Sparsely methylated mitochondrial cell free DNA released from cardiomyocytes contributes to systemic inflammatory response accompanied by atrial fibrillation

Figures

• 

  Figure 1. Elevation of total cfDNA in AF patients. (a) Comparison between non-AF and AF. (b) Comparison among the 4 subgroups. Mann–Whitney test was performed for a, Kruskal–Wallis test followed by post-hoc Steel–Dwass for b. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Figure 2. mt-cfDNA, rather than n-cfDNA, was increased in AF patients. Comparison between non-AF and AF for n-cfDNA (a) and mt-cfDNA (b). Comparison among the 4 subgroups for n-cfDNA (c) and m-cfDNA (d). Mann–Whitney test was performed for (a) and (b), Kruskal–Wallis test followed by post-hoc Steel–Dwass for (c) and (d). *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Figure 3. Total cfDNA, n-cfDNA, and mt-cfDNA in in vitro AF mimicking model. (a) Total cfDNA, (b) n-cfDNA copy number, (c) mt-cfDNA copy number in an in vitro tachycardia pacing model (n = 3 in each pacing duration). Absolute n-cfDNA and mt-cfDNA copy numbers were calculated using qPCR, using the created standard curves. Unpaired t test was performed. ANOVA test were followed by post-hoc Tukey for a in comparison among pacing groups (†). *p < 0.05, **p < 0.01, ***p < 0.001, † in comparison among pacing groups.

• 

  Figure 4. Total cfDNA, n-cfDNA, and mt-cfDNA in an in vivo AF mimicking model. (a) Total cfDNA, (b) n-cfDNA copy number, (c) mt-cfDNA copy number in an in vivo tachycardia pacing model (n = 7). Absolute n-cfDNA and mt-cfDNA copy number were calculated using qPCR using the created standard curves. * p < 0.05.

• 

  Figure 5. mt-cfDNA, but not n-cfDNA, promoted IL-1β and IL-6 expression in macrophages. (a) Total cfDNA (25 ng/mL, 250 ng/mL or 500 ng/mL) (n = 4–8), (b) n-cfDNA (500 ng/mL), and mt-cfDNA (500 ng/mL) (n = 5–8) were applied to J774.1, then pro-inflammatory cytokines expression was analyzed using qPCR. Nuclear and mitochondrial DNA were independently extracted and fragmented to simulate n-cfDNA and mt-cfDNA. ANOVA followed by post-hoc Tukey was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

• 

  Figure 6. Sparsely methylated mt-cfDNA induced IL-1β and IL-6 expression in macrophages via TLR9. (a) Comparison of IL-1β and IL-6 expression levels between TLR9 inhibitor vs. ODN control under mitochondrial DNA exposure (500 ng/mL) (n = 4–6). (b) Comparison of IL-1β and IL-6 expression levels between fully methylated mitochondrial DNA (500 ng/mL) versus totally unmethylated mitochondrial DNA (500 ng/mL) (n = 6). Unpaired t test was performed for (a) and (b) *** p < 0.001.

• 

  Figure 7. Receiver operating curve (ROC) curve analysis for AF incidence and progression. ROC analysis for AF incidence (a) (non-AF vs. AF), and for AF progression (b) (non-AF and paroxysmal AF vs. persistent AF) using total cfDNA, n-cfDNA, and mt-cfDNA. AUC, area under the curve.