Atrial Fibroblasts‐Derived Extracellular Vesicles Exacerbate Atrial Arrhythmogenesis

Figures

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  Figure 1. Exosomes derived from Ang II‐treated fibroblasts promoted AF development. A) Representative transmission electron microscopy images of exosomes derived from hACFs (n = 3 per group). Scale bar: 100 nm. B) Particle size distribution detected by NanoSight tracking analysis (n = 2 per group). C and D) Western blot analysis of the exosome markers Alix (p>0.9999) and CD81 (p = 0.849) in cytosol lysis and exosomes of hACFs, GAPDH as control (n = 6 per group). E) Scheme for hACFs exosome preparation, injection, and AF induction test in rats. F) Representative simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in Exo‐Ctl and Exo‐Ang II rats after programmed intracardiac stimulation (red line). G) Incidence of pacing inducible AF in two groups of rats (n = 7 per group, p = 0.0291). H) AF duration in Exo‐Ctl and Exo‐Ang II rats (n = 7 per group, p = 0.0041). I) AERP in Exo‐Ctl and Exo‐Ang II rats (n = 7 per group, p = 0.0347). J) LA area in Exo‐Ctl and Exo‐Ang II rats (n = 6 per group, p = 0.7074). Exo‐Ctl, control hACFs‐derived exosomes; Exo‐Ang II, angiotensin II‐treated hACFs‐derived exosomes; Exo, exosomes; Cyto, cytosol lysis; ECG, electrocardiography; SR,…

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  Figure 2. Exosome miR‐224‐5p derived from atrial fibroblasts is a potential molecule enhancing AF inducibility. A) Representative simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in Exo‐Ctl, Exo‐Ang II, and Exo‐siDicer+Ang II rats after programmed intracardiac stimulation (red line). B) Incidence of pacing inducible AF in three groups of rats (n = 7 or 8 per group, Exo‐Ctl versus Exo‐Ang II p = 0.0014, Exo‐Ang II vs Exo‐siDicer+Ang II p = 0.0256). C) AF duration in Exo‐Ctl, Exo‐Ang II, and Exo‐siDicer+Ang II rats (n = 7 or 8 per group, Exo‐Ctl vs Exo‐Ang II p = 0.0002, Exo‐Ang II vs Exo‐siDicer+Ang II p = 0.017). D) Heatmap of differential miRNAs between control and Ang II‐treated cultured primary adult rat ACFs (n = 3 per group). E) Heatmap of differential exosome miRNAs secreted from control and Ang II‐treated cultured primary adult rat ACFs (n = 2 per group). F and G) miR‐224‐5p expression in both primary adult rat ACFs (n = 6 per group, Exo‐Ctl versus Exo‐Ang II p = 0.0035, Exo‐Ang II vs Exo‐siDicer+AngII p = 0.0031) and ACFs‐derived exosomes by using qRT‐PCR test (n = 6 per group, Exo‐Ctl vs Exo‐Ang II p = 0.0002, Exo‐Ang II vs Exo‐siDicer+Ang II p =…

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  Figure 3. Rat ACFs‐derived exosome miR‐224‐5p exacerbates AF susceptibility in vivo. A) Validation of cellular (n = 8 per group, p = 0.0061) and B) exosome miR‐224‐5p in cultured primary adult rat ACFs transfected with negative control or miR‐224‐5p mimics (n = 9 per group, p = 0.0005). C) WGA and PKH‐26 staining in atrial tissues after exosomes were transferred in rats (n = 3 per group). Scale bar: 25 µm. D) Representative traces of simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in Exo‐NC and Exo‐Mimic injected rats after programmed intracardiac stimulation (red line). E and F) Incidence rate (n = 8 per group, p = 0.007) and duration of AF in Exo‐NC and Exo‐Mimic rats (n = 8 per group, p = 0.0019). G and H) Validation of cellular (n = 7 or 8 per group, Exo‐NC vs Exo‐Ang II p = 0.0001, Exo‐Ang II vs Exo‐Inhibitor+Ang II p < 0.0001) and exosome miR‐224‐5p expressions in cultured primary adult rat ACFs transfected with negative control or miR‐224‐5p inhibitor (n = 7 or 8 per group, Exo‐NC vs Exo‐Ang II p < 0.0001, Exo‐Ang II vs Exo‐Inhibitor+Ang II p < 0.0001). I) WGA and PKH‐26 label staining in atrial tissues after exosomes were transferred in rats (n = 3 per…

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  Figure 4. miR‐224‐5p promotes AF pathogenesis and abbreviated APD of atrial cardiomyocytes in rats. A) Scheme for negative control and agomiR‐224‐5p injection to SD rats, and AF induction test. B) Atrial samples labeled with miR‐224‐5p‐probe (red) and Nuclei probe (Blue) respectively (n = 3 per group). Scale bar: 50 µm. C–E) Representative traces of simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in NC and AgomiR‐224‐5p rats after programmed intracardiac stimulation (red line), and incidence rate (n = 8 per group, p = 0.0101) and duration of AF (n = 8 per group, p = 0.0044). F) Quantification of AERP in NC and AgomiR‐224‐5p rats (n = 8 per group, p = 0.0184). G) Representative recordings of APD in isolated ACMs of NC and AgomiR‐224‐5p rats, and quantification of APD50 (n = 10 or 11 per group, p < 0.0001). H) Scheme for negative control and antagomiR‐224‐5p injection to SD rats, and AF induction test. I) Atrial samples labeled with miR‐224‐5p‐probe (red) and Nuclei probe (Blue) respectively (n = 3 per group). Scale bar: 50 µm. J–L) Representative simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in NC and AntagomiR‐224‐5p rats with…

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  Figure 5. miR‐224‐5p directly targets CACNA1c mRNA in atrial cardiomyocytes. A) Heatmap of differential mRNAs in atria of NC and AntagomiR‐224‐5p rats (n = 3 per group). B) GO analysis of differentially expressed mRNAs. C) Luciferase activities of WT and MT after treatment with NC and mimics of miR‐224‐5p in cultured rat neonatal ACMs (n = 6 per group, NC vs mimic‐miR‐224‐5p p = 0.0144). D) RISK pulldown assay showing CACNA1C level in cultured rat neonatal ACMs treated with WT and MT miR‐224‐5p by qRT‐PCR test (n = 3 per group, Input vs miR‐224‐5p p = 0.6718, miR‐224‐5p vs MT‐miR‐224‐5p p = 0.0022). E) Representative Western blot and quantification of whole tissue Cav1.2 protein level in NC and AgomiR‐224‐5p rats (n = 6 per group, p<0.0001). F) Representative recordings and I‐V curve of I Ca,L in isolated atrial cardiomyocytes of NC and AgomiR‐224‐5p rats (n = 4 or 7 per group). G) Representative Western blot and quantification of total whole tissue Cav1.2 protein level in NC and AntagomiR‐224‐5p rats (n = 7 per group, p = 0.043). H) Representative recordings and I‐V curve of I Ca,L in isolated atrial myocytes of NC and AntagomiR‐224‐5p rats (n = 4 per group). The bar graph data are…

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  Figure 6. Fibroblasts‐specific miR‐224‐5p knock‐in increases AF susceptibility in mice. A–C) Representative traces of simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in WT and FMKI mice after programmed intracardiac stimulation (red line), and incidence rate (n = 8 per group, p = 0.0101) and AF duration (n = 8 per group, p = 0.0039). D and E) Representative recordings of APD and quantification of APD50 in isolated atrial cardiomyocytes of WT and FMKI mice (n = 8 per group, p = 0.0446). F and G) Representative recordings and I‐V curve of I Ca,L in isolated atrial cardiomyocytes of WT and FMKI mice (n = 8 per group). H) Isolated atrial cardiomyocytes stained with Cav1.2 (green), α‐Actinin (red), and DAPI (Blue) respectively (n = 3 per group), Scale bar: 20 µm. I) Western blot testing Cav1.2 protein level in the atrium of WT and FMKI mice (n = 7 per group, p = 0.0002). J–L) Representative traces of simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in FMKI treated AAV9 virus mice after programmed intracardiac stimulation (red line), and incidence rate (n = 7 or 8 per group, p = 0.0406) and AF duration (n = 7 or 8 per group, p = 0.0296).…

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  Figure 7. Knockdown of CFs‐miR‐224‐5p attenuated spontaneous AF incidence in Crem mice. A) Study design for Crem mice injected with NC or AAV9‐shmiR‐224‐5p. B) Representative surface ECG recordings in WT, Crem, and Crem+AAV9 mice. C and D) Spontaneous AF incidence rate (n = 7 or 8 per group, WT vs Crem p = 0.0047, Crem vs Crem+AAV9 p = 0.0406) and AF episode duration (n = 7 or 8 per group, WT vs Crem p = 0.0025, Crem vs Crem+AAV9 p = 0.0224). E) miR‐224‐5p expression of isolated ACFs (n = 6 per group, WT vs Crem p = 0.0001, Crem vs Crem+AAV9 p = 0.0003) and ACFs‐derived exosomes from mice (n = 6 per group, WT vs Crem p = 0.0006, Crem vs Crem+AAV9 p = 0.0013). F and G) Representative echocardiography images, quantification of LA area (n = 6 per group, WT vs Crem p = 0.0094, Crem vs Crem+AAV9 p = 0.1648) and LVEF (n = 6 per group, WT vs Crem p = 0.006, Crem vs Crem+AAV9 p = 0.1417) in WT, Crem and Crem+AAV9 mice. H) HE (up) and Picrosirius staining (down) of the whole heart in three groups of mice, Scale bar: 1 mm; Scale bar: 0.5 mm. I) qRT‐PCR testing CACNA1C expression in the left atrium of WT, Crem, and Crem+AAV9 mice (n = 4 per group, WT vs Crem p = 0.0023, Crem vs Crem+AAV9 p = 0.0104).…

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  Figure 8. Plasma exosomes from AF patients are enriched with miR‐224‐5p and exacerbate AF susceptibility in rats. A) Representative IF images of isolated primary atrial fibroblasts from patient right atria staining with miR‐224‐5p (green), α‐SMA (red), and DAPI (Blue), respectively (n = 3 per group), Scale bar: 20 µm. B) qRT‐PCR test of miR‐224‐5p expressions in isolated primary human ACFs (n = 3 per group, p = 0.0063) and their released exosomes (n = 3 per group, p = 0.0325). (C) qRT‐PCR test of miR‐224‐5p and miR‐224‐3p expressions in the circulating exosomes obtained from SR and AF patients (n = 3 per group, p = 0.0117). D) Scheme for Exo‐SR and Exo‐AF transferred SD rats, and AF induction test. E) Representative traces of simultaneous recordings of surface ECG (lead I) and intracardiac electrograms in Exo‐SR and Exo‐AF rats after programmed intracardiac stimulation (red line). F) Incidence rate of pacing‐induced AF occurrence and AF duration in Exo‐SR and Exo‐AF rats (n = 8 or 9 per group, p = 0.0023). G) AF duration in Exo‐SR and Exo‐AF rats (n = 8 or 9 per group, p = 0.0055). H) Circulating levels of exosome miR‐224‐5p in SR and AF patients (SR n = 125, AF n = 130, p < 0.0001). I)…