Inactivation of the NLRP3 inflammasome mediates exosome-based prevention of atrial fibrillation

Figures

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  Figure 1. Dose dependent effects of intramyocardial injection of human extracellular vesicles (EVs) on cardiac electrophysiology and atrial fibrillation inducibility in a model of rat sterile pericarditis. (A) Increasing doses of EVs or vehicle control were injected into the atria of middle-age female Sprague-Dawley rats at the time of talc application. A sham control group was included to demonstrate background inducibility. Three days after surgery, all rats underwent invasive electrophysiological testing prior to sacrifice for histological and molecular characterization. (B) Effect of EV treatment on the probability of inducing AF after burst pacing. A two-sided Chi square test was performed with no penalty for multiple comparisons. *P<0.05 vs. talc + vehicle. #P<0.05 vs. sham. (C) Effect of pericarditis and EV treatment on the duration of the longest AF episode recorded. Data is displayed using a logarithmic abscissa and was analyzed using Kruskal-Wallis testing for multiple comparisons. #P<0.05 vs. sham. (D) Effect of pericarditis and EV treatment on electrocardiographic and electrophysiological function. One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons…

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  Figure 2. Effects of intramyocardial injection of human extracellular vesicles (EVs) on cardiac connexin distribution and atrial activation using a model of rat sterile pericarditis. (A) Representative images from sham, talc + vehicle and talc + EV treated animals showing connexin 40 and 43 localization at cell poles and intercalated disks (n= 4 random fields per group). Scale bar 20 μm. (B) Effect of pericarditis and EV treatment on p wave duration (n=10-19 animals per group). One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. talc + vehicle and #P vs. sham.

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  Figure 3. Intramyocardial injection of human extracellular vesicles (EVs) reduce inflammatory infiltrates and oxidative stress in a model of rat sterile pericarditis. (A) Representative images from sham, talc + vehicle and talc + EV treated animals showing inflammatory infiltrates using hematoxylin and eosin staining. Scale bar 2 or 0.2 mm, indicated. (B) Random field analysis showing that increasing EV dose had an immediate and progressive inhibitory effect on inflammatory infiltrates (hematoxylin and eosin staining) within talc treated animals (n=5). Dotted line indicates baseline (sham). (C) Effect of pericarditis and EV treatment on myeloperoxidase activity (n=5). One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. talc + vehicle treated animals. #P<0.05 vs. sham. EV, extracellular vesicles.

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  Figure 4. Intramyocardial injection of human extracellular vesicles (EVs) reduced inflammatory cytokine content in a model of rat sterile pericarditis. Effect of increasing EV doses on cytokine levels within atrial tissue (n=3-5). One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. talc + vehicle. IL, interleukin; monocyte chemoattractant protein 1, MCP-1; platelet-derived growth factor AB, PDGF-AB; transforming growth factor beta 1, TGFβ1; tumor necrosis factor alpha, TNFα.

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  Figure 5. Intramyocardial injection of human extracellular vesicles (EVs) reduce fibrosis and cardiomyocyte size in a model of rat sterile pericarditis. (A) Effect of pericarditis and EV treatment on hydroxyproline content (n=5-10) and Masson's Trichrome fibrosis (n=3-7). (B) Effect of pericarditis and EV treatment on atrial mass normalized to body weight (n=5-15). (C) Effect of pericarditis and intramyocardial injection of 109 EVs on cardiomyocyte size quantified using histological analysis of plasma membrane border (n=4 biological replicates with 5 technical replicates) after staining with wheat germ agglutinin (WGA) and 4′,6-diamidino-2-phenylindole (DAPI). Scale bar 20 μm. (D) Effect of pericarditis and intramyocardial injection of 109 EVs on the nucleus to cytoplasm ratio quantified using histological analysis (n= 5 random fields with 10 cells per field) after staining with WGA and DAPI. Scale bar 20 μm. One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. talc + vehicle. #P<0.05 vs. sham. EV, extracellular vesicles; veh, vehicle.

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  Figure 6. Intramyocardial injection of human extracellular vesicles (EVs) reduce collagen complexity and pro-fibrotic myofibroblasts within the atria of a model of rat sterile pericarditis. (A) Effect of pericarditis and EV treatment on collagen I and collagen III content (n=3). (B) Effect of pericarditis and EV treatment on alpha smooth muscle actin (αSMA) content within treated atria (left panel) and the number of αSMA+ cells (n=5). (C) Representative images of the effect of pericarditis and EV treatment on αSMA+ cells. Scale bar 20 μm. One-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. sham animals, #P<0.05 vs. talc + vehicle treated animals. EV, extracellular vesicles; DAPI, 4′,6-diamidino-2-phenylindole; veh, vehicle.

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  Figure 7. Intramyocardial injection of human extracellular vesicles (EVs) reduce activation of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in a model of rat sterile pericarditis. (A) Schematic outline of the inflammatory effect of post operative pericarditis on NLRP3 activation, atrial remodeling and EV effects (red boxes). (B) Effect of pericarditis and EV treatment on the atrial content of NLRP3 and downstream mediators (interleukin-1 beta (IL-1β) and IL-18). NLRP3 data was analyzed using a one-way ANOVA with individual-mean comparisons by Šídák's multiple comparisons test. *P<0.05 vs. talc + vehicle treated animals. Relationship between NLRP3 and IL-1β or IL-18 abundance within atrial tissue was analyzed using simple linear regression. (C) Rank order plot highlighting the abundance of miRNAs (left panel) and proteins (right panel) found in EVs suggested to directly inhibit NLRP3 activation. (D) In vitro effect of explant-derived cell (EDC) EVs on caspase-1 activity, a measure of NLRP3 activation, in THP-1 cells (macrophages) and primary cultured atrial fibroblasts after activation by lipopolysaccharide (LPS) and…