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Figure 1. miRNA expression levels.Relative expression levels of miRNAs normalized to the geometric mean of miR‐26b, U47, and RNU6B. For miRNAs indicated in red, interactions with KCNK3 were predicted. For miRNAs indicated in black, an involvement in the pathophysiology of atrial fibrillation was previously described. Data shown as median with min to max (n=61). miRNA indicates microRNA; and Rel., relative.
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Figure 2. Differently expressed miRNAs.
A, Heat map of miRNAs fold change (log2) relative to median of sinus rhythm. Values in red are more and in blue are less abundant. B and C, Fold change (log2) of miRNA expression in paroxysmal atrial fibrillation (n=21; B) and chronic atrial fibrillation (n=17; C) compared with SR samples (means; error bars, standard error; *, P<0.05; **, P<0.01; ***, P<0.001 from t test followed by Bonferroni correction for n=23 comparisons). (D and E) Log2 fold changes for pAF (D) and cAF (E) relative to SR and negative log10‐scaled P values of t tests (horizontal dashed line, P=0.01). (F) Differential expression of miRNAs in cAF relative to SR. Expression levels relative to housekeeping genes and absolute values of log2 fold changes were analyzed to assess which miRNAs are abundant as well as differentially expressed (red font, predicted interaction with KCNK3; black font, previously described association with AF; means of n=61 patients±SEM are indicated). AF indicates atrial fibrillation; cAF, chronic atrial fibrillation; miRNA, microRNA; pAF, paroxysmal atrial fibrillation; and SR, sinus rhythm.
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Figure 3. Effect of heart rhythm on expression of KCNK3 and selected miRNAs.(A through F) miRNA and KCNK3 expression levels in sinus rhythm (n=23), paroxysmal atrial fibrillation (n=21), and chronic atrial fibrillation (n=17); miRNA measurements were normalized to the geometric mean of miR‐26b, U47, and RNU6B, KCNK3 measurements to IPO8; boxes: medians, 25%‐ and 75%‐percentiles; whiskers: minimal and maximal values; P values of 2‐tailed t tests with P<0.05, regarded as significant, are indicated. (G through K) Expression levels of KCNK3 relative to miRNA expression levels (n=61; r, p, Spearman rank order correlation coefficients and P values). cAF indicates chronic atrial fibrillation; miRNA, microRNA; pAF, paroxysmal atrial fibrillation; and SR, sinus rhythm.
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Figure 4. Expression of KCNK3 in transfected hiPSCs.Human induced pluripotent stem cells (hiPSC) were transfected with miRNA mimics, inhibitors, or controls. Expression was measured by real‐time PCR (qPCR; A and B) or Western blot (C through F) 48 hours after transfection. qPCR data normalized to IPO8 expression after transfection with miRNA inhibitors (A) or miRNA mimics (B) with respective controls (n=1–6). Representative Western blots of inhibitors (C) and mimics (D). Human TASK‐1 (hTASK‐1) protein relative to GAPDH for miRNA inhibitors (E) or miRNA mimics (F) with respective controls (means of n=5; error bars, SEM; P values of 2‐tailed t tests with P<0.05, regarded as significant, are indicated). miRNA indicates microRNA; qPCR, quantitative polymerase chain reaction; Rel., relative; and TASK‐1, tandem of P domains in a weak inward rectifying K+ channel (TWIK)–related acid sensitive K+ channel 1.
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Figure 5. TASK‐1 is regulated by miR‐34a.Effects of miRNA mimics and inhibitors on resting membrane potential (RMP) and transmembrane current in Xenopus laevis oocytes after co‐injection with TASK‐1 were quantified. A, Current measurements 48 hours after co‐injection of all tested miRNAs with TASK‐1 (means of n=21–28; error bars, SEM). (B and C) Current measurements 24–72 hours after co‐injection of TASK‐1 with miR‐34a mimic (B, green) or miR‐34a inhibitor (C, green) in comparison to controls (grey; means of n=14–29; error bars, SEM). (D and E) Current‐voltage‐relation 48 hours after co‐injection of TASK‐1 with miR‐34a mimic (D, green) or miR‐34a inhibitor (E, green) in comparison to controls (grey, n=21–28). (F) RMP measurements 48 hours after co‐injection of all tested miRNAs with TASK‐1 (n=21–28). (G and H) Measurements of RMP 24 to 72 hours after co‐injection of TASK‐1 with miR‐34a inhibitor (G, green) or miR‐34a mimic (H, green) in comparison to controls (grey, means of n=14–29; error bars, SEM; P<0.05 of 2‐tailed t tests were indicated). miRNA indicates microRNA; and TASK‐1, tandem of P domains in a weak inward rectifying K+ channel (TWIK)–related acid sensitive K+ channel 1.
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Figure 6. Associations between clinical parameters and miRNA expression.
A, Clusters of rank‐order correlation coefficients (Spearman) for numeric or rank‐ordered parameters and miRNA expression. Dendrogram groups of miRNAs and clinical parameters are indicated by colored bars and grey separation lines (miRNA groups: maroon, orange, blue; parameter groups: purple, grey, yellow; grey squares, P<0.05; black squares, P<pcrit,BH=7.6⋅10‐4 from Benjamini‐Hochberg adjustment for multiple‐testing; (B) Clusters of log2 fold changes of miRNA expression dependent on presence vs absence of binary features (grey squares, P<0.05 from 1‐way ANOVA; black squares, P<pcrit,BH=2.2⋅10‐6 from Benjamini‐Hochberg adjustment for multiple‐testing; dendrogram groups are indicated by colored bars and grey separation lines. ACE indicates angiotensin‐converting enzyme; AF, atrial fibrillation; ALT/GPT, alanine transaminase/glutamic‐pyruvic transaminase; AST/GOT, aspartate transaminase/glutamic oxaloacetic transaminase; AT1, angiotensin II receptor type 1; AVR, aortic valve replacement; BMI, body mass index; CABG, coronary artery bypass graft; COL1A2, collagen alpha‐2(I) chain; COPD, chronic obstructive pulmonary disease; CRP, C‐reactive protein; DDR2, discoidin domain‐containing receptor 2; DOAC, direct oral anticoagulant drug; EDD, end‐diastolic diameter; ESD, end‐systolic diameter; GFR (CKD‐E), glomerular filtration rate from Chronic Kidney Epidemiology Collaboration formula; HCT, hydrochlorothiazide intake; MDRD, Modification of Diet in Renal Disease; GGT, gamma‐glutamyl transferase; LA; left atrium; LDH, lactate dehydrogenase; LV, left ventricle; LVEF, LV ejection fraction; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; miRNA, microRNA; PCI, percutaneous coronary intervention; RBC, red blood cell count; RDW‐CV, red cell distribution width coefficient of variation; S100A4, S100 calcium‐binding protein A4; and TASK‐1, tandem of P domains in a weak inward rectifying K+ channel (TWIK)–related acid sensitive K+ channel 1.
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Figure 7. Analysis of circulating miR‐34a levels.
A through D, Circulating miR‐34a level were analyzed from peripheral blood samples of SR (n=5, ie 3 patients from the study population and 2 additional matched SR controls), pAF (n=5 patients from the study population), and cAF (n=6 patients from the study population). Circulating levels of miR‐34a tended to be higher in patients with increased atrial miR‐34a tissue levels (C) or with increased atrial KCNK3 mRNA levels (D). Means of n=5–6 are presented relative to the internal standard miR‐16 (for circulating miR‐34a), the aforementioned pool (tissue levels of miR‐34a) or IPO8 (KCNK3); error bars, SEM; P values of 2‐tailed t tests with P<0.05, regarded as significant, are indicated. cAF indicates chronic atrial fibrillation; miRNA indicates microRNA; pAF, paroxysmal atrial fibrillation; and SR, sinus rhythm.
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