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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 origin 4 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 cellderived 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 wereThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Bone marrow adipocyte formation plays a role in bone homeostasis and whole body energy metabolism. However, the transcriptional landscape and signaling pathways associated with adipocyte lineage commitment and maturation are not fully delineated. Thus, we performed global gene expression profiling during adipocyte differentiation of human bone marrow stromal (mesenchymal) stem cells (hMSCs) and identified 2,589 up-regulated and 2,583 down-regulated mRNA transcripts. Pathway analysis on the up-regulated gene list untraveled enrichment in multiple signaling pathways including insulin receptor signaling, focal Adhesion, metapathway biotransformation, a number of metabolic pathways e.g. selenium metabolism, Benzo(a)pyrene metabolism, fatty acid, triacylglycerol, ketone body metabolism, tryptophan metabolism, and catalytic cycle of mammalian flavin-containing monooxygenase (FMOs). On the other hand, pathway analysis on the down-regulated genes revealed significant enrichment in pathways related to cell cycle regulation. Based on these data, we assessed the effect of pharmacological inhibition of FAK signaling using PF-573228, PF-562271, and InsR/IGF-1R using NVP-AEW541 and GSK-1904529A on adipocyte differentiation. hMSCs exposed to FAK or IGF-1R/InsR inhibitors exhibited fewer adipocyte formation (27–58% inhibition, P<0005). Concordantly, the expression of adipocyte-specific genes AP2, AdipoQ, and CEBPα was significantly reduced. On the other hand, we did not detect significant effects on cell viability as a result of FAK or IGF-1R/InsR inhibition. Our data identified FAK and insulin signaling as important intracellular signaling pathways relevant to bone marrow adipogenesis.

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