oxidative stress and atrial fibrillation: a long journey in to the clinic? (prof. barbara casaidei)
DESCRIPTION
Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with significant morbidity and increased mortality. To date, the mechanisms responsible for the new onset of AF are only partially understood and even less is known of the processes that underlie the progression from paroxysmal to persistent AF and influence the response to treatment. In the absence of therapeutic approaches targeting the signalling pathways involved in the substrate that supports AF, current management is mainly focussed on relieving symptoms and preventing embolic stroke. There is therefore a pressing need to deepen our understanding of the pathogenesis of AF and identify mechanisms that could be targeted by novel therapeutic interventions. Our work has shown that atrial NOX2 activity is an independent predictor of post-operative AF in patients undergoing cardiac surgery and that short-term statin therapy or ex-vivo incubation inhibits myocardial NOX2 activityin humans and suppresses AF induction in a mouse model of myocardial specific NOX2 overexpression. The impact of atrial NOX2 inhibition by statins on post-operative AF and perioperative irreversible myocardial damage is now being tested in a large randomised clinical trial (STatinsIn Cardiac Surgery (STICS),TRANSCRIPT
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 MedicineBHF Centre of Research ExcellenceUniversity of Oxford
VHIR Seminar30 April 2013
AF: epidemiology & treatment
• Most common sustained clinical arrhythmia (1:4 lifetime risk)
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)
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)
Mortality after incident AF in 1993-2007Piccini et al. Circulation: Cardiovascular Quality and Outcomes. 2012
1993 1997 2003 20070
5
10
15
20
25
30
35
Mortality trends for coronary heart disease in the USA
Prevalence of AF 1993-2007
‘AF begets AF’Wijffels et al, 1995
Short APD & loss of rate adaptation promote re-entry
200 ms
SRAF
Targeting ion channels (conduction and excitability)
Therapeutic strategies aimed at molecular
targets that are upstream or
independent of ion channels
Oxidative stress and atrial remodelling in AF
ATRIAL FIBRILLATION
ATRIAL FIBRILLATION
RAPID ATRIALACTIVATION
RAPID ATRIALACTIVATION
ELECTRICALREMODELLING
ELECTRICALREMODELLINGAF begets AF
+
OXIDATIVE
STRESS
OXIDATIVE
STRESS
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)
.NAD(P)H
p22phox
racp67phox
p47phox
O2
NAD(P)+
H+
NOX2
.O2-
NC p67phoxp47phox
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 cellsKim et al, Circ Res 2005
Atrial NOX2 activity is increased in patients with (mostly) PAF
*
0
2
4
6
SR PAF
NO
X2
acti
vity
(R
LU
/sec
/mg
pro
tein
)
Kim et al, Circ Res 2005
NOX2 activity is increased in the LA of goats after 2 weeks of AF
0.0
0.1
0.2
0.3
0.4
Right Atrium Left Atrium
*
O2
- (RLU
/s/
g pr
otei
n)
Reilly et al. Circulation 2011
RA LA RA LA
NOX2
GAPDH
SR AF
R² = 0.25
0
0.2
0.4
0.6
0.8
1
0 50 100 150 200 250
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)
Atria
l NO
X2 a
ctivi
ty (R
LU/m
g pr
otei
n)
• 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?
• 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
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
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
Plasma markers of systemic oxidative stress and post-operative AF
Post-Op
* *
TBARS Protein Carbonyls0
0.2
0.4
0.6
0.8
1
Pre-Op
Pre-Op
SRAF
Pla
sma
mar
kers
of
oxi
dat
ive
stre
ss
(nm
ol/
ml)
SRAF
SR AF
SR AF
Post-Op
* *
Atrial NOX2 activity is independently associated with a higher incidence of post-operative AF (n=170)
OR 95% CI PAge 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 0.16TBARS (post) 4.90 0.48-48.96 0.18Carbonyls (pre) 0.55 0.02-13.08 0.71Carbonyls (post) 2.12 0.20-22.11 0.53Diabetes 0.89 0.31-2.60 0.83Beta-blockers 2.63 0.76-9.13 0.13ACEI/ARB 1.56 0.59-4.10 0.37
Kim et al JACC 2008
Atrial NOX2 activity is an independent predictor of new-onset AF after cardiac surgery (n=281)
Cox-regression (HR[95%CI], P):Lowest tertile: RefMid tertile: 3.15[1.06-9.4], P=0.039Highest tertile: 6.43[2.10-19.69], P=0.001
Post-operative days
1
0.8
0.6
0.4
0.2
00 4 8 121062 14 16
AF
-fre
e s
urv
iva
l
Antoniades et al. JACC 2011
Tertiles of atrial NOX2 activity HighestMidLowest
Atrial NOX2 activity is a predictor of in-hospital outcome after cardiac surgery
HighestMidLowest
Atr
ial
NO
X2
acti
vity
(RLU
/sec
/mg
prot
ein)
12x104
8x104
4x104
2x104
0
6x104
10x104
P=0.0001
0 1 ≥2Inotropic support (days)
Post-operative days
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20 25 30
Ho
spit
al d
isch
arg
e
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
• 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?
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
WT nNOS-KO0
1
2
3
4
5
Num
ber
of
AF
epi
sode
s
WT mNOX2 Tg
WT nNOS-KO0
5
10
15
20
Pro
babili
ty o
fA
F in
duc
tion
(%)
0.2 secondsCha
nnel
4-E
CG
Volts
3
2
10.2 secondsC
hann
el 4
-EC
GVo
lts
3
2
1
Cha
nnel
4-E
CG
Vol
ts
3
2
10.2 secondsC
hann
el 4
-EC
GV
olts
3
2
10.2 seconds
AF
Normal sinus rhythm
n=10-13
Myocardial-specific overexpression of NOX2 increases AF inducibility in vivo in mice
WT mNOX2 TgRecalde et al, unpublished data
P Niethammer et al. Nature 2009
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?
.
.
.
p22phox
O2
NOX2
NOX2 NADPH oxidases: activated by cytokines and AngII
NADPH NADP + H+
O2-
rac
p67phox
p47phox
p67phoxp47phox
p22phox
O2
NOX2
rac
STATINS
Statin-mediated inhibition of NOX2
Postop AF Postop SR
p47phox
GTP-Rac1
- +
Total Rac1
p47phox
GAPDH
GTP-Rac1
Total Rac1
p67phox
AtorvastatinMevalonateAtorvastatinMevalonate - +
++--
- +++
--
0
1
2
3
AtorvastatinMevalonate -
--
++
+--
-++
+
* * *#G
TP
-Rac
1/to
tal R
ac1
Postop AF Postop SR
p67phoxp47phox
p22phox
O2
NOX2
rac
STATINS
Atrial Rac1 activity is increased in patients who develop post-operative AF and is inhibited by atorvastatin
0.2 secondsCha
nnel
4-E
CG
Volts
3
2
10.2 secondsC
hann
el 4
-EC
GVo
lts
3
2
1
Cha
nnel
4-E
CG
Vol
ts
3
2
10.2 secondsC
hann
el 4
-EC
GV
olts
3
2
10.2 seconds
Atorvastatin treatment inhibits AF inducibility in mNOX2 Tg mice
Recalde et al, unpublished data
Effect of peri-operative statin treatment on post-operative AF
Chen et al. J Thorac Cardiovasc Surg. 2010
• 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.
Effect 3-day preoperative treatment with atorvastatin (20 mg od vs. placebo) on atrial and vascular redox state in 42 CABG patients
IMA
NO
X2 A
ctiv
ity
Antoniades et al. JACC 2011Antoniades et al. Circulation 2011
Atr
ial N
OX2
Act
ivity
2x104
4x104
6x104
8x104
10x104
(RLU
/sec
/mg
prot
ein)
3d-Placebo 3d-Atorva0
**
Primary ObjectivesTo 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).
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
First 850 patients (mean age 59±10 yrs, LVEF 60±8%)
Previous MI
Previous stroke/TIA
Diabetes
Heart Failure
Beta-blockers
Antiplatelets/Anticoagulants
ACEI/ARB
Nitrates
STATINS
On pump surgery
0 10 20 30 40 50 60 70 80 90 100
RosuvasatinPlacebo
%
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?
Svetlana ReillyXing LiuAlice RecaldeRicardo CarnicerRaja Jayaram
Keith ChannonCharis Antoniades(Cardiovascular Medicine)
Rana SayeedMario PetrouRavi DeSilva (Cardiothoracic Unit)
Blanca RodriguezAlfonso Bueno(Computer Science)
Rory CollinsZhengming ChenJonathan Emberson
Zhe ZhengLixin Jiang(CTSU & Fuwai Hospital)
Manuela Zaccolo(DPAG, Oxford)
Uli SchottenSander Verheule(Maastricht University)
Ajay ShahPhil Eaton(King’s College)
Emilio Hirsch(University of Turin)
Stroke, Cholesterol and Statins
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.
STARR Statin Treatment on Atrial Refractoriness & Reperfusion injury
Effects of rosuvastatin on atrial fibrillation occurrence: ancillary results of the GISSI-HF trial
Maggioni et al. Eur Heart J 2009
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?
Atrial sources of oxidative stress change with the duration of AF and the presence of atrial structural remodelling
0.0
0.1
0.2
0.3
0.4
Right Atrium Left Atrium
**
O2
- (RLU
/s/
g p
rote
in)
6M AF (n=10)
0.0
0.1
0.2
0.3
0.4
0.5
Right Atrium Left Atrium
**
O2
- (RLU
/s/
g p
rote
in)
AV block (n=9)
0.00
0.05
0.10
0.15
0.20
0.25
Sinus Rhythm Chronic Atrial Fibrillation(Right Atrium)
**
O2- (R
LU/s
/ g
pro
tein
)
Chronic AF (n=26) vs. SR in humans (n=72)
p22phox
GAPDH
GAPDH
CAFP-op AFSR
NOX2
Reilly et al. Circulation 2011
L-Arginine + O2
Citrulline + NO
Increased atrial superoxide release in chronic AF is associated with an increase in Mito complexes and uncoupled NOS activity
- +
NOX5GAPDH
- +
NOX4GAPDH
- +SR CAFNOX2
GAPDH
GAPDH
VDAC
Mito
chon
dria
l co
mpl
exes
V
IV
III
II
I
SR CAF
GAPDH
VDAC
V
IV
III
II
I
O2
.O-2
0.0
0.1
0.2
0.3
0.4
Right Atrium Left Atrium
**
O2
- (RLU
/s/
g p
rote
in)
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
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
†
BH
4 (p
mo
l/mg
)
0
10
20
30
40
50
0 100 200 300 400 500 600 7004 4.25 4.5 5 5.25 5.5Minutes
mV
*4.3
SR
6M-AF
00
10
20
30
40
50
0 100 200 300 400 500 600 7004 4.25 4.5 5 5.25 5.5Minutes
mV
*4.3
SR
6M-AF
0
10
20
30
40
50
0 100 200 300 400 500 600 7004 4.25 4.5 5 5.25 5.5Minutes
mV
*4.3
SR
6M-AF
0
10
20
30
40
50
0 100 200 300 400 500 600 7004 4.25 4.5 5 5.25 5.5Minutes
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-or
nith
ine
(%)
-10
0
10
20
30
40
50
60
0 30 60 90 12017 1816
SR
6M-AF
16.25 16.5 17.25Minutes
mV
-10
0
10
20
30
40
50
60
0 30 60 90 12017 1816
SR
6M-AF
16.25 16.5 17.25-10
0
10
20
30
40
50
60
0 30 60 90 12017 1816
SR
6M-AF
16.25 16.5 17.25Minutes
mV
0
-10
0
10
20
30
40
50
60
0 30 60 90 12017 1816
SR
6M-AF
16.25 16.5 17.25Minutes
mV
-10
0
10
20
30
40
50
60
0 30 60 90 12017 1816
SR
6M-AF
16.25 16.5 17.25-10
0
10
20
30
40
50
60
0 30 60 90 12017 1816
SR
6M-AF
16.25 16.5 17.25Minutes
mV
0
L-ornithine
Prevention of coronary events by statins is due to sustained LDL cholesterol lowering
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.
A higher preoperative serum level of the proinflammatory and prothrombotic CD40-ligand (sCD40L) is associated with a greater
risk of postoperative AF
AFSR
sCD4
0L (n
g/m
l)
01234567 P<0.05
In hospitalAFSR
At 6 weeks
P<0.05
01234567
sCD4
0L (n
g/m
l)N=147 patientsRR for highest vs lowest sCD40L tertile = 3.81, 95% CI: 1.12-12.95, P<0.05
Antoniades et al. Circulation 2008
Platelet activation and prothrombotic
state may p
recede (and promote?) the
onset of AF
Gudbjartsson et al. Nat Genet. 2009