MicroRNA-21 mediated cross-talk between cardiomyocytes and fibroblasts in patients with atrial fibrillation

Figures

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  Figure 1. Circulating miR-21 correlates with left atrial LVAs and treatment outcome. (A) Representative high-density bipolar voltage maps from patients undergoing AF catheter ablation. No/minor LVAs: 37.1%, moderate LVAs: 22.3%, Extensive LVAs: 40.6% (semi-quantitative illustration, no scale bar available). (B) Left atrial LVAs strongly correlate with treatment outcome after ablation. Patients in SR after 12 months: 73.3% having no/minor LVAs, 51.4% having moderate LVAs and 18.2% having extensive LVAs. n = 37–66. (C) Circulating miR-21 positively correlates with left atrial LVAs. Patients with low miR-21 serum concentration had low LVAs and patients with high miR-21 had significantly more LVAs. n = 21–40. (D) Circulating miR-21 in arterial serum correlates with treatment outcome after catheter ablation. 39.1% of patients with high miR-21 serum levels were in stable SR 12 months after ablation, whereas 48.9% with moderate miR-21 concentration and 60% with low miR-21 concentration were free from AF 12 months after ablation. n = 10–45. Data are expressed as mean ± SEM. **p < 0.01. LVAs = low voltage area.

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  Figure 2. AF model with cultured murine cardiomyocytes (HL-1 cells) and fibroblast (NIH/3 T3 cells). (A) Sinus rhythm was simulated using a regular wave form at 25 V 6 ms 1 Hz. (B) For the simulation of AF, a faster, irregular pacing protocol was used (25 V 6 ms 5 Hz, 80% variability). (C) After 4.5 h of pacing, increased miR-21 expression under tachyarrhythmic conditions could be observed in the HL-1 cardiomyocytes. (D) miR-21 concentration in the medium of cardiomyocytes that were held under tachyarrhythmic conditions was strongly increased, whereas no release of miR-21 to the medium under SR conditions could be observed. (E) No increase in miR-21 expression in NIH/3 T3 fibroblast was observed, when treated with control, SR and AF paced medium from HL1 cardiomyocyte cells. (C–E) Representative data out of 3 similar experiments. Data are expressed as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01. CtrlPM, control pacing medium; SRPM, sinus rhythm pacing medium; AFPM, atrial fibrillation pacing medium.

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  Figure 3. miR-21 released from cardiomyocytes induces pro-fibrotic pathways in NIH/3 T3 fibroblasts. (A) The medium of tachyarrhythmic paced cardiomyocytes was transferred to fibroblasts and incubation was done for 72 h, then the pathway analysis was performed. A fibrosis profiler array showed a strong induction of pro-fibrotic pathways and collagen expression in the NIH/3 T3 cells incubated with AF medium. Each sample type represents a pool of one 6 well plate. The induction is especially true for the fibrosis markers α-SMA (B) and Ctgf (C). (B,C) Representative data out of 3 similar experiments. Data are expressed as mean ± SEM. ****p < 0.0001, *p < 0.05. Cav1: caveolin-1, Ccl11: c-c motif chemokine 11, Col1a2: collagen type I alpha 2, Ctgf: connective tissue growth factor, Dcn: decorin, Edn: endothelin, Egf: epidermal growth factor, End: endoglin, Hgf: hepatocyte growth factor, Inhbe: inhibin subunit beta E, Itga3: integrin subunit alpha3, Itgav: integrin alpha V, Itgb5: integrin beta 5, Ltbp1: latent transforming growth factor-β-binding protein-1, Mmp2: matrix metalloproteinase-2, Mmp9: matrix metalloproteinase-9, Serpine1: serpin family E member 1, Serpinh1: serpin family H member 1,…

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  Figure 4. The HDAC1 inhibitor mocetinostat attenuates miR-21 expression and collagen production in AF. (A) A luciferase assay was performed with miR-21, miR-21 with DMSO, miR-21 and miR-21 inhibitor, different concentration of mocetinostat. (B) Mocetinostat attenuated activation of pro-fibrotic pathways and collagen expression in NIH/3T3 fibroblasts incubated with medium from AF- paced cells. Anti-mir-21, that specifically antagonizes miR-21 in the medium, showed similar effects. Each sample type represents a pool of one 6 well plate. Real time PCR (C,D), Western blot analysis (E–G) and immunofluorescence staining (H,I) antibody against Ctgf and α-SMA and counterstaining with DAPI, representative pictures shown in (H) of the fibrosis markers Ctgf and SMA in fibroblasts incubated with SR and AF medium, could confirm the results of the profiler array. Scale bars = 50 μm. (A,C–G) Representative data out of three similar experiments. Data are expressed as mean ± SEM. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Abbreviations same as Figure 3.

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  Figure 5. Central illustration. The proposed pathway in miR-21 mediated adverse atrial remodeling. miR-21 is released from cardiomyocytes under tachyarrhythmic conditions and triggers pro-fibrotic pathways in fibroblasts, mainly through the SMAD7/TGF-b pathway. Mocetinostat, an HDAC1 inhibitor, attenuates miR-21 dependent adverse atrial remodeling and collagen creation by antagonizing the STAT3-pathway. Tgf-β: transforming growth factor beta 1, Tgf-β-R: transforming growth factor beta receptor, HDAC1: Histone deacetylase 1, Smad7: SMAD family member 7, Smad2/3: SMAD family member 2/3, Smad4: SMAD family member 4, STAT3: signal transducer and activator of transcription 3, α-SMA: alpha smooth muscle actin, CTGF: connective tissue growth factor.