Oxidative stress and atrial fibrillation: a long journey in to the clinic? (Prof. Barbara Casaidei)

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 is associated with a significantly increased mortality and high medical costs (ca. 3% of the UK NHS budget)



• 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



• An increase in atrial NOX2 activity precedes AF

• Is increased atrial NOX2 activity sufficient to create a substrate for AF?

.

.


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




• 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


**

O2

- (RLU

/s/

g p

rote

in)

6M AF (n=10)

0.0

0.1

0.2

0.3

0.4

0.5


**

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


**

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

Oxidative stress and atrial fibrillation: a long journey in to the clinic? (Prof. Barbara Casaidei)

Health & Medicine

atrial nox2 oxidases

atrial remodelling

atrial oxidative stress

af inductionin

history of af

af mihm

usaprevalence of af

incident af