":2022,"id":250077,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":27,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9598,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":891509,"name":"András Saftics","orcid":"0000-0001-7129-6142","position":1,"is_corresponding":false},{"id":891510,"name":"Sarah Abuelreich","orcid":"0000-0002-0476-5946","position":2,"is_corresponding":false},{"id":891511,"name":"Parul Sahu","orcid":"0000-0001-8864-4043","position":3,"is_corresponding":false},{"id":404382,"name":"H. Immo Lehmann","orcid":"0000-0003-4379-283X","position":4,"is_corresponding":false},{"id":886862,"name":"Adam L. Maddox","orcid":"0000-0002-5942-6538","position":5,"is_corresponding":false},{"id":892240,"name":"Reem Bagabas","orcid":null,"position":6,"is_corresponding":false},{"id":97061,"name":"James L. Januzzi","orcid":"0000-0002-8338-1798","position":7,"is_corresponding":false},{"id":237817,"name":"Kendall Van Keuren‐Jensen","orcid":"0000-0001-8833-6323","position":8,"is_corresponding":false},{"id":251876,"name":"Ravi V. Shah","orcid":"0000-0002-4471-7156","position":9,"is_corresponding":false},{"id":251878,"name":"Saumya Das","orcid":"0000-0002-4521-4606","position":10,"is_corresponding":false},{"id":407384,"name":"Tijana Jovanović‐Talisman","orcid":"0000-0003-1928-4763","position":11,"is_corresponding":false},{"id":778185,"name":"Kathleen M. Lennon","orcid":"0000-0003-3043-331X","position":0,"is_corresponding":true}],"reference_count":10,"raw_metadata":null,"created_at":"2026-07-19T00:24:28.242323Z","pmid":"35988159","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}