Increased Arrhythmic Risk in Obesity Is Transduced by Adipose Tissue–Derived Extracellular Vesicles

Figures

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  Figure 1. Altered Electrical Properties of Myocytes in Obese Individuals (A) Action potential in human right atrial myocytes isolated from patients undergoing open heart surgeries. (B) Action potential duration measured at 90% repolarization (APD90). Atrial myocytes from lean patients (body mass index [BMI] <25 kg/m2; n = 6) showed significantly shorter APD90 compared to atrial myocytes from overweight/obese patients (BMI ≥25 kg/m2; n = 9) measured at 1-Hz pacing rate. (C) Resting membrane potential (RMP) of atrial myocytes. No statistically significant difference was found between the RMP of lean and overweight/obese groups. Each data point was derived from average of 2 to 4 recorded myocytes per patient, with bars showing mean ± SEM. ∗P < 0.05. ns = not significant.

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  Figure 2. Alterations in Electrical and Calcium Cycling Properties in Cardiomyocytes Treated With VAT EVs (A) Fluorescence signal representing change in membrane voltage of induced pluripotent stem cell–derived atrial cardiomyocytes (iPSC-aCMs) during pacing at 1 Hz. Treatment of iPSC-aCMs with visceral adipose tissue–derived extracellular vesicles (VAT EVs) (red) prolongs APD compared to control (black). (B) IPSC-aCMs treated with VAT EVs (red, 21 wells from 4 VAT donors) have significantly prolonged APD compared to control (black, 15 wells, treated with iPSC-aCM–derived EVs). APD was measured during pacing at 1 Hz. (C) Representative membrane voltage recordings of induced pluripotent stem cell–derived ventricular cardiomyocytes (iPSC-vCMs), paced at 1 Hz, showing APD prolongation upon treatment with VAT EVs (red) compared to control (black). (D) IPSC-vCMs treated with VAT EVs (red, 19 wells from 5 VAT donors) have significantly prolonged APD compared to control (black, 7 wells, treated with iPSC-vCM–derived EVs). APD was measured during pacing at 1 Hz. (E) Example of arrhythmia observed in paced iPSC-aCMs treated with VAT EVs. (F) IPSC-aCMs treated with VAT EVs (4 events out of 21 wells)…

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  Figure 3. VAT EVs Are Profibrotic and Proinflammatory (A) Quantitative real-time polymerase chain reaction (PCR) of iPSC-derived cardiac fibroblasts (CFs) demonstrates significant upregulation of markers of myofibroblasts upon treatment with VAT EVs (red, 11 wells from 4 VAT donors) compared to control (black, 5 wells, treated with iPSC-CF–derived EVs). (B) IPSC-CFs treated with VAT EVs secrete significantly more collagen into culture media (red, 12 wells from 6 VAT donors) compared to control (black, 5 wells, treated with iPSC-CF–derived EVs). (C) Quantitative real-time PCR analysis of macrophage polarization markers in THP-1–derived macrophages treated with VAT EVs (red bars, 8 wells from 3 VAT donors) compared to control macrophages treated with macrophage-derived EVs (black bars, wells). VAT EV treatment shifted the macrophage activation spectrum toward a proinflammatory state, with significant upregulation of proinflammatory activation markers (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β], interleukin-6 [IL-6], monocyte chemoattractant protein-1 [MCP-1]) and downregulation of anti-inflammatory or reparative activation markers (arginase 1 [Arg1], cluster of differentiation…

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  Figure 4. Characterization of Adipoq-Cre+/Exomap+ Mice (A) Line diagram displaying transfer vector (top) used to create Exomap mouse line (adapted from Fordjour et al26). The Exomap mouse line was crossed with Adipoq-Cre line (Adiopoq) where a Cre recombinase is expressed under control of adiponectin promoter/enhancer region. In the absence of Cre recombinase, mitochondrial red fluorescence protein (mt-tdTomato) is expressed. In the presence of Cre recombinase, green fluorescent protein (mNeoGreen)-tagged human cluster of differentiation 81 (hCD81-mNG) is expressed and localized to the plasma membrane. (B) Bright field (top), and fluorescence images (middle, green channel, bottom, red channel) of freshly mounted inguinal white adipose tissue (iWAT). Only iWAT from double positive mouse (Adipoq-Cre+/Exomap+, left column), express hCD81-mNG on plasma membrane with absence of red mt-tdTomato fluorescence, whereas iWAT of Adipoq-Cre+ line (middle column) does not contain either green hCD81-mNG or red mt-tdTomato fluorescence signal. iWAT of Exomap+ mouse only expresses red mt-tdTomato fluorescence. Scale bar represents 72 μm. (C) Western blot of iWAT lysates from Adipoq-Cre+/Exomap+,…

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  Figure 5. Adipose Tissue–Derived EVs Are Deposited in the Hearts (A) Western blot of iWAT lysate probed with anti-mNeonGreen antibody demonstrates presence of hCD81-mNeonGreen protein in iWAT tissue of both lean and obese mice (bottom row), indicating successful Cre recombination and expression of hCD81-mNeonGreen recombinant protein in iWAT tissue. There is no significant difference between normalized amount of hCD81-mNeonGreen (using vinculin as a loading control) in iWAT between lean and obese groups. (B) Western blot of lysate from left atria probed with anti-mNeonGreen antibody demonstrates presence of hCD81-mNeonGreen of both lean and obese mice (bottom row), indicating presence of adipose tissue-specific EVs tagged with hCD81-mNeonGreen. (C) Western blot of lysate from left ventricles probed with anti-mNeonGreen antibody (bottom row) demonstrates presence of adipose tissue-specific EVs tagged with hCD81-mNeonGreen in left ventricle (bottom row). Normalized amount of hCD81-mNeonGreen is significantly higher in left ventricles of obese compared to lean mice (3 lean mice and 3 obese mice, each with 2 technical replicates). ∗P < 0.05, ∗∗P < 0.01. (D to F) Immunostaining of left…

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  Figure 6. Treatment With VAT EVs Alter Transcriptional Landscape Bulk RNA sequencing of iPSC-aCMs (A and B), iPSC-vCMs (C and D), iPSC-CFs (E and F), and macrophages (G and H), comparing VAT EV–treated groups to their respective controls. A, C, E, G Hierarchical clustering was performed on Ctrl and treatment (VAT EVs) groups based on the differentially expressed genes. The horizontal axis is composed of all the samples in both groups, and vertical axis includes all differentially expressed genes. Control samples are denoted in blue squares and treatment samples in red squares. Dark blue to dark red color gradient shows lower to higher expression. B, D, F, H Volcano plot was created by all differentially expressed genes. The y-axis shows the false discovery rate (FDR)–adjusted P value, and the x-axis displays the log2 fold-change (FC) value. The red dots represent the differentially expressed genes with FDR-adjusted P ≤ 0.1 and absolute FC ≥1.2, whereas green dots represent nonsignificantly differentially expressed genes. Abbreviations as in Figures 1, 2, and 3.

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  Figure 7. TWAS of QT Interval and Atrial Fibrillation (A) Transcriptome-wide association studies (TWAS) of QT interval identified 32 genes that attained genome-wide significance. The red line above indicates genome-wide TWAS significance (P < 5.56 × 10-5), and the red line below indicates genome-wide association studies (GWAS) significance (P < 5 × 10-8). (B) Eleven unique QT interval–associated genes were differentially expressed in in vitro studies and linked (via Hi-C) to a genome-wide significant (P < 5 × 10-8) regulatory GWAS single nucleotide polymorphism (SNP) in the same cell type or tissue in which differential expression was observed (atrium, ventricle, fibroblast, or macrophage). The threshold for genome-wide significance (P < 5 × 10-8) is denoted in black, and the threshold for nominal significance (P < 0.05) is denoted in gray. (C) TWAS of atrial fibrillation identified 24 genes that attained genome-wide significance. (D) Atrial fibrillation TWAS associations with differentially expressed genes in in vitro studies and linked (via Hi-C) to a genome-wide significant (P < 5 − 10-8) regulatory GWAS SNP in the same cell type or tissue in which differential expression was observed…

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  Figure 8. TRPC3 Inhibition Rescues VAT EV–Induced APD Prolongation (A) Representative action potential traces and quantification in atrial cardiomyocytes showing complete rescue of APD prolongation by Pyr3 treatment. (B) The action potential morphology is restored to near-control levels, with normalized repolarization kinetics. (black, ctrl EVs 9 wells; gray ctrl EVs + Pyr3 6 wells; red, VAT EVs 6 wells; blue, VAT EVs + Pyr3, 6 wells) (C and D) Similar analysis in ventricular cardiomyocytes demonstrating significant rescue of APD prolongation by transient receptor potential canonical 3 (TRPC3) inhibition (black, ctrl EVs n = 6 wells; gray ctrl EVs + Pyr3 5 wells; red, VAT EVs 19 wells; blue, VAT EVs + Pyr3, 5 wells) ∗P < 0.05, ∗∗∗P < 0.001. Pyr3 = pyrazole 3; other abbreviations as in Figures 1 and 2.