SUPPLEMENTAL MATERIAL MITOCHONDRIAL DYSFUNCTION AS AN ARRHYTHMOGENIC SUBSTRATE: A TRANSLATIONAL PROOF-OF-CONCEPT STUDY IN PATIENTS WITH METABOLIC SYNDROME DEVELOPPING POST-OPERATIVE ATRIAL FIBRILLATION. Montaigne et al. SUPPLEMENTAL METHODS In vitro mitochondrial complex activities Frozen atrial tissues were pulverized and homogenized, and protein samples were prepared for measurements of citrate synthase and electron transport chain complex activities as previously described (1). Briefly, citrate synthase activity was assayed by monitoring the reaction of sodium oxaloacetate, acetyl-coenzyme A and 5,5′-dithio-bis-(2 nitrobenzoic) acid at 412 nm. Complex I was determined by following the rotenone-sensitive ubiquinone-1 (Q1)-stimulated NADH oxidation. Complex II+III was assayed by monitoring the rate of reduced cytochrome c formation using succinate as substrate. Complex III was assayed by ubiquinol-2 (Q2H2)-stimulated cytochrome c (cyt c) reduction and verified by inhibition with antimycin A. Complex IV was assayed by measuring ferrocytochrome c oxidation, and further confirmed by inhibition with potassium cyanide. Mitochondrial complex activities were expressed as activities normalized to citrate synthase activity. ROS production and antioxidant enzymes ROS production and antioxidant enzymes (superoxide dismutase (SOD), catalase) were performed using standardized detection methods. In brief, concentration of reactive oxygen species were evaluated in atrial preparations by electron paramagnetic resonance spectroscopy (EPR) using the spin probe 1-hydroxy -3-methoxycarbonyl -2,2,5,5tetramethylpyrrolidine (CMH) by INSERM U644, University of Rouen, France (2). SOD and Catalase activities were evaluated at the Centre of Biology CHRU de Lille as previously described (3). In brief, SOD activity was estimated by measuring the inhibition of xanthine oxidase-mediated cytochrome c reduction. Absorbance was monitored for 2 min at 418 nm. KCN (5 mM) was added to inhibit Cu-ZnSOD activity. SOD activity was expressed as units/mg of protein. One unit of SOD activity was defined as the activity required to inhibit cytochrome c reduction by 50%. Catalase activity was monitored by recording H2O2 decrease at 240 nm in a mixture containing 50 mM potassium phosphate (pH 7) and 25 mM H2O2. The decomposition of H2O2 was monitored for 1 min at 25°C. One unit of catalase activity was defined as 1 μmol of H2O2 degraded per minute per milligram of protein. Transcriptomic study Total RNA was purified from atrial biopsies using the RNeasy kit (Qiagen). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer and RNA preparation exhibiting a RNA Integrity Number (RIN) >7.5 were further processed for hybridization onto Agilent microarrays. mRNAs were converted into complementary RNAs and labeled with Cy5 using the One-color labeling kit (Agilent). cRNAs were hybridized onto Agilent SurePrint G3 Human Gene Expression 8x60K v2 using the Gene Expression Hybridization kit. Signals were collected with the Agilent Microarray scanner and raw data were processed using the Agilent Genespring software GX 11. After uploading into the Genespring software suite and quality controls using the Genespring Feature Extraction QC metrics, the Robust Multichip Analysis (RMA) was used as the summarization algorithm, and the 75th percentile normalization method was used to exclude genes expressed at noise level (lower cut-off: 20%). Gene-level expression analysis were performed and conditions (n=8 for each group) were compared using a Student t-test to determine the statistical significance of gene expression variations. Biological pathway analysis of up- or down-regulated genes (FC>1.2, p<0.05) was performed on the exported gene lists using the Database for Annotation, Visualization and Integrated Discovery (DAVID) web portal (4). The Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to get a representative scheme of genes altered in functional pathways (5). Raw data were deposited on the ArrayExpress with the following accession number E-MTAB-1243. SUPPLEMENTAL TABLES Supplemental Data Table 1 - Characteristics of matched patients in post-operative sinus rhythm and post-operative atrial fibrillation. SR Patients (n = 8) POAF Patients (n = 8) P value Age (year) Gender (Male) n (%) BMI (kg/m²) Waist circumference (cm) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Heart Rate (bpm) 68 ± 7 6 (75) 30 ± 4 115 ± 12 133 ± 18 73 ± 7 66 ± 8 69 ± 7 6 (75) 32 ± 7 117 ± 15 131 ± 17 74 ± 10 73 ± 13 0.78 1 0.42 0.54 0.82 0.77 0.25 Comorbidities Hypertension n (%) Diabetes n (%) Dyslipidemia n (%) Active smoker n (%) Euroscore 8 (100) 8 (100) 6 (75) 2 (25) 3.6 ± 1.3 7 (87.5) 8 (100) 7 (87.5) 1 (12.5) 3.5 ± 1.6 0.99 1 0.99 0.99 0.72 9.2 ± 2.5 7.5 ± 1.6 0.36 ± 0.04 1.4 ± 0.4 10.3 ± 1.3 6.8 ± 0.7 0.32 ± 0.09 1.5 ± 0.5 0.31 0.27 0.25 0.64 ECG and echocardiographic data P-wave duration (ms) LVEF (%) Dilated LA (%) 112 ± 23 61 ± 8 3 (37.5) 100 ± 21 57 ± 9 3 (37.5) 0.30 0.31 1 Time on bypass (min) 80 ± 37 82 ± 16 0.81 Preoperative medication Statins n (%) Diuretics n (%) ACE-inhibitors or ARBs n (%) Beta-blockers n (%) Antiplatelet agent n (%) Oral anticoagulant n (%) Insulin n (%) Metformin n (%) Sulfonylureas n (%) Fibrates n (%) 8 (100) 2 (25) 4 (50) 7 (87.5) 8 (100) 0 (0) 0 (0) 4 (50) 3 (37.5) 0 (0) 7 (87.5) 3 (37.5) 5 (62.5) 8 (100) 8 (100) 0 (0) 0 (0) 4 (50) 2 (25) 0 (0) 0.99 0.98 0.99 0.99 1 1 0.98 - Variables Laboratory data Creatinine (mg/L) Glycated hemoglobin: A1C (%) HDL (g/L) TG (g/L) Continuous variables are given as the mean ± SD, or the median [25th–75th percentiles] if non-normally distributed. SR, sinus rhythm; BMI, body mass index; HDL, high density lipoprotein; LDL, low density lipoprotein; TG, triglycerides; LVEF, left ventricular ejection fraction; LA, left atrial; ARBs, angiotensin receptor blockers; ACE, angiotensin converting enzyme. SUPPLEMENTAL FIGURE LEGEND Supplemental data Figure 1: Gene expression study. (A) Volcano plot illustrating the differences between atrial samples from 8 patients with no POAF matched with 8 patients with POAF occurrence for significant expression changes assessed by paired t-tests. The FC cut-off was set at 1.2 and the p value at 0.05. (B) Top ten up and down regulated genes. (according to HUGO Gene Nomenclature Committee database). SUPPLEMENTAL FIGURE Supplemental data Figure 1: SUPPLEMENTAL REFERENCES (1) Birch-Machin MA, Turnbull DM. Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Methods Cell Biol. 2001;65:97-117. 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