Atrial myocyte-derived exosomal microRNA contributes to atrial fibrosis in atrial fibrillation

Figures

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  Fig. 1. Atrial myocytes secrete exosomes and exert a profibrotic effect through their miRNA cargo. A Electron microscopy image of atrial myocyte-derived exosomes. The image shows small vesicles approximately 100 nm in diameter; scale bar: 200 nm. B, C Western blot characterization of atrial myocyte-derived exosomes based on the expression of the exosomes markers ALIX, CD63 and CD81 (*P < 0.05 and **P < 0.01 compared with the Ctr group). D Confocal imaging analysis of exosomes uptake experiment. Purified atrial myocyte-derived exosomes were labeled with PKH67 (green), and incubated with atrial fibroblasts. Scale bar: 10 μm. E, F Western blot analysis of the expression levels of collagen I, collagen IIIand α-SMA in atrial fibroblasts treated with atrial myocytes-derived exosomes. (*P < 0.05 and **P < 0.01 compared with EXO-siNC; #P < 0.05 and ##P < 0.01 compared with EXO-Pacing). G Immunofluorescence staining showing the relative expression levels of α-SMA (green) in atrial fibroblasts; nuclei were stained with DAPI (blue). Scale bar: 10 μm

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  Fig. 2. miRNA profiles of atrial myocytes and patient serum-derived exosomes. A Sequencing of differentially expressed exosomal miRNAs between the tachypacing and control groups of atrial myocytes. B Sequencing of differentially expressed exosomal miRNAs between AF and SR groups of patients. C qRT‒PCR of selected differentially expressed miRNAs in atrial myocyte-derived exosomes. D miR-210-3p was detected in exosomes of patient serum (***P < 0.001 compared with the SR group)

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  Fig. 3. Effects of AgomiR-210-3p on atrial function and structure in rats. A qRT-PCR analysis of the expression of miR-210-3p in the rats heart (n = 7; ***P < 0.001 compared with the Ctr and NC groups). B Representative echocardiographic images captured 4 weeks after the AgomiR-210-3p infusion treatment. C The LAD were calculated at 4 weeks in rats (n = 7; *P < 0.05). D Representative electrocardiogram (ECG) traces after atrial burst pacing (left) at 3000 beats/min. E Probability of AF induction by atrial burst pacing of the induced AF episodes (n = 7; *P < 0.05). F, G TGFβ1, α-SMA and Collagen I protein levels were determined in atrial tissues (n = 7; *P < 0.05)

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  Fig. 4. Effect of miR-210-3p KO on cardiac features and function in Ang II-infused rats. A Representative echocardiographic images captured 4 weeks after the Ang II infusion in rats that received KO-miR-210-3p treatment. B The LAD was calculated at 4 weeks in rats (n = 7; *P < 0.05 for the comparison of the WT + Ang II and KO + Ang II groups). The EF%, FS% and RAD calculated at 4 weeks in rats (n = 7; ns for the comparison between the WT + Ang II and KO + Ang II groups). C Representative ECG traces after atrial burst pacing (left) at 3000 beats/min. D Probability of AF induction by atrial burst pacing of the induced AF episodes (n = 7; *P < 0.05 for the comparison of the WT + Ang II and KO + Ang II groups). E AERP measurement in rats (n = 7; ns for the comparison of the WT + Ang II and KO + Ang II groups). F The atrial myocyte cross-sectional area was calculated with Image J software. Scale bar: 50 μm (n = 7; **P < 0.01). G Atrial fibrosis (the ratio of the fibrotic area to total atrium area) was measured using Image J software. Scale bar: 50 μm (n = 7; ***P < 0.001). H Representative images of HE staining of atrial samples; the results are graphed for representative bright field images.…

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  Fig. 5. GPD1L is a target of miR-210-3p and may mediate its effects on fibrosis. A The site predicted GPD1L as a target gene. B RNA-Seq analysis of the differentially expressed mRNAs in the WT and miR-210-3p KO groups of rat hearts. Levels of the GPD1L (C) and Col11a2 (D) mRNAs were analyzed in atrial myocytes by qRT-PCR (*P < 0.05 and **P < 0.01). E The GPD1L protein expression level in the mimic and inhibitor groups of atrial myocytes (*P < 0.05 compared with the inhibitor NC; ***P < 0.001 compared with the mimic NC). F The GPD1L protein expression level in WT and KO groups of rats (n = 7; *P < 0.05). G The GPD1L protein expression level in atrial fibroblasts treated with atrial myocyte-derived exosomes in Ctr and EXO-Pacing groups (***P < 0.001). H Wild-type sequence (WT-GPD1L) and mutated sequence (MUT-GPD1L) for the miR-210-3p binding site. I Dual-luciferase expression is shown for GPD1L (***P < 0.001 compared with mimic NC)

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  Fig. 6. miR-210-3p promotes fibroblast proliferation and collagen synthesis by targeting GPD1L. A GPD1L was silenced by siRNAs in atrial fibroblasts. The protein B, C expression levels of Collagen III, TGFβ1 and α-SMA and mRNA (D) expression levels of α-SMA, Collagen I and TGFβ1 in atrial fibroblasts (*P < 0.05, **P < 0.01, and ***P < 0.001). E CCK8 assay to detect the effect of miR-210-3p and GPD1L on the proliferation of atrial fibroblasts (##P < 0.05 compared with NC, **P < 0.05 compared with miR-210-3p mimic + GPD1L). F Immunofluorescence staining to detect the relative expression levels of α-SMA (green) and DAPI (blue) in atrial fibroblasts. Scale bar: 50 μm

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  Fig. 7. The miR-210-3p-mediated GPD1L/PI3K/AKT signaling pathway contributes to fibrosis and the changes are reversed by LY294002. A KEGG pathway analysis of signaling molecules downstream of GPD1L. B, C Western blot analysis of the levels of the PI3K, P-AKT and AKT proteins in atrial fibroblasts (*P < 0.05, **P < 0.01, and ***P < 0.001). D, E Western blot analysis of the levels of the TGFβ1, Collagen I and Collagen III proteins in atrial fibroblasts (*P < 0.05). F CCK-8 assay of atrial fibroblast proliferation (##P < 0.01 compared with NC and **P < 0.01 compared with si-GPD1L + LY294002). G Immunofluorescence staining showed the relative expression levels of α-SMA (green) and DAPI (blue) in atrial fibroblasts. Scale bar: 50 μm