This document summarizes research on the role of oxidative stress and the enzyme NADPH oxidase 2 (NOX2) in atrial fibrillation (AF). The key points are: 1. Atrial NOX2 activity is increased in patients with AF and correlates with the extent of electrical remodeling caused by AF. Increased NOX2 activity also precedes the onset of AF. 2. Experiments in animal models show that increased atrial NOX2 activity, through genetic overexpression of NOX2, is sufficient to create a substrate for AF induction. 3. Pharmacological inhibition of NOX2, such as with statins, prevents AF in animal models and reduces the risk of postoperative AF

1. Oxidative stress and atrial fibrillation:
a long journey into the clinic?
Barbara Casadei MD DPhil FRCP
British Heart Foundation Professor of Cardiovascular Medicine
& Hon Consultant Cardiologist
Department of Cardiovascular Medicine
BHF Centre of Research Excellence
University of Oxford
VHIR Seminar
30 April 2013

2. AF: epidemiology & treatment
• Most common sustained clinical arrhythmia (1:4 lifetime risk)

3. AF: epidemiology & treatment
• Most common sustained clinical arrhythmia (1:4 lifetime risk)
• AF is associated with a significantly increased mortality and
high medical costs (ca. 3% of the UK NHS budget)

4. AF: epidemiology & treatment
• Most common sustained clinical arrhythmia (1:4 lifetime risk)
• AF is associated with a significantly increased morbidity and
with high medical costs (ca. 3% of the NHS budget)
• Available treatment is suboptimal (targeted to symptoms and
prevention of thromboembolism)

5. Mortality after incident AF in 1993-
2007
Piccini et al. Circulation: Cardiovascular Quality and Outcomes. 2012
0
5
10
15
20
25
30
35
1993 1997 2003 2007
Mortality trends for coronary
heart disease in the USA
Prevalence of AF 1993-2007

6. ‘AF begets AF’
Wijffels et al, 1995
Short APD & loss of rate
adaptation promote re-entry
200 ms
SR
AF

7. Targeting ion channels
(conduction and excitability)
Therapeutic
strategies aimed at
molecular targets
that are upstream
or independent of
ion channels

8. Oxidative stress and atrial remodelling in AF
ATRIAL
FIBRILLATION
RAPID ATRIAL
ACTIVATION
ELECTRICAL
REMODELLINGAF begets AF
+
OXIDATIVE
STRESS

9. Atrial oxidative stress and AF
• There is evidence of oxidative injury in atrial samples
from patients with AF (Mihm et al. Circ 2001)
• There is a correlation between oxidative stress and
atrial ERP shortening in animal models (Carnes et al. Circ
Res 2004)
• Treatment with anti-oxidant/anti-inflammatory agents
prevents atrial electrical remodelling and AF induction
in a dog model of atrial tachypacing (Carnes et al. Circ Res
2001, Shiroshita-Takeshita et al. Circ Res 2004)

10. .NAD(P)H
p22phox
rac
p67phox
p47phox
O2
NAD(P)+
H+
NOX2
.O2
-
NC p67phox
p47phox
Test
Phase
Stain
Contrast
Immunolocalization in human atrial myocytes
atrial myocytesRAA
 47kD
 67kDAnti-p67phox
Anti-p47phox
Anti-p22phox
+
 22kD
Immunoblotting
A NOX2 oxidase in human atrial cells
Kim et al, Circ Res 2005

11. Atrial NOX2 activity is increased
in patients with (mostly) PAF
*
0
2
4
6
SR PAF
NOX2activity
(RLU/sec/mgprotein)
Kim et al, Circ Res 2005

12. NOX2 activity is increased in the LA of goats after 2
weeks of AFC
ontrol
Apo
R
otL-N
AM
E
O
xy
C
ontrol
Apo
R
otL-N
AM
E
O
xy
0.0
0.1
0.2
0.3
0.4
Right Atrium Left Atrium
*
O2
-
(RLU/s/mgprotein)
Reilly et al. Circulation 2011
RA LA RA LA
NOX2
GAPDH
SR AF

13. Elevated atrial NOX2 activity is associated with shortening of the
atrial effective refractory period
Reilly et al. unpublished data
Atrial effective refractory period (ms, measured at CL=300 ms)
AtrialNOX2activity(RLU/mgprotein)

14. • NOX2 oxidases are present in the human
atrial myocardium
• Atrial NOX2 activity is increased in AF and
correlated with the extent of the AF-induced
atrial electrical remodelling
• Does an increase in atrial NOX2 activity
precede AF?


?
Atrial NOX2 activity and AF: cause or effect?

15. •Atrial fibrillation is a frequent complication of most types of
cardiac surgery, occurring in 35-50% of patients within the first
2-5 postoperative days
•The inflammatory reaction associated with cardiac surgery
and cardiopulmonary bypass has been implicated in the
genesis of this arrhythmia
•NOX2 activity is stimulated by cytokines
Post-operative atrial fibrillation

16. Hypotheses:
• Atrial NOX2 oxidases may “sense” systemic
inflammation and translate it into a local
increase in oxidative stress leading to
arrhythmogenesis
•Atrial NOX2 activity may predict the occurrence
of postoperative AF

17. Study protocol
• 170 patients undergoing first-time on pump CABG
• Exclusion criteria: history of AF, valvular disease, use of
anti-arrhythmic drugs other than beta-blockers
• Atrial NADPH-stimulated superoxide production was
measured by lucigenin-enhanced chemiluminescence in a
sample of the RAA obtained prior to the initiation of CPB.
• Plasma markers of protein & lipid oxidation (TBARS, protein
carbonyls, isoprostanes) were measured in blood samples
obtained after the induction of anaesthesia and 10 minutes
after the administration of protamine.
Kim et al JACC 2008

18. Plasma markers of systemic oxidative stress and
post-operative AF
Post-Op
* *
0
0.2
0.4
0.6
0.8
1
TBARS Protein Carbonyls
Pre-Op
Pre-Op
SR
AF
Plasmamarkersof
oxidativestress
(nmol/ml)
SR
AF
SR AF
SR AF
Post-Op
* *

19. Atrial NOX2 activity is independently associated with a higher
incidence of post-operative AF (n=170)
OR 95% CI P
Age 1.01 0.95-1.08 0.69
Atrial NOX2
activity
(RLU/sec/mg protein)
2.61 1.61-4.23 0.00003
TBARS (pre) 0.12 0.01-2.29 0.16
TBARS (post) 4.90 0.48-48.96 0.18
Carbonyls (pre) 0.55 0.02-13.08 0.71
Carbonyls (post) 2.12 0.20-22.11 0.53
Diabetes 0.89 0.31-2.60 0.83
Beta-blockers 2.63 0.76-9.13 0.13
ACEI/ARB 1.56 0.59-4.10 0.37
Kim et al JACC 2008

20. Atrial NOX2 activity is an independent predictor of new-onset AF
after cardiac surgery (n=281)
Cox-regression (HR[95%CI], P):
Lowest tertile: Ref
Mid tertile: 3.15[1.06-9.4], P=0.039
Highest tertile: 6.43[2.10-19.69], P=0.001
Post-operative days
1
0.8
0.6
0.4
0.2
0
0 4 8 121062 14 16
AF-freesurvival
Antoniades et al. JACC 2011
Tertiles of atrial NOX2 activity
Highest
Mid
Lowest

21. Atrial NOX2 activity is a predictor of in-hospital outcome after
cardiac surgery
Highest
Mid
Lowest
AtrialNOX2activity
(RLU/sec/mgprotein)
12x104
8x104
4x104
2x104
0
6x104
10x104
P=0.0001
0 1 ≥2
Inotropic support (days)
Post-operative days
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25 30
Hospitaldischarge
Cox-regression (HR[95%CI], P):
Lowest: Ref
Mid: 0.78[0.53-1.17], P=0.234
Highest: 0.61[0.39-0.93], P=0.02
Antoniades et al. JACC 2011
Tertiles of atrial NOX2
activity

22. • NOX2 oxidases are present in the human atrial
myocardium
• Atrial NOX2 activity is increased in AF and
correlated with the extent of the AF-induced atrial
electrical remodelling
• An increase in atrial NOX2 activity precedes AF
• Is increased atrial NOX2 activity sufficient to
create a substrate for AF?

.
.
Atrial NOX2 activity and AF: cause or effect?

23. LA
RA
oesophagus
electrodes
trachea
Octapolar
transeosophageal
catheter
2% isoflurane
ECG electrode 1
ECG electrode 2
ECG electrode 3
ECG electrode 4
Atrial burst stimulation in the mouse

24. WT nNOS-KO
0
1
2
3
4
5
Numberof
AFepisodes
WT mNOX2 Tg
WT nNOS-KO
0
5
10
15
20
Probabilityof
AFinduction(%)
Channel4-ECG
Volts
3
2
1
0.2 seconds
Channel4-ECG
Volts
3
2
1
0.2 seconds
AF
Normal sinus rhythm
n=10-13
Myocardial-specific overexpression of NOX2 increases AF
inducibility in vivo in mice
WT mNOX2 Tg
Recalde et al, unpublished data

25. P Niethammer et al. Nature 2009

26. Atrial NOX2 activity and AF: cause or effect?
• NOX2 oxidases are present in the human atrial
myocardium
• Atrial NOX2 activity is increased in AF and
correlated with the extent of the AF-induced atrial
electrical remodelling
• An increase in atrial NOX2 activity precedes AF
• Increased atrial NOX2 activity is sufficient to create a
substrate for AF
• Do pharmacological interventions that inhibit
NOX2 activity prevent AF?

.
.
.

27. p22phox
O2
NOX2
NOX2 NADPH oxidases:
activated by cytokines and AngII
NADPH NADP + H+
O2
-
rac
p67phox
p47phox

28. p67phox
p47phox
p22phox
O2
NOX2
rac
STATINS
Statin-mediated inhibition of NOX2

29. Postop AF Postop SR
p47phox
GTP-Rac1
- +
Total Rac1
p47phox
GAPDH
GTP-Rac1
Total Rac1
p67phox
Atorvastatin
Mevalonate
Atorvastatin
Mevalonate - +
++
-
-
- +
++
-
-
0
1
2
3
Atorvastatin
Mevalonate -
-
-
+
+
+
-
-
-
+
+
+
* * *#
GTP-Rac1/totalRac1
Postop AF Postop SR
p67phox
p47phox
p22phox
O2
NOX2
rac
STATINS
Atrial Rac1 activity is increased in patients who develop post-
operative AF and is inhibited by atorvastatin

30. WT NOX2 Tg WT NOX2 Tg
0
10
20
30
Control ATV
AFprobability
*
#
#
WT mNOX2Tg WT mNOX2Tg
0
5
10
15
20
AFepisodes/animal
Control ATV
* Channel4-ECG
Volts
3
2
1
0.2 seconds
Channel4-ECG
Volts
3
2
1
0.2 seconds
Atorvastatin treatment inhibits AF inducibility in mNOX2 Tg mice
Recalde et al, unpublished data

31. Effect of peri-operative statin treatment on
post-operative AF
Chen et al. J Thorac Cardiovasc Surg. 2010

32. •A double-blind, randomised, placebo-controlled
trial of perioperative Rosuvastatin (20 mg od)
•Treatment is started 3-7 days before surgery and
continued until the 5th post-operative day
•1800 patients undergoing cardiac surgery
•1350 patients recruited so far.
Prevention of myocardial damage and post-operative
atrial fibrillation in patients undergoing cardiac surgery.

33. Effect 3-day preoperative treatment with atorvastatin (20 mg od vs.
placebo) on atrial and vascular redox state in 42 CABG patients
IMANOX2Activity
Antoniades et al. JACC 2011
Antoniades et al. Circulation 2011
AtrialNOX2Activity
2x104
4x104
6x104
8x104
10x104
(RLU/sec/mgprotein)
3d-Placebo 3d-Atorva
0
**

34. Primary Objectives
To establish whether perioperative administration of Rosuvastatin
leads to a reduction in:
• the incidence of post-operative AF (as assessed by
continuous ECG monitoring)
• perioperative myocardial injury (as assessed by serial
Troponin measurements).

35. Secondary Objectives
• In-hospital clinical outcomes:
– Hospital and intensive care unit stay
– Major in-hospital cardiac or cerebrovascular events
• Biomarkers:
– Myocardial oxidase activity and serum CD40L, NT-
proBNP, lipids, and renal function.
• LV function by echocardiography

36. First 850 patients (mean age 59±10 yrs, LVEF 60±8%)
0 10 20 30 40 50 60 70 80 90 100
Previous MI
Previous stroke/TIA
Diabetes
Heart Failure
Beta-blockers
Antiplatelets/Anticoagulants
ACEI/ARB
Nitrates
STATINS
On pump surgery
Rosuvasatin
Placebo
%

37. What will we learn?
• Is aggressive statin treatment in the
perioperative period beneficial?
• Are statins cardioprotective and anti-
arrhythmic in these patients?
• Are the pleiotropic effects of statins
(e.g., NOX2 inhibition) clinically
relevant?

38. Svetlana Reilly
Xing Liu
Alice Recalde
Ricardo Carnicer
Raja Jayaram
Keith Channon
Charis Antoniades
(Cardiovascular
Medicine)
Rana Sayeed
Mario Petrou
Ravi DeSilva
(Cardiothoracic Unit)
Blanca Rodriguez
Alfonso Bueno
(Computer Science)
Rory Collins
Zhengming Chen
Jonathan Emberson
Zhe Zheng
Lixin Jiang
(CTSU & Fuwai Hospital)
Manuela Zaccolo
(DPAG, Oxford)
Uli Schotten
Sander Verheule
(Maastricht University)
Ajay Shah
Phil Eaton
(King’s College)
Emilio Hirsch
(University of Turin)

40. Stroke, Cholesterol and Statins

41. To assess the effects of Atorvastatin 80mg od on the atrial effective
refractory period (over 5 post-operative days) and myocardial
ischemia/reperfusion injury in paired right atria samples in patients
undergoing cardiac surgery
60 of a planned 80 patients have been randomised (mean age 65
yrs, range from 41 to 80 yrs, 79% males) to atorvastatin 80 mg od
vs. placebo. 40% developed post-op AF.
Statin Treatment on Atrial
Refractoriness & Reperfusion injury

42. Effects of rosuvastatin on atrial fibrillation occurrence: ancillary
results of the GISSI-HF trial
Maggioni et al. Eur Heart J 2009

43. Does atrial ROS production or its
enzymatic sources vary with the duration
and substrate of AF?
…and might this affect the efficacy of
statins in the prevention of AF?

44. Atrial sources of oxidative stress change with the duration of AF and the
presence of atrial structural remodelling
C
ontrol
Apo
R
otL-N
AM
E
O
xy
C
ontrol
Apo
R
otL-N
AM
E
O
xy
0.0
0.1
0.2
0.3
0.4
Right Atrium Left Atrium
*
*
O2
-
(RLU/s/mgprotein)
6M AF (n=10)
C
ontrol
Apo
R
otL-N
AM
E
O
xy
C
ontrol
Apo
R
otL-N
AM
E
O
xy
0.0
0.1
0.2
0.3
0.4
0.5
Right Atrium Left Atrium
**
O2
-
(RLU/s/mgprotein)
AV block (n=9)
Control
Apo
RotL-Nam
e
Oxy
Control
Apo
RotL-Nam
e
Oxy
0.00
0.05
0.10
0.15
0.20
0.25
Sinus Rhythm Chronic Atrial Fibrillation
(Right Atrium)
*
*
O2
-
(RLU/s/mgprotein)
Chronic AF (n=26) vs. SR in humans (n=72)
p22phox
GAPDH
GAPDH
CAFP-op AFSR
NOX2
Reilly et al. Circulation 2011

45. L-Arginine + O2
Citrulline
+ NO
Increased atrial superoxide release in chronic AF is associated with an
increase in Mito complexes and uncoupled NOS activity
- +
NOX5
GAPDH
- +
NOX4
GAPDH
- +SR CAF
NOX2
GAPDH
GAPDH
VDAC
Mitochondrial
complexes
V
IV
III
II
I
SR CAF
GAPDH
VDAC
V
IV
III
II
I
O2
.O-
2
C
ontrol
Apo
R
otL-N
AM
E
O
xy
C
ontrol
Apo
R
otL-N
AM
E
O
xy
0.0
0.1
0.2
0.3
0.4
Right Atrium Left Atrium
*
*
O2
-
(RLU/s/mgprotein)

46. Summary
•The main source of superoxide production in the human
atrial myocardium is a membrane-bound NOX2 oxidase
•Both atrial rac1 and NOX2 activity are increased in
animal models of short-term AF and in patients who
develop AF after cardiac surgery
•Atrial NOX2 activity is an independent predictor of post-
operative AF
•An increase in myocardial NOX2 activity is sufficient to
create a substrate for AF induction
•Atrial NOX2 is a promising pharmacological target for AF
prevention

47. NOS uncoupling in the RA myocardium is due to an ipsilateral reduction in
BH4 and increase in arginase activity
RA LA RA LA RA LA RA LA
0.0
0.5
1.0
1.5
2.0
2.5
*
SR AVB6M-AF2W-AF
†
BH4(pmol/mg)
0
10
20
30
40
50
0 100 200 300 400 5004 4.25 4.5 5 5.25
Minutes
mV
*4.3
SR
6M-AF
00
10
20
30
40
50
0 100 200 300 400 5004 4.25 4.5 5 5.25
Minutes
mV
*4.3
SR
6M-AF
0
10
20
30
40
50
0 100 200 300 400 5004 4.25 4.5 5 5.25
Minutes
mV
*4.3
SR
6M-AF
0
10
20
30
40
50
0 100 200 300 400 5004 4.25 4.5 5 5.25
Minutes
mV
*4.3
SR
6M-AF
0
BH4
RA LA RA LA RA LA RA LA
0
5
10
15
20
25
*
SR AVB6M-AF2W-AF
†
#
L-ornithine(%)
10
20
30
40
50
60
SR
6M
mV
10
20
30
40
50
60
SR
6M
10
20
30
40
50
60
SR
6M
mV
10
20
30
40
50
60
SR
6M
mV
10
20
30
40
50
60
SR
6M
10
20
30
40
50
60
SR
6M
mV
L-ornithine

48. Prevention of coronary events by statins is due to
sustained LDL cholesterol lowering

49. Secondary Objectives
• In-hospital clinical outcomes:
– Hospital and intensive care unit stay
– Major in-hospital cardiac or cerebrovascular events
• LV function by echocardiography
• Biomarkers:
– Myocardial oxidase activity, markers of inflammation, NT-
proBNP, lipids, and renal function.

50. A higher preoperative serum level of the proinflammatory and
prothrombotic CD40-ligand (sCD40L) is associated with a greater
risk of postoperative AF
AFSR
sCD40L(ng/ml)
0
1
2
3
4
5
6
7 P<0.05
In hospital
AFSR
At 6 weeks
P<0.05
0
1
2
3
4
5
6
7
sCD40L(ng/ml)
N=147 patients
RR for highest vs lowest sCD40L tertile = 3.81, 95% CI: 1.12-12.95, P<0.05
Antoniades et al. Circulation 2008

51. Gudbjartsson et al. Nat Genet. 2009