High-resolution respirometry

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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.
(2) Mellin V, Isabelle M, Oudot A, Vergely-Vandriesse C, Monteil C, Di Meglio B, Henry
JP, Dautreaux B, Rochette L, Thuillez C, Mulder P. Transient reduction in myocardial
free oxygen radical levels is involved in the improved cardiac function and structure after
long-term allopurinol treatment initiated in established chronic heart failure. Eur Heart J.
2005;26(15):1544-50.
(3) Vives-Bauza C, Starkov A, Garcia-Arumi E. Measurements of the antioxidant enzyme
activities of superoxide dismutase, catalase, and glutathione peroxidase. Methods Cell
Biol. 2007;80:379-393.
(4) Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene
lists using DAVID Bioinformatics Resources. Nature Protoc. 2009;4(1):44-57.
(5) Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M. KEGG for integration and
interpretation of large-scale molecular datasets. Nucleic Acids Res. 2012;40:D109-D114.
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