Online Appendix for the following JACC article

TITLE: A Randomized Controlled Trial to Prevent Postoperative Atrial Fibrillation by

Antioxidant Reinforcement

AUTHORS: Ramón Rodrigo, MSc, Panagiotis Korantzopoulos, MD, PhD, Mauricio

Cereceda, MD, René Asenjo, MD, Jaime Zamorano, MD, Eli Villalabeitia, MD, Cristián Baeza

MD, Rubén Aguayo, MD., Rodrigo Castillo, MD, PhD, Rodrigo Carrasco, MD. and Juan G.

Gormaz, PhD.

1

APPENDIX 2

3

Supplementary Introduction 4

5

Postoperative atrial fibrillation (POAF) is the most common arrhythmia following cardiac surgery (1). 6

Despite improvement in anesthesia, surgical techniques, and medical therapy POAF occurs in 20–40% 7

of patients, even when proven and recommended preventive drug therapies such as beta-blockers and/or 8

amiodarone are used (2-5). Patients developing POAF are at increased risk of presenting hemodynamic 9

instability, persistent congestive heart failure and cerebrovascular complications, all contributing to the 10

increased pulmonary ventilator derived complications, intensive care unit readmission, together with 11

increased treatment costs and length of hospital stay (6-11). 12

13

2

The pathogenesis of postoperative atrial fibrillation is complex and multifactorial, including 14

preoperative, intraoperative, and postoperative factors (12). Classical preoperative factors comprise age, 15

gender, obesity, systemic arterial hypertension, diabetes mellitus, chronic obstructive pulmonary 16

disease, history of paroxistic atrial fibrillation, previous myocardial infarction, left ventricular 17

dysfunction, and discontinuation of beta adrenergic drugs prior to surgery (13-15), among others. 18

Intraoperative stress plays a key role due to the occurrence of reperfusion, inflammation, or hemostasis 19

impairment (16,17). Accumulating clinical and basic evidence suggests that one of the leading causes of 20

POAF includes ischemia-reperfusion injury as a main source of oxidative damage (18-24). Oxidative 21

stress results from an imbalance between the generation of reactive oxygen species (ROS) and the 22

endogenous antioxidant defense system so that the latter becomes overwhelmed (1, 25), being 23

postulated as a key factor in the development of other cardiovascular alterations including atherogenesis 24

(26), essential hypertension (27) and heart failure (28). Atrial NADPH oxidase is a major source of 25

ROS and its activity is increased during cardiac ischemia-reperfusion processes (29,30), being 26

independently associated with an increased risk of POAF (31). Furthermore, patients undergoing POAF 27

have significantly more elevation in total serum peroxide levels compared with patients who maintain 28

sinus rhythm (32). 29

30

In addition, oxidative stress may lead to inflammation in cardiac tissue by triggering pro-inflammatory 31

signaling pathways by ROS-dependent activation of transcriptional factors, such as nuclear factor 32

kappaB (NF-κB) (33-35), being postulated that oxidative stress may be a unifying link between 33

cardiovascular risk factors, inflammation, and subsequent development of cardiovascular disease (36). 34

In fact a marked postoperative elevation of systemic inflammatory biomarkers (37) have been 35

demonstrated in patients who develop POAF after on-pump cardiac surgery compared with patients who 36

maintain sinus rhythm after the surgical procedure (38-43). Subsequently, inflammation could positive 37

feedback oxidative stress (44,45). As a result, oxidative stress and inflammation should be unavoidably 38

3

present in all cardiac surgeries with extracorporeal circulation, being atrial tissue subjected to an 39

oxidative challenge likely to participate in the mechanism of POAF. 40

41

According the relationship between oxidative stress and POAF, direct antioxidants have been proposed 42

in various clinical trials as a possible prophylactic therapy for decreasing POAF incidence (46). 43

Administering the ROS scavenger edaravone to patients with acute myocardial infarction immediately 44

prior to reperfusion significantly reduced infarct size and reperfusion arrhythmias (47). Further, it was 45

found that oral vitamin C in combination with β-blockers is more effective than β-blockers alone in the 46

prevention of POAF (48). More recently it was reported a significant reduction of the incidence of 47

POAF in patients undergoing on-pump surgery who were treated with N-acetylcysteine alone (49) or in 48

combination with β-blockers (50). Moreover, it was shown that vitamin added to β-blockers not only 49

prevents POAF, but also reduces intensive care unit length of stay, and shortens the time interval for 50

conversion into sinus rhythm (51). 51

52

On the other hand, the finding that that low-to-moderate ROS levels, not enough to generate cellular 53

injury, can act as a signal enhancing the endogenous antioxidant response (52), favored the development 54

of non-hypoxic prophylactic strategies aimed to reduce oxidative damage associated with ischemia-55

reperfusion in cardiac surgeries (1,18). The cell signaling of this process occurs through activation of 56

nuclear factor-erythroid 2-related factor 2 (Nrf2) transcription factor (53). It was shown that this 57

mediator up-regulates several housekeeping genes including endogenous antioxidants enzymes such as 58

catalase (CAT) and glutathione peroxidase (GSH-Px) in rat heart (54) and primary cardiomyocytes (55). 59

Therefore, it should be expected that innocuous substances highly prone to peroxidation, such as n-3 60

PUFAs, can increase myocardial ROS in a dose-dependent regulation manner being enough to activate 61

the Nrf2 pathway (53) but not cell death pathways, thus decreasing the vulnerability of myocardial 62

tissue to a subsequent oxidative challenge (1,18). 63

64

4

Based on the biological roles of n-3 PUFAs, these compounds were previously tested as antiarrhythmics 65

agents in animal models and isolated cardiomyocytes, showing promissory results (56-62). However, up 66

to date, the therapeutic role of n-3 PUFAs in POAF prevention is not clear, as some studies showed 67

significant prevention of POAF (63-65) but others could not demonstrate such beneficial effect (66-69). 68

Although, all these studies have a similar design, the major difference is found in the DHA:EPA ratio in 69

the supplementation formulae. Thus, the efficacy in POAF prevention could be partly explained on the 70

basis of the ability of formulations to counteract the ischaemia reperfusion-derived oxidative stress, 71

beyond the pleiotropic effects of n-3 PUFAs. This paradigm supports the view of a role for oxidative 72

stress as an important event contributing to POAF development in all cardiac surgeries with 73

extracorporeal circulation. However, oxidative stress could paradoxically also mediate an antioxidant 74

response, according to the time-course activation and levels of ROS. Therefore, it is reasonable to 75

assume that a reinforcement of the antioxidant defense system of cardiac muscle should result in a 76

protective effect of myocardium against this damage. 77

78

The following hypothesis will be tested with the present trial: In patients scheduled for cardiac surgery, 79

supplementation with n-3 polyunsaturated fatty acids (from 7 days prior to surgery until hospital 80

discharge) plus antioxidant vitamins C and E (from 2 days prior to surgery until hospital discharge) will 81

reduce the occurrence of POAF. This effect will be associated with levels of atrial and blood biomarkers 82

accounting for increased antioxidant potential and decreased inflammatory response and oxidative 83

stress. 84

85

Expanded Methods 86

87

Samples 88

Blood samples were drawn during the morning hours at a fasting state on the day of enrollment (day -7), 89

following 5 days of n-3 PUFAs exposure (day -2), 15 minutes before starting extracorporeal circulation 90

5

(Time 0), 6-8 hours after the surgical operation (day +1), and on postoperative day 5 (day +5). The 91

plasma supernatants and red blood cell lysates were stored at -70ºC. Right atrial appendage samples 92

(approximately 400 mg) were obtained immediately before starting extracorporeal circulation, frozen in 93

liquid nitrogen and stored at -70ºC. 94

95

Biochemical analyses 96

Determinations of Cu-Zn superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase 97

(GSH-Px) activities were performed as published elsewhere (70). Plasma antioxidant status was 98

assessed by the reduction of ferric iron (ferric reducing ability of plasma, FRAP), with a detection limit 99

of 10 µmol/L (70). The intracellular redox status was assessed by measuring the thiol index, calculated 100

from the reduced to oxidized glutathione (GSH/GSSG) ratio in atrial tissue and erythrocytes (70). 101

Xanthine oxidase (XO) activity was measured using the Amplex red xanthine/xanthine oxidase assay kit 102

from Invitrogen (Eugene, Oregon), using the manufacturer’s recommended protocol and normalized to 103

protein concentration. Lipid peroxidation was measured through the determination of malondialdehyde 104

(MDA) in plasma and atrial tissue by high performance liquid chromatography (HPLC), with a 105

detection limit of 0.48 µmol/L as previously reported (70). Protein carbonylation in atrial tissue was 106

assayed by reacting 2,4-dinitrophenylhydrazine with protein carbonyls (71). Inflammation was assessed 107

through measurements of plasma high sensitivity C-reactive protein (hs-CRP) (72) using an ELISA kit 108

(Magiwell CRP kit United Biotech, Mountain View) and leukocyte cell count. The plasma biomarkers 109

of oxidative stress and inflammation measured on day +1 were corrected for hemodilution, as assessed 110

by hematocrit fall, in view of the infusion of pump priming and other fluids during and after surgery. 111

Biochemical determinations in atrial tissue could not be performed in all patients due to either technical 112

difficulties in obtaining tissue samples or low appendage sample size. All reagents were purchased from 113

Sigma–Aldrich, Merck or Riedel-de Häen and were of the highest commercial grade available. The 114

capsules of n-3 PUFAs, vitamin C (L-ascorbic acid), vitamin E, and respective placebos were provided 115

by PROCAPS® Laboratories (Colombia) and Gynopharm CRF (Chile) Laboratories. 116

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117

Molecular Determinations 118

Protein expression analysis was assessed by western blot for the NADPH oxidase p47 phox subunit and 119

the nuclear fraction NF-κB p50 subunit. Western blot analysis for the NADPH oxidase p47 phox 120

subunit in the atrial tissue cytosolic fraction was performed with a specific rat IgG monoclonal antibody 121

to human p47 phox protein (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The constitutive control 122

evaluation was performed with a mouse specific monoclonal anti-human β-Actin antibody (Santa Cruz 123

Biotechnology, Santa Cruz, CA, USA). Western blot analysis of the nuclear fraction NF-κB p50 subunit 124

in atrial tissue was performed with specific rat IgG monoclonal antibody to human p50 protein (Santa 125

Cruz Biotechnology, Santa Cruz, CA, USA). The constitutive control was a specific rat IgG monoclonal 126

antibody to human C23 nucleolin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Samples of right 127

atrial appendage was pulverized and homogenized using a Polytron homogenizer in 1.5 ml cold lysis 128

buffer, which contained 40 mM Tris•HCl (pH 7.5), 1mM EDTA, 1 mM DTT, 0.1 mM Na3VO4, 10 129

µg/ml aprotinin, 5 µg/ml pepstatin, 20 µg/ml leupeptin, 1 mM bezamidine. The homogenates were 130

centrifuged at 800g for 10 min at 4°C. The supernatants were removed and centrifuged at 20000g for 131

20 min at 4°C to remove mitochondria, lysosomes and peroxisomes. The new supernatants were 132

removed and centrifuged at 105000g for 60 min at 4°C to separate cytosolic extract (supernatants) from 133

microsomic fraction (pellets). The pellets were resuspended in 1 ml of cold lysis buffer, aliquoted and 134

stored at -80°C until used to perform p47 phox western blot analysis. The original first pellets 135

(centrifuged at 800g for 10 min at 4°C) were resuspended in 1 ml of cold lysis buffer and were 136

centrifuged at 1400g for 5 min at 4°C. The supernatant was removed and the clean nuclear pellet was 137

re-suspended in high-salt buffer (20 mM HEPES, pH 7.9, 400 mM NaCI, 1 mM EDTA, 1 mM EGTA, 138

1 mM DTT, 10 µg/ml aprotinin, 5 µg/ml pepstatin, 20 µg/ml leupeptin, 1 mM bezamidine) and placed 139

on a shaking platform for 20 minutes at 4°C to disrupt nuclear membranes. The nuclear fraction 140

was collected after centrifugation at 13,500g for 5 minutes at 4°C, aliquoted, and frozen immediately. 141

All extracts were stored at -80°C until used to perform p50 analysis western blot analysis. The protein 142

7

content was measured by using the Lowry protein assay (73). After being boiled with denaturation 143

buffer (Tris.HCL pH 6.8, glicerol%, 2.5% β-mercaptoethanol) for 5 minutes at 95°C, samples of fifty 144

micrograms protein per lane from cytoplasmic (p47 phox analysis) or nuclear extracts (p50 analysis) 145

were subjected to electrophoresis on 10% or 15% SDS-polyacrylamide gel at 110 V during 120 min and 146

transferred to nitrocellulose membrane at 75 mA/cm2 during the night in the cooling room. For 147

blocking procedure, the membrane was incubated in 5% fat free milk in a buffer, which contained 20 148

mM Tris•HCl (pH 7.4), 150 mM NaCI, and 0.05% Tris-buffered saline plus Tween-20 (TBS-T) at 20°C 149

during 60 minutes. Membranes were washed three times during 5 min each time using TBS-T and then 150

incubated at 20°C during 120 minutes with a primary antibody against human p47 phox (1:2,000), or 151

human p50 (1:1,000 dilution). Subsequently the membranes were washed 3 times in TBS-T solution and 152

then the blots were incubated with a horseradish peroxidase-conjugated secondary immunoglobulin 153

[anti-rat for p47phox analysis (1:5,000 dilution) and anti-rat for p50 (1:5,000 dilution)] at 20°C for 1 154

hour. After washed 3 times with TBS-T, the proteins were developed with an enhanced 155

chemiluminescence (ECL) western blot detection kit according to the manufacturer’s instruction 156

(Amersham Pharmacia Biotech). In all determinations, the membrane was stained with Ponceau stain to 157

verify the transfer efficiency across the test samples and reblotted with antibody against a constitutive 158

protein [anti-human β-actin for p47phox analysis (1:500, dilution), and anti-human C23 nucleolin for 159

p50 analysis (1:1000 dilution)] after being stripped to verify the uniformity of protein loading. The 160

specificity of protein bands was corroborated by protein standards (Precision Plus Protein Standards, 161

Bio-Rad) in all cases. All bands were quantified by densitometry using IMAGEJ (NIH Image, USA). 162

163

Gene expression analysis was assessed by quantitative RT-PCR for Cu-Zn SOD, CAT, GSH-Px and 164

NADPH oxidase p47 phox subunit. Total RNA was extracted using Trizol reagent (Invitrogen,Carlsbad, 165

CA) according to the manufacturer's instructions.Real-time quantitative reverse transcription–166

polymerase chain reaction (RT-PCR) for NADPH oxidase p47 phox subunit and antioxidant enzymes 167

was performed using the TaqMan system (Prism 7700 Sequence Detection System, PE Biosystems). 168

8

PCRs were performed in a 20 µl volume containing SYBR Green PCR Master Mix (1× concentration), 169

500 nM forward primer, 500 nM reverse primer, and cDNA equivalent to that generated from 20 ng 170

total RNA. A cycle threshold (Ct) was calculated for each sample using the GeneAmp 7700 software. 171

Standard curves were generated for each gene using a reference cDNA containing equal proportions of 172

cDNA isolated from the two groups. Standard curves covered a range of cDNA equivalent to that 173

generated from 1 to 200 ng RNA for all genes. For p47 phox the primers were: 5’-174

ACCCAGCCAGCACTATGTGT-3’ and 3’-AGTAGCCTGTGACGTCGTCT-5’, using the following 175

conditions: initial denaturation at 94 ºC for 5 min, followed by 27 cycles of denaturation at 94 ºC for 30 176

seconds, annealing at 55 ºC for 30 seconds, and primer extension at 72 ºC for 10 minutes and 30 177

seconds. For Cu-Zn SOD the primers were: 5’-GTAATGGACGAGTGAAGGTGTG-3’ and 3´-178

CAATTACACCACAAGCCAACCG-5’, using the following conditions: initial denaturation at 98 ºC 179

for 5 minutes, followed by 40 cycles of denaturation at 63 ºC for 30 seconds, annealing at 55 ºC for 40 180

seconds, and primer extension at 74 ºC for 5 minutes and 30 seconds. For CAT the primers were: 5´-181

TCCGGGATCTTTTTAACGCCATTG-3’ and 3´-TCGAGCACGGTAGGGACAGTTCAC-5’, using 182

the following conditions: initial denaturation at 94 ºC for 10 min, followed by 40 cycles of denaturation 183

at 60 ºC for 25 seconds, annealing at 55 ºC for 20 seconds, and primer extension at 72 ºC for 10 minutes 184

and 30 seconds. For GSH-Px the primers were: 5´-GCGGCGGCCCAGTCGGTGTA-3’ and 3´-185

GAGCTTGGGGTCGGTCATAA-5’, using the following conditions: initial denaturation at 94 ºC for 186

10 min, followed by 40 cycles of denaturation at 63 ºC for 30 seconds, annealing at 50 ºC for 30 187

seconds, and primer extension at 72 ºC for 5 minutes and 30 seconds. GAPDH was used as a 188

housekeeping gene in atrial tissue. For GAPDH the primers were: 5′-GTGAAGGTCGGTGTCAAC-3′; 189

3′-CTCCTTGGAGGCCATGT-5′. The Breakpoint sequences determination was performed by direct 190

sequencing of RT-PCR products with Big Dye terminator method (ABI PRISM big dye terminator 191

cycle sequencing ready reaction kit, Perkin-Elmer). DNA binding analysis was assessed by 192

electromobility shift assay (EMSA) for NF-κB DNA binding as previously reported by our group (74). 193

194

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195

196

Supplementary Results 197

198

Oxidative stress related biomarkers 199

The blood antioxidant potential represented by the plasma antioxidant capacity (FRAP) (extracellular) 200

and the thiol index in erythrocytes (intracellular) are presented in Appendix Figures 1A and 1B 201

respectively. Xanthine oxidase contribution to atrial oxidative stress is showed in Appendix Figures 1C 202

and 1D. Following 5 days of n-3 PUFAs exposure (day -2), plasma FRAP level displayed a 21.1% drop 203

compared to the basal value (p<0.01), being 19.1% lower than the respective placebo value (p<0.01), 204

but the FRAP level increased 2 days after antioxidant vitamin supplementation (Time 0). In turn, in the 205

placebo group, plasma FRAP levels presented no significant differences between day -7 and Time 0. 206

Early after the surgical operation (day +1), the placebo group presented a 37.3% drop in plasma FRAP 207

levels with respect to Time 0 (p<0.01), being 28.4% lower than the respective value in the supplemented 208

patients (p<0.01). At discharge (day +5), the plasma FRAP values of the placebo patients were 12.6% 209

lower than those of the supplemented patients (p<0.01) (Appendix Fig. 1A). In erythrocytes, following 210

5 days of n-3 PUFAs treatment (day -2), the thiol index was 15.3% lower than its basal value (p<0.01) 211

and 18.4% lower than that of placebo group value (p<0.01). Two days after initiating antioxidant 212

vitamin supplementation (Time 0), the thiol index was not significant different between the two groups, 213

but significantly increased values with respect to baseline (p<0.01) were found. Early after the operation 214

(day +1), the placebo group had a 40.5% drop in the erythrocyte thiol index compared to preoperative 215

values (p<0.01), being 33.2% lower than respective values in the supplemented patients (p<0.01). At 216

discharge, no significant differences in the erythrocyte thiol index were evident between the 217

supplemented and the placebo groups, nor were there any significant differences between the group and 218

basal values (Appendix Fig. 1B). Before reperfusion XO did not show no significant difference between 219

10

the groups (Appendix Fig. 1C), but it was 33.7% higher in patients who developed postoperative atrial 220

fibrillation, compared either with supplemented or placebo patients in sinus rhythm (p<0.05) (appendix 221

Fig. 1D). 222

223

Inflammation-related biomarkers 224

Inflammation-related parameters studied in the protocol are shown in Appendix Figure 2. The presence 225

of the NF-B p50subunit in the nuclear fractions of the atrial tissue of placebo, and supplemented 226

patients, as assessed by western blotting analysis, indicated that supplemented patients had 45.3% lower 227

levels of this protein compared with placebo patients (p<0.05) (Appendix Fig. 2A).The atrial tissue 228

nuclear fraction NF-B DNA binding was compared between atrial fibrillation and sinus rhythm. 229

Patients developing atrial fibrillation demonstrated 52.7% higher binding than those in sinus rhythm 230

(p<0.05) (Appendix Fig. 2B1). The suppression of the EMSA NF-B in control bands by 100-fold 231

molar excess of the respective unlabeled DNA probes confirmed the specificity of the binding 232

determinations (Appendix Fig. 2B2). 233

234

235

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236

Appendix Figure 1. Oxidative

Stress-related Biomarkers in Blood

Throughout the Protocol and Atrial

Tissue Xanthine Oxidase Activity

on the Day of Surgery (Time 0). (A)

Plasma antioxidant capacity (n=50 for

each group). (B) Erythrocyte thiol

index (n=50 for each group). (C)

Effect of supplementation on xanthine

oxidase activity (n=12 for each

group). (D) Association between the

occurrence of atrial fibrillation and

xanthine oxidase activity (n=6 for

each group). *p<0.01 versus

placebo. †p<0.01 versus basal.

‡p<0.01 versus preoperative.

§p<0.05

versus atrial fibrillation. XO =

xanthine oxidase. AF+ = atrial

fibrillation. AF- = sinus rhythm. Day-7

= moment of starting the n-3 PUFAs

supplementation or placebo. Day-2 =

moment of starting the antioxidant

vitamins supplementation or placebo.

Time 0 = day of surgery. Day+1 = 6-8

h after surgery. Day+5 =

postoperative 5th day.

12

237

238

239

240

Appendix Figure 2. Western Blotting and EMSA NF-κB Analyses of the Nuclear Fraction

of Atrial Tissue in Supplemented vs Placebo Patients Plus Atrial Fibrillation vs Sinus

Rhythm Patients. (A) Western blotting analysis for the nuclear NF-κB p50 subunit of the

supplemented and placebo patients. Bar graphs show the densitometric quantification of

relative protein levels (n=12 for each group). (B1) Electrophoretic mobility shift assay (EMSA)

for NF-κB in atrial tissue from atrial fibrillation and sinus rhythm patients. Bar graphs show the

densitometric quantification of specific NF-κB-DNA binding (n=6 for each group). (B2)

Specificity of the NF-κB binding is shown in a competition experiment with a control sample

without (C) and with 100-fold molar excess of the unlabeled DNA probe (CP) in EMSA

analysis. *p<0.05 versus placebo. †p<0.05 versus atrial fibrillation. AF+ = atrial fibrillation. AF-

= sinus rhythm.

13

Supplementary Discussion 241

242

This study is based on a paradigm stating that a reinforcement of the antioxidant potential should 243

decrease the vulnerability of cardiac tissue to oxidative challenges. Following cardiomyocyte exposure 244

to a moderate pro-oxidant status, a response on up-regulation for antioxidant enzymes expression should 245

be enhanced via Nrf2 transcription factor (30). The pro-oxidant status induced by n-3 PUFAs was 246

confirmed at day -2 by the elevation of plasma MDA levels, in the supplemented group (Fig. 3A), as 247

well as by the reduction of FRAP (Online Appendix Fig. 1A) and the erythrocyte thiol index (Online 248

Appendix Fig. 1B) in the supplemented group. Of note, there was a positive correlation between lipid 249

peroxidation in plasma and atrial tissue (Fig. 3C), while the lipid peroxidation (Fig. 3A) and 250

inflammation biomarkers (Figs. 3D-3E) increased significantly early after surgery in both groups but 251

less markedly in the supplemented group. 252

253

As the incidence of POAF was significantly lower in the supplemented group, our data provide further 254

support to the involvement of both oxidative stress and inflammation in the pathogenesis of this 255

arrhythmia, as previously reported by other authors (46). Our findings show that the supplementation 256

enhanced the expression and activity of the antioxidant enzymes CAT, SOD, and GSH-Px in atrial 257

tissue (Figs. 4A-4C). These results are being reported in humans for the first time, but are in agreement 258

with the previous studies in animal and cell cultured models (54,55), and could be attributed to a 259

genomic antioxidant response triggered by n-3 PUFAs exposure. 260

261

Concerning the sources of ROS, NADPH oxidase has been reported as the main source of atrial 262

superoxide production in myocardial tissue (75). In this context, previous clinical trials demonstrated 263

that patients undergoing postoperative atrial fibrillation have higher preoperative NADPH oxidase 264

activity compared with patients in sinus rhythm (31). These authors report an independent association 265

between this enzyme activity and the risk of postoperative atrial fibrillation, thus suggesting that this 266

14

oxidase system may be a key mediator for the development of atrial fibrillation after cardiac surgery. In 267

addition, it has been reported a higher XO activity in a porcine AF model, respect to a group in sinus 268

rhythm (76,77). These findings are in agreement with data here reported (Fig. 4D-4E and Online 269

Appendix Fig. 1D). 270

271

Cardioprotection by vitamins C plus E 272

Vitamin C, a potent water-soluble antioxidant, may exert cardioprotective effects through 273

multiple mechanisms (78). In specific, it prevents the oxidation of tetrahydrobiopterin, a 274

coupling cofactor of nitric oxide synthase that is highly sensitive to oxidation, representing a 275

potential source of superoxide anion. In addition to being a free-radical scavenger, it is 276

implicated in the reduction of oxidized vitamin E and oxidized glutathione. Furthermore, 277

ascorbic acid depletion may lead to the oxidation of myoglobin and to the formation of the 278

toxic ferryl moiety able to induce lipid peroxidation (79). In turn, vitamin E is a major peroxyl 279

radical scavenger in biological lipid phases, such as membranes (80). The antioxidant effect of 280

vitamin E, mainly α-tocopherol, has been ascribed to its ability to act chemically as a lipid-281

based free radical chain-breaking molecule through its conversion into α-tocopheroxyl radical. 282

It is of interest to mention that α-tocopherol can be restores with redox-active reagents such as 283

vitamin C (81), thus giving rise to a synergistic effect of both antioxidant vitamins. However, 284

despite the scientific rationale, epidemiological and retrospective studies on cardioprotection by 285

antioxidants have been persuasive, clinical trials designed for long-term administration have 286

failed to demonstrate beneficial effects (30). Although, oral doses of vitamin C fail to protect 287

the heart against peroxynitrite, beneficial effects other than superoxide scavenging, such as 288

improvement of microcirculatory reperfusion has been reported in patients undergoing elective 289

percutaneous coronary intervention (82). 290

15

291

Cardioprotection by n-3 PUFAs 292

The administration of n-3 PUFAs supplementation, performance in three of the four similar randomized 293

placebo-controlled trials (RPCT) failing to demonstrate POAF reduction, used the same pharmaceutical 294

supplement (Omacor, Pronova). This formulation contains ~465 mg EPA and ~375 mg DHA per 295

capsule (66,68,69) with an EPA:DHA ratio equal to 1.24. The fourth trial reported the utilization of a 296

supplement with 1240 mg EPA and 1000 mg DHA (Omega Forte, Lysi) with the same 1.24 ratio (67). 297

In contrast, successful trials in preventing POAF including the present study and two previous RPCT 298

(63,65) used pharmaceutical supplements containing ~294 mg EPA and ~588 mg DHA per capsule 299

with an EPA:DHA ratio equal to 0.5. In support of this data, a recent meta-regression analysis showed a 300

trend toward a benefit from n-3 PUFAs supplementation when the EPA:DHA ratio was 0.5 (83). 301

302

Human atrial tissue exposed to EPA:DHA shows a more rapid incorporation of DHA, reaching the 303

DHA half-maximal uptake during the first week of fish oil supplementation (84). These authors reported 304

rapid DHA incorporation kinetics into the human heart atrial tissue during the period of time examined 305

in the present trial, likely accounting for a relevant functional cardiac effect in our patients. Moreover, 306

as this process depends on the EPA:DHA ratio, we could expect even further DHA incorporation into 307

the membrane phospholipids due to the use of a 1:2 EPA:DHA ratio in the present study. Indeed, in rats 308

supplemented with fish oil having a 1:4 EPA:DHA ratio, it takes only 2 days for DHA to reach its half-309

maximal incorporation into myocardial tissue (85). To our knowledge, no previous trials have been 310

performed to study the correlation between the anti-arrhythmic and metabolic effects of EPA or DHA in 311

humans. Nevertheless, from studies in animal models, the antiarrhythmic effect of dietary fish oil has 312

been partly attributed to the selective DHA accumulation in myocardial cell membranes (86). In this 313

context, Ramadeen et al. very recently demonstrated that DHA, but not EPA, attenuated atrial 314

fibrillation vulnerability and reduced atrial structural remodeling and fibrosis in a dog experimental 315

model (62). 316

16

In summary, this study presents the highest reduction in postoperative atrial fibrillation compared with 317

the two other related trials (63,65) with the same EPA:DHA ratio used in the present study. These 318

optimal results can be explained on the basis of the genomic antioxidant response induced by n-3 319

PUFAs together with the additional non-enzymatic antioxidant reinforcement provided by the 320

antioxidant vitamins C and E. 321

322

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