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AndréL,GouziF,ThireauJ,etal.Carbonmonoxideexposureenhancesarrhythmiaaftercardiacstress:involvementofoxidativestress

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ORIGINAL CONTRIBUTION

Carbon monoxide exposure enhances arrhythmia after cardiacstress: involvement of oxidative stress

Lucas Andre • Fares Gouzi • Jerome Thireau • Gregory Meyer • Julien Boissiere •

Martine Delage • Aldja Abdellaoui • Christine Feillet-Coudray • Gilles Fouret •

Jean-Paul Cristol • Alain Lacampagne • Philippe Obert • Cyril Reboul •

Jeremy Fauconnier • Maurice Hayot • Sylvain Richard • Olivier Cazorla

Received: 3 February 2011 / Revised: 22 July 2011 / Accepted: 24 July 2011

� Springer-Verlag 2011

Abstract Arrhythmias following cardiac stress are a key

predictor of death in healthy population. Carbon monoxide

(CO) is a ubiquitous pollutant promoting oxidative stress

and associated with hospitalization for cardiovascular dis-

ease and cardiac mortality. We investigated the effect of

chronic CO exposure on the occurrence of arrhythmic

events after a cardiac stress test and the possible involve-

ment of related oxidative stress. Wistar rats exposed

chronically (4 weeks) to sustained urban CO pollution

presented more arrhythmic events than controls during

recovery after cardiac challenge with isoprenaline in vivo.

Sudden death occurred in 22% of CO-exposed rats versus

0% for controls. Malondialdehyde (MDA), an end-product

of lipid peroxidation, was increased in left ventricular tis-

sue of CO-exposed rats. Cardiomyocytes isolated from

CO-exposed rats showed higher reactive oxygen species

(ROS) production (measured with MitoSox Red dye),

higher diastolic Ca2? resulting from SR calcium leak and

an higher occurrence of irregular Ca2? transients (mea-

sured with Indo-1) in comparison to control cells after a

high pacing sequence. Acute treatment with a ROS scav-

enger (N-acetylcysteine, 20 mmol/L, 1 h) prevented this

sequence of alterations and decreased the number of

arrhythmic cells following high pacing. Chronic CO

exposure promotes oxidative stress that alters Ca2?

homeostasis (through RYR2 and SERCA defects) and

thereby mediates the triggering of ventricular arrhythmia

after cardiac stress that can lead to sudden death.

Keywords Calcium � Electrocardiography � Signal

transduction � Tachyarrhythmias

Introduction

The clinical importance of an arrhythmic phenotype fol-

lowing cardiac stress is well recognized. Ventricular

ectopic beats (VEB) during recovery represent a better

predictor of increased risk of death in healthy populations

than ventricular ectopy triggered only during exercise [18].

However, the origin and factors that influence the occur-

rence of post-cardiac stress ectopic beats are still unknown.

Mitochondria could play a key role since mitochondrial

dysfunction produces reactive oxygen species (ROS) that

F. Gouzi and J. Thireau equally contributed to this work.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00395-011-0211-y) contains supplementarymaterial, which is available to authorized users.

L. Andre � F. Gouzi � J. Thireau � J. Boissiere � A. Abdellaoui �A. Lacampagne � J. Fauconnier � M. Hayot � S. Richard �O. Cazorla (&)

INSERM U1046 Physiologie & Medecine Experimentale Coeur

Muscles, Universite Montpellier1, CHU Arnaud de Villeneuve,

34295 Montpellier, France

e-mail: olivier.cazorla@inserm.fr

L. Andre � F. Gouzi � J. Thireau � J. Boissiere � A. Abdellaoui �A. Lacampagne � J. Fauconnier � M. Hayot � S. Richard �O. Cazorla

INSERM U1046, Universite Montpellier2,

34295 Montpellier, France

G. Meyer � J. Boissiere � P. Obert � C. Reboul

EA-4278, Universite Avignon et des Pays de Vaucluse,

84000 Avignon, France

M. Delage � J.-P. Cristol

Department of Biochemistry, Lapeyronie Hospital,

34295 Montpellier, France

C. Feillet-Coudray � G. Fouret

INRA UMR 866, 34060 Montpellier, France

123

Basic Res Cardiol

DOI 10.1007/s00395-011-0211-y

can promote calcium (Ca2?) leak through Ryanodine

receptors (RyR), leading to irregular Ca2? transients.[3, 6,

10, 24, 42].

Air pollution promotes oxidative stress [5, 9] and is

involved in the development of arrhythmic events that

trigger heart attack and increase the risk of cardiovascular

death [26, 44]. Epidemiological studies have associated air

pollution with decreased heart rate variability, increased

propensity toward arrhythmic events [43] and sudden car-

diac death [16, 32, 46]. Carbon monoxide (CO), an ubiq-

uitous air pollutant that is found in many common sources

(secondhand smoke, vehicular exhaust, industrial emis-

sions…), is closely associated with hospital admissions for

cardiovascular disease [23] and cardiac mortality [40]. In

urban areas, ambient CO usually varies between 2 and

40 ppm, but under certain circumstances, such as during

heavy traffic, its concentration may reach levels as high as

150–200 ppm [51]. We have recently shown in an exper-

imental animal model that chronic exposure to CO levels,

which mimic pollution in urban areas, induces a heart

failure, like remodeling of Ca2? homeostasis in ventricular

myocytes in basal conditions accounting for a high

occurrence of in vivo premature ventricular contractions in

healthy rats [4]. The pro-arrhythmogenic phenotype in CO-

exposed rats was further enhanced under b-adrenergic

stimulation. These defects were suspected to be associated

with increased tissular oxidative stress. However, the link

between CO exposure and the related oxidative stress on

the occurrence of arrhythmic events after a cardiac stress

test is unknown.

We investigated in a rat model the signaling path-

way(s) that associates chronic CO exposure and post-car-

diac stress arrhythmic phenotype. In vivo chronic CO

exposure increased the number of ventricular ectopic beats

and the risk of sudden cardiac death during recovery from a

pharmacological cardiac challenge. Moreover, we found

that ROS production was significantly higher in cardio-

myocytes isolated from chronically exposed rats than in

controls after a protocol of rapid pacing change that altered

Ca2? homeostasis resulting in an arrhythmic phenotype.

Methods

Detailed information on the methodology is available as

online-only data supplement.

Animal model of chronic CO exposure

Experiments complied with the guide for Care and Use of

Laboratory Animals published by the US National Insti-

tutes of Health (NIH Publications No. 85-23, revised 1996)

and the European Union Council Directives for care of

laboratory animals, and were approved by the French

Ministry of Agriculture.

Male Wistar rats (13 week/old, n = 36) were exposed to

CO in airtight container for 4 weeks as previously descri-

bed [4]. Briefly, rats were exposed to 30 ppm CO for a

12-h period that also included 5 peaks at 100 ppm (1 h

each) to reproduce environmentally relevant air quality

variations [51]. For the remaining 12 h, rats were exposed

to filtered air (\1 ppm CO). CO content was continuously

monitored with an infrared aspirative CO analyzer

(CHEMGARD, NEMA 4 Version, MSA). Rats exposed to

CO pollution were compared with rats exposed only to

filtered ambient air (CO \ 1 ppm; control, n = 27).

Experiments were performed 24 h after the last CO expo-

sure to avoid CO acute effects.

ECGs were recorded by Holter telemetry during the

recovery from a b-adrenergic challenge following a bolus

of 1 mg kg-1 isoprenaline (iso) injected intraperitoneally

(IP). Ventricular ectopic beats including isolated, sequen-

tial or repetitive extra-systoles, and ventricular tachycardia

([20 successive VEB) were counted over a 20-min period

starting when the heart rate was 15% below the maximal

iso-induced rate to avoid rhythmic storm.

Cell isolation

Single intact rat ventricular cardiomyocytes were isolated

by enzymatic digestion from the inner sub-endocardial

layer of the left ventricle (LV) as previously described

[33].

Mitochondrial ROS production

Mitochondrial ROS production was measured using

MitoSOX Red [12]. Single rat cardiomyocytes were

loaded with 5 lmol/L MitoSOX Red at room temperature

for 15 min. MitoSOX Red fluorescence was measured at

583 nm following excitation at 488 nm using a Zeiss

LSM 510 inverted confocal microscope with a 409 lens.

In order to reproduce an exercise test protocol, cardio-

myocytes were subjected to workload changes. Cardio-

myocytes were paced at 0.5 Hz for 5 min, followed by

5 min at high pacing (4 Hz, HP), and then back to 0.5 Hz

for 5 min. MitoSOX Red fluorescence was measured at

the end of each phase. For each cell, the value in quies-

cent cells was set at 0% and background noise was

subtracted.

Anti-oxidant and mitochondrial complex activities

Catalase, total superoxide dismutase (SOD), Complex I and

V activities were measured in rat cardiomyocytes as

described elsewhere [14].

Basic Res Cardiol

123

Ca2? imaging in intact cardiomyocytes

Cardiomyocytes were loaded with Indo-1 AM (10 lM

molecular probes, France) at room temperature for

30 min to monitor intracellular Ca2?. Cells were illumi-

nated at 305 nm using a xenon arc lamp. Sarcomere

length (SL) and Indo-1 fluorescence emitted at 405 and

480 nm were simultaneously recorded (IonOptix system,

Hilton, USA). In some experiments, cells were pre-trea-

ted with the ROS scavenger N-acetylcysteine (20 mmol/L

for 1 h).

For Ca2? sparks, myocytes were loaded for 30 min

with the fluorescent Ca2? indicator Fluo-4 AM (5 lM,

molecular probes). Spontaneous Ca2? sparks (in quies-

cent cells) were recorded in line-scan mode (1.54 ms/

line, 512 pixels 9 3,000 lines) with a Zeiss LSM 510

confocal microscope (639 lens) [13]. Data were ana-

lyzed using ImageJ 1.41 coupled with SparkMaster.

Ca2?-spark frequency was calculated for each cardio-

myocytes, based on scans performed on 10 successive

images collected at 3–4 different line locations and

represented as the number of sparks per lm per second

(events lm-1 s-1).

Western blotting and protein kinase A activity

Proteins were separated using 2–20% SDS–PAGE and

blotted onto PVDF membranes (Protran, Schleichen and

Schuele, Dassel, Germany). Membranes were incubated

with the primary anti-RyR-2 (Covalab, France), -Phospho

Ser2809-RyR-2 (A010-30, Badrilla, UK), -SERCA-2a

(A010-20, Badrilla, UK) and -PLB antibodies (Badrilla,

UK) overnight at 4�C. Immunodetection was carried out

using the ECL Plus System (Amersham Pharmacia,

England).

Malondialdehyde (MDA), an end-product of lipid per-

oxidation, was measured in myocardial tissue isolated

either from the sub-endocardium of hearts that have been

unpaced for 5 min and washed out in Ca2? free solution or

from hearts mounted in Langendorff apparatus and paced

to 7 Hz for 15 min prior rapid dissection. Total protein

samples (5 lg) from each rat heart were dotted onto

nitrocellulose membranes, and dot blot analyses were

performed. The membranes were incubated with the pri-

mary anti-MDA antibody (Academy Biomedical, Houston,

TX, USA) for 1 h at room temperature. Immunodetection

was revealed with ECL. Optical density of each dot was

quantified after scanning and MDA content was expressed

relative to Ponceau S staining.

Protein kinase A (PKA) activity was measured using

a non-radioactive PKA activity assay kit according to

the manufacturer’s recommendations (Assay Designs,

France).

Statistical analysis

Data were analyzed using one-way or 2-way ANOVA

between groups. When significant interactions were found,

a Bonferroni post-hoc test was applied with P \ 0.05

(Statview 5.0). For comparing the number of arrhythmic

cells between control and CO-exposed rats a Chi-square

test was used. Data are presented as mean ± SEM.

Results

Impact of CO on in vivo ventricular arrhythmic events

after pharmacological cardiac challenge in a rat model

To investigate the impact of chronic CO exposure in the

occurrence of arrhythmic events during recovery after a

cardiac challenge, we used a previously characterized

animal model of chronic CO exposure [4]. Rats exposed to

CO for 4 weeks (n = 9) and controls (exposed to filtered

air) (n = 9) were challenged in vivo with a IP bolus of

1 mg kg-1 isoprenaline, a dose that induced a homoge-

neous and rapid increase of the heart rate in all animals. We

then measured the number of VEB occurring during the

recovery phase when the heart rate started to recuperate

(Fig. 1a). All animals from control and CO-exposed groups

exhibited VEB. Although the mean heart rate was com-

parable in control and CO-exposed rats (367 ± 1 and

365 ± 1 bpm, respectively), CO-exposed animals pre-

sented about 2.5 times more VEB than controls (Fig. 1a).

Qualitative analysis of the arrhythmic events showed that

control rats developed exclusively isolated ventricular

extra-systoles, whereas CO-exposed rats presented more

complex arrhythmic events, such as salvos of extra-systoles

(Fig. 1b). Moreover, in CO-exposed animals, we observed

one animal with non-sustained ventricular tachycardia and

two animals with sustained ventricular tachycardia that

degenerated into ventricular fibrillation and led to death

during the recovery period (Fig. 1c). These findings con-

firm that chronic CO exposure is associated with higher

occurrence of VEB and cardiac death during recovery after

cardiac challenge.

Impact of CO on arrhythmic events in rat ventricular

cardiomyocytes after high-frequency pacing

We then investigated the cellular mechanisms of the CO-

associated ventricular arrhythmia observed in vivo using

LV cardiomyocytes isolated from CO-exposed (n = 4) and

control rats (n = 5). Cardiomyocytes, which had been

loaded with Indo-1 to measure cytosolic Ca2?, were elec-

trically stimulated at 0.5 Hz (pre-HP) and submitted to an

abrupt increase in stimulation frequency to 4 Hz (high

Basic Res Cardiol

123

pacing, HP) followed by a return to 0.5 Hz (post-HP)

(Fig. 2a). Occurrence of cellular arrhythmic events was

quantified by measuring the number of irregular Ca2?

transients. During the pre-HP and HP phases, no irregular

Ca2? transient was observed in cardiomyocytes from both

control and CO-exposed rats. Conversely, during the post-

HP period, irregular Ca2? transients (Fig. 2a) occurred

spontaneously in about 10% of control cardiomyocytes and

30% of cardiomyocytes isolated from CO-exposed rats

(Fig. 2b).

Since diastolic intracellular Ca2? overload can trigger

spontaneous Ca2? waves and irregular Ca2? transients

[47], we then measured the diastolic Ca2? concentration in

these cells. During pre-HP, diastolic Ca2? levels were

significantly higher in cardiomyocytes from CO-exposed

rats than in control animals (Fig. 2c). Rapid pacing

increased the diastolic Ca2? concentration in both groups,

but more in controls than in CO-exposed rats and thus the

maximal diastolic Ca2? level in the two groups was com-

parable at HP. After HP, the diastolic Ca2? concentration

decreased to almost pre-HP levels in both groups, but

remained significantly higher in cardiomyocytes from CO-

exposed rats than in control cells (Fig. 2c). Calcium

homeostasis was further studied by measuring spontaneous

Ca2?-sparks, as an index of diastolic sarcoplasmic reticu-

lum (SR) Ca2? leak via RyR2. Ca2?-sparks frequency was

higher in CO-exposed rats than in control animals

(Fig. 3a). SR Ca2? leak in CO-exposed rats was also

addressed by measuring tetracaine-sensitive changes in

diastolic Ca2? as previously described by Shannon et al.

[41] (Fig. 3b). After high pacing, stimulation was stopped,

Na?/Ca2? exchanger (NCX) was blocked and diastolic

Ca2? was measured after 1 min. Diastolic Ca2? was higher

in CO-exposed rats. The protocol was repeated in presence

of tetracaine during the quiescent phase to block RyR.

Diastolic Ca2? decreased only in CO-exposed rats. The

difference of [Ca2?] between the two protocols, which

reflects the SR Ca2? leak, was higher in CO-exposed rats.

SR Ca2? load measured after caffeine pulse was decreased

in CO-exposed rats (Fig. 3c). Furthermore, cardiomyocytes

from CO-exposed rats (n = 5) showed also reduced Serca-

2a expression, which might result in a weaker Ca2?

Fig. 1 Arrhythmic phenotype

following cardiac challenge in

CO-exposed rats. a Isoprenaline

was injected in awaken rats

equipped for surface ECG

recordings. Heart beat

frequency increased rapidly,

remained stable (60 min on this

example) and then decreased to

basal values. Spontaneous

rhythmic disorders were

investigated over a period of

20 min starting at a heart rate

15% below the maximal

isoprenaline-induced heart rate.

b Ventricular ectopic beats

(VEB) were counted. An

example of isolated extra-

systoles is shown (left panel). In

CO-exposed rats we observed

more complex arrhythmic

events such as salvos of extra-

systoles (right panel).C Ventricular tachycardia and

fibrillation were observed in

three CO-exposed rats that led

to death in two of them (n = 9

rats per group). *P \ 0.05

versus control rats

Basic Res Cardiol

123

reuptake capacity during relaxation. Phospholamban, the

regulatory protein of SERCA, was similarly expressed in

control and CO rats, but after high pacing its phosphory-

lation on PKA site (ser16) was increased in CO rats (no

change in PThreo17-PLB/PLB, CaMKII-dependent), which

could be an adaptive process to compensate at least in part

for the reduced SERCA content (Fig. 3d). In addition,

higher phosphorylation of RyR2 at the PKA-dependent

Ser2809 site might also increase Ca2? leak from the SR via

RyR2 during diastole, in comparison to control cells

(n = 5)(Fig. 3d). The lack of arrhythmias before hp indi-

cates that the concentration of diastolic ca2? per se is not

sufficient to trigger arrhythmia and suggests that other

underlying mechanisms occurring during high pacing are

involved.

Involvement of oxidative stress in CO-induced cellular

arrhythmias

Recent studies show that increased contraction frequency

induces ROS formation in rat cardiomyocytes [22]. In

addition, mitochondrial dysfunction and subsequent ROS

production are involved in the development of arrhythmias

[3, 10]. Since CO is known to disturb mitochondrial

function and promote oxidative stress [37], we measured

the level of ROS using MitoSOX Red in cardiomyocytes

from CO-exposed (n = 4) and control (n = 4) rats at the

end of each pacing phase (Fig. 4a). ROS production was

similar in control cells at low and high pacing (about

?10%). In CO cells, ROS production was similar at low

pacing (about ?10% at pre-HP and post-HP), but increased

during high-frequency stimulation (?40%) by fourfold

compared to low pacing and control cells (Fig. 4b).

ROS production results from an imbalance between pro-

oxidant activity and anti-oxidant defenses. We thus eval-

uated in CO-exposed (n = 4) and control (n = 4) cardio-

myocytes the activity of two anti-oxidant enzymes:

superoxide dismutase (SOD), which converts superoxide

anion (O2�-) into hydrogen peroxide (H2O2), and Catalase,

which converts H2O2 into H2O. Both SOD and Catalase

activities were reduced in cells from CO-exposed rats

(Fig. 4c). Chronic CO exposure also significantly

decreased the activity of Complex I and IV of the electron

transport chain (Fig. 4d). This change might be a sign of

mitochondrial malfunction that could contribute to further

increase ROS production [25]. To assess oxidative stress in

the heart, we measured MDA content which is the end-

product of lipid peroxidation by ROS in control and CO-

exposed rats. The measurement has been performed in

‘‘unpaced’’ hearts maintained for 10 min in Ca2? free

solution or in heart mounted in a Langendorff apparatus

and paced for 15 min at 7 Hz prior rapid dissection

(Fig. 4e). In ‘‘unpaced’’ hearts the content of MDA tended

to increase in the CO-group although it did not reach sig-

nificance. In ‘‘paced’’ hearts, the amount of MDA was

higher in CO-exposed rats (Fig. 4e).

Taken together, these results suggest that chronic CO

exposure leads to reduced anti-oxidant defenses and altered

electron transport chain activity, resulting in ROS over-

production, particularly after high-frequency pacing.

Higher ROS production and associated increase in diastolic

Ca2? concentration seem essential to initiate the Ca2?

A

/F48

0)

High pacing0.8

0.7

0.6

CC

a2+

(F40

5

20 s

0.5

0.4

BA

rryt

hmic

cel

ls%

tota

l num

ber) Control

CO

0 40

0.45

0.50

0.55

0.60

* *

olic

Ca2

+(F

405/

F48

0)

10

20

30

40

*

HP

A (%

Post-HPPre-HP0.35

.

Dia

sto

HP Post-HPPre-HP0

Fig. 2 CO exposure induces

irregular Ca2? transient after

high pacing. a Typical example

of irregular Ca2? transients in

cardiomyocytes from CO-

exposed rats after high pacing

(HP). b Number of arrhythmic

control and CO-exposed

cardiomyocytes before (pre-

HP), during (HP) and after high

pacing (post-HP). c Steady-state

diastolic Ca2? levels in control

and CO cardiomyocytes

measured before HP, during HP

and after HP (15–25 cells/4

control and 5 CO-exposed

hearts). *P \ 0.05 versus

control rats

Basic Res Cardiol

123

waves responsible for cellular arrhythmias in CO-exposed

cardiomyocytes.

Finally, in order to further test the relative importance of

oxidative stress in the arrhythmic process, we incubated

CO-exposed cardiomyocytes with 20 mmol/L N-acetyl-

cysteine (NAC) (a ROS scavenger) for 1 h before testing

the effect of high-frequency pacing. NAC treatment (1)

reduced the number of arrhythmic cells after HP to a level

similar to that of control cardiomyocytes (Fig. 5a), and (2)

normalized both diastolic Ca2? concentration (Fig. 5b) and

PKA-dependent phosphorylation of RyR2 (Fig. 5c) in CO-

exposed cardiomyocytes. Consistently, PKA activity,

which was increased in co-exposed cardiomyocytes, was

normalized by NAC treatment (Fig. 5d).

A3

m/s

ec

*Control

0

1

2

Spa

rks/

100µ

m

Control CO

CO

10 µ

m

CB

4

6

8

10

leak

(ra

tio*1

00)

*

0.2

0.3

0.4

Ca

2+ c

onte

nt

*0.5

0.6

0.7

0.8

#*

control CO*

tolic

Ca2

+ra

tiohi

gh p

acin

g

500 ms

0

2S

R C

a2+

0.0

0.1

SR

Control COControl CO

3 40 4

0.3

0.4

Dia

staf

ter

0Ca, 0Na 0Ca, 0Na+ tetracaine

0

1

2

*

0

1

2

3

4

*

0.0

0.2

.

Control CO

Ser

ca c

onte

nt

RyR

con

tent

P-R

yR /

RyR

D

Control CO Control CO

Control

RyR

Serca

P-RyRCO

2

4

6

8

PLB

con

tent

PLB

PThreo17-PLB

PSer16-PLB

2

4

6

*

er16

-PLB

/ P

LB

0.5

1.0eo

17-P

LB /

PLB

0Control CO

CSQ 0Control CO

PS

e

0.0Control CO

PT

hre

Fig. 3 CO-associated alterations of Ca2? homeostasis. a Typical

examples of spontaneous Ca2? sparks in cardiomyocytes from control

and CO-exposed rats. The frequency of sparks appearance increased

by three times in CO-exposed myocytes (30–38 cells/3 control and

CO-exposed hearts). b SR Ca2? leak in CO-exposed rats was also

addressed by measuring tetracaine-sensitive changes in diastolic

Ca2?. After high pacing for 3 min, stimulation was stopped, Na?/

Ca2? exchanger was blocked by a solution of 0Ca2?/0Na? and

diastolic Ca2? was measured after 1 min. The protocol was repeated

in presence of tetracaine (1 mM) during the quiescent phase to block

RyR (left panel). The difference of [Ca2?] between the two protocols

reflects the SR Ca2? leak (right panel) (12 cells/3 hearts). *P \ 0.05

versus control rats, #P \ 0.05 versus without tetracaine. c SR Ca2?

load measured after caffeine pulse (10 mM) was decreased in CO-

exposed rats (12 cells/3 hearts). d Expression/phosphorylation of

RyR-2, Serca-2a and PLB in control and CO-exposed cardiomyocytes

were analyzed by western blotting. RyR-2, Serca-2a and PLB

contents were normalized to Calsequestrin (CSQ). After high pacing

the level of PKA-dependent Ser2809 RyR-2 phosphorylation (P-RyR)

was normalized to total RyR-2 and the PKA-dependent Ser16-

phosphorylated PLB and CamKII-dependent threo17-phosphorylated

PLB were normalized to PLB (5 hearts each). *P \ 0.05 versus

control rats

Basic Res Cardiol

123

Discussion

Air pollution is suspected to be involved in sudden death

by increasing ventricular arrhythmic events. VEB preva-

lence during recovery from cardiac stress is a valuable

predictor of mortality. In this study, we find higher VEB

occurrence during recovery after cardiac challenge in an

experimental rat model of chronic exposure to urban

atmospheric CO pollution that leads in some cases to

sudden death. Moreover, higher ROS production (and thus

oxidative stress) seems to be essential together with

increased diastolic Ca2? concentration to initiate the Ca2?

waves responsible for the arrhythmic phenotype in single

cardiomyocytes isolated from CO-exposed rats. The

Control

Post-HPPre-HPA

CO

B CControl 12060

O2-

prod

uctio

n(%

res

ting

leve

l) CO

20

40

60

80

100

120

**

0

10

20

30

40

50 *

HP Post-HPPre-HP

Act

ivity

(U

/mg

prot

ein)

SOD Catalase00

3 3

*

E

MDA

Ponceau

Unpaced

MDA

Ponceau

Paced

0

1

2

0

1

2

Control CO

MD

A c

onte

nt

Control CO

MD

A c

onte

nt

D

Control

CO

3

4

5

6

20

30

400

600

800

Act

ivity

(U

/g p

rote

in)

Complex IVComplex I

Act

ivity

(U

/g p

rote

in)

Act

ivity

(U

/g p

rote

in)

Citrate synthase0

1

2

3

0

10 *

0

200

400

*

Fig. 4 Effects of CO pollution

on mitochondrial ROS

production. a Typical images

showing Mitosox Red

fluorescence (indicator of

superoxide production) in

cardiomyocytes from control

and CO-exposed rats at rest and

after high pacing (HP).

b Production of O2�2 in control

and CO-exposed

cardiomyocytes measured at the

end of each pacing phase

before, during and after HP

relative to the value at rest

(n = 22–30 cells/4 control and

4 CO-exposed hearts).

c Myocardial anti-oxidant

defences were evaluated by

measuring SOD and Catalase

activity in cardiomyocytes from

control and CO-exposed rats (4

hearts per group). d Myocardial

citrate synthase (CS), Complex I

and Complex IV activities in

cardiomyocytes from control

and CO-exposed rats (4 hearts

per group). e Malondialdehyde

(MDA) content in myocardial

tissue isolated from the sub-

endocardium of hearts that have

been either unpaced for about

5 min in Ca2? free solution or

paced to 7 Hz for 15 min using

Langendorff apparatus.

*P \ 0.05 versus control rats

Basic Res Cardiol

123

variations in diastolic Ca2? handling were normalized to

control levels, when CO-exposed cardiomyocytes were

treated with a ROS scavenger before high pacing.

CO exposure increases ROS production after high

pacing

Air pollution is known to promote oxidative stress [9, 43].

Traffic-related air pollutants, such as particles, CO and

nitrogen species, have been associated with increased

systemic inflammation and oxidative stress [9, 43]. The

relative contribution of each pollutant was not investigated.

In order to control the level and origin of CO, we used an

experimental model in which rats were exposed to CO

levels that mimic those recorded in urban areas. This ani-

mal model allowed us to study precisely the effects of

chronic CO exposure on ROS production and its conse-

quences on heart rhythm independently of the other

pollutants.

One key finding of this study is that chronic CO expo-

sure potentiates at high-frequency pacing severe mito-

chondrial alterations leading to exacerbated ROS

production in cardiomyocytes and related oxidative stress

at the tissue level. The deleterious effects of CO were not

observed at low pacing in cells and in unpaced hearts. This

suggests that despite reduced antioxidant defences, the cell

is able to control the level of ROS production under basal

conditions. Moreover, the activity of other cytosolic anti-

oxidant enzymes such as peroxiredoxin or glutathione

peroxidase-1 have not been tested and could compensate

for the decrease in SOD and catalase activities [54]. The

effect of CO directly at the cardiomyocyte level might be

due to the fact that, under condition of chronic CO expo-

sure, CO competes with oxygen for binding to hemoglobin

(Hb), resulting in decreased cardiomyocyte oxygenation.

Tissue hypoxia could be worsened by CO binding also to

cardiac myoglobin [37], leading to disturbed cardiac

mitochondrial oxidation [20]. This is, however. unlikely in

our experimental conditions due to the modest reduction of

oxygen in arterial blood resulting from CO binding to Hb

(data not shown). Moreover, since we did not observe

electrocardiographic signs of ischemia in CO-exposed rats,

the involvement of the vascular system in vivo due to CO-

induced vasoconstriction linked to ROS production [30]

can also be excluded. Alternatively, CO binding to Cyto-

chrome p-450 might inhibit directly the Complex IV of the

electron transport chain, leading to ROS production [18,

37]. This hypothesis is supported by the reduced Complex

IV activity we observed in cardiomyocytes from CO-

exposed rats (Fig. 4d).

The present results contrast with the described cyto-

protective effect of CO at low dose and acute treatment in

Fig. 5 Effect of acute NAC

treatment on CO-associated

Ca2? handling alterations.

a Percentage of arrhythmic

cardiomyocytes isolated from

control (5 hearts), CO-exposed

(5 hearts) and CO-exposed rats

treated with NAC for 1 h (CO-

NAC) (5 hearts) during

recovery after high pacing

(15–25 cells). b Diastolic Ca2?

levels in control, CO-exposed

and CO-NAC cardiomyocytes

after high pacing (35–50 cells).

c Level of PKA-dependent

Ser2809 RyR-2 phosphorylation

normalized to total RyR-2

(n = 5 hearts). d PKA activity

was measured in cardiac tissue

from control and CO-exposed

and CO-NAC rats. *P \ 0.05

versus control rats

Basic Res Cardiol

123

cardiovascular diseases [17] with anti-oxidant, anti-

inflammatory, and anti-apoptotic properties [36]. However,

our model of chronic moderate CO exposure (30 ppm/day

plus five 1-h peaks at 100 ppm, 4 weeks) highlights the

potential harmful effects of low-dose CO gas inhalation or

synthetic CO-releasing molecules on cardiac function if

used as chronic treatments.

CO-mediated ROS production triggers irregular Ca2?

transients

Excessive ROS production plays a pivotal role in triggering

cellular and ventricular arrhythmic events through changes

in RyR2 activity [13, 48]. In CO-exposed cardiomyocytes,

arrhythmias were observed in association with increased

diastolic Ca2? level and ROS overproduction induced by

high pacing. This supports the concept of a crosstalk

between Ca2? and ROS as a central element in CO-medi-

ated Ca2? handling disorders in which Ca2?/ROS poten-

tiate each other [15]. Our results are in line with a previous

study in isolated guinea-pig cardiomyocytes showing that

perfusion of 1 mM H2O2 increased after 5 min diastolic

Ca2? and few minutes later, cells became automatic [21].

Acute treatment with the broad range antioxidant NAC

prevented diastolic Ca2? overload and irregular Ca2?

transients in CO-exposed cardiomyocytes (Fig. 5a). This is

consistent with the beneficial effects of NAC treatment on

RyR2-mediated diastolic SR Ca2? leak and arrhythmias

reported in Duchenne muscular dystrophy cardiomyocytes

[13]. Antioxidant strategies are also beneficial in various

rhythmic pathologies. In a sheep model, an anti-oxidant

cocktail (vitamin C and deferoxamine) reduced the number

of ventricular tachycardia/fibrillation events induced by

ischemia–reperfusion [28].

Oxidation of ion transport systems that participate in

cardiac excitation–contraction coupling can potentially

lead to Ca2? overload [11, 52]. However, other post-

translational modifications of RyR2 may be involved since

ROS can affect also protein phosphorylation through

inhibition of protein phosphatases [38] and activation of

PKA [34, 8]. Indeed, CO-exposed cardiomyocytes show

PKA-dependent hyper-phosphorylation of RyR2 that is

reverted following NAC treatment (Fig. 3c). Although this

is controversial, PKA-dependent RyR2 hyper-phosphory-

lation may lead to SR Ca2? leak that contributes to

increasing diastolic Ca2? levels [50, 31], which may be

involved in the contribution of sustained sympathetic

activation on arrhythmogenesis [35]. Additional pathway

may involve the Ca2?/calmodulin-dependent serine/threo-

nine kinase-d (CaMKIId), which is activated by Ca2?-

calmodulin at high Ca2? concentration, particularly when

heart rate increases. The impact of RyR2 phosphorylation

by CaMKII is controversial ranging from stabilizing RyR2

activity [53] to RyR2 disturbance resulting in Ca2? leak

[2]. A recent study showed that RyR2 phosphorylation by

CaMKIId is required for normal force–frequency response

in mice [29]. Its possible involvement in arrhythmia in CO-

exposed rats should be carefully investigated.

The reduced expression of SERCA-2a (responsible for

Ca2? reuptake in the SR during diastole) in CO-exposed

cardiomyocytes may also contribute to increasing dia-

stolic Ca2? concentration and the reduction in SR Ca2?

load. However, in our study acute NAC treatment nor-

malized diastolic Ca2? level indicating that a reversible

post-translational modification is involved. We show here

that the phosphorylation level of PLB on PKA site is

increased in CO-exposed rats, which should at least in

part compensate for the decrease in SERCA content.

Further phosphorylation on PLB after NAC treatment is

unlikely, considering the decrease of PKA activity in

presence of NAC. Other direct or indirect pathways

induced by ROS over-production may impact on SERCA-

2a function after chronic CO exposure, since previous

reports have shown that ROS inhibit SERCA-2a activity

and expression [1]. This pathway will require further

studies.

It has been reported that ‘‘millimolar concentrations of

ROS can cause increase in the Ca2? and Na? permeabil-

ity’’ but there is no consensus regarding potential effects on

Ca2? channels and on the Na?/Ca2? exchanger [19]. We

can just point out that decreased activities of IcaL and NCX

would not be in favor of proarrhythmogenicity. Although

ROS were reported to increase the late sodium current [45,

49] favoring Na? and Ca2? overloads responsible for

DADs/EADs, we have no evidence for prolonged AP

repolarization and no change in L-type Ca2? current in

subendocardial cells [4]. Finally, most of studies report

acute effects of moderate to high concentrations of H2O2,

whereas the present study describes effects resulting from

remodeling after long-term exposure to CO enhanced

during/after high pacing. Considering that we did not focus

on these mechanisms in the present study, we cannot

completely exclude their contribution to the increase dia-

stolic Ca2?.

CO level in human and incidence on cardiovascular

disease

Diastolic Ca2? overload induces oscillations of the cell

membrane potential that trigger delayed after-depolariza-

tions [47]. We provide direct evidence that sustained CO

exposure promotes irregular Ca2? transients in vitro and

arrhythmic events in vivo during recovery after cardiac

challenge in an experimental animal model. Ventricular

arrhythmias occurring during recovery after a cardiac stress

is common even in healthy subjects as indicated by the

Basic Res Cardiol

123

presence of VEB in all control rats (&12 VEB/h). How-

ever, these events are exclusively isolated VEB. The fact

that CO-exposed rats had 2.5 times more VEB (&36 VEB/

h) and, more importantly, more complex events such as

repetitive extra-systoles and ventricular tachycardia (3/9

CO animals and none in control rats) indicate that CO

exposure is pro-arrhythmogenic. Nevertheless, the link

between human CO levels and arrhythmias remains to be

determined. In an exploratory investigation, we measured

various functional parameters in non-smoker healthy vol-

unteers who performed an incremental maximal exercise

test on ergocycle (Supplemental data Figure S1A). Age,

gender, VO2max, and maximal heart rate were not corre-

lated with the number of arrhythmic events during recovery

after exercise (Supplemental data, Table S1). Although the

air was the same in the hospital, volunteers have been

exposed prior to their arrival at the hospital to various

levels of environmental CO (secondhand smoke, vehicular

exhaust, industrial emissions…) linked to different lifestyle

(living/working in the urban and/or non-urban locations of

the Montpellier region). In order to assess CO exposure, we

measured carboxyhemoglobin level (HbCO), which is

recognized as a biological marker of recent CO exposure

[39]. Conversely, we found a positive correlation

(R2 = 0.76, P \ 0.05) between the blood level of HbCO

and the number of VEB (Figure S1B). Similarly, the blood

level of HbCO was linearly correlated (R2 = 0.82,

P \ 0.01) with that of isoprostane, an index of oxidative

stress, consistent with a pro-oxidative effect of CO expo-

sure (Figure S1C). Since oxidative stress plays a critical

role in arrhythmogenesis, we then evaluated whether the

number of VEB after exercise was also associated with the

concentration of isoprostane (Figure S1D). Although a

positive linear correlation between isoprostane concentra-

tion and VEB number did not reach statistical significance

(P = 0.07), most probably due to the small number of

subjects available for this analysis (n = 17), the results

suggest, however, a link between (CO-induced) oxidative

stress and the occurrence of VEB in healthy human sub-

jects. Those results have to be confirmed in more subjects,

but they are in line with a recent study performed in a large

cohort of patients from the Framingham Heart study [7]. In

this 4 years follow-up study, a close relation between

exhaled CO, considered as an indicator of total blood CO

concentration, and risk of developing future cardiovascular

diseases and metabolic syndrome was established. This

work was based on the hypothesis that the measured CO

reflected the endogenous CO production without consid-

ering exogenous environmental CO. The effect of CO

pollution on the post-exercise arrhythmic phenotype could

be even more deleterious in elderly populations [27] or in

populations with pathologies, such as metabolic syndrome

or heart failure, which are known to generate oxidative

stress. This remains to be determined in epidemiological

studies.

Conclusions

The present study provides evidence that sustained expo-

sure to urban CO pollution directly influences the occur-

rence of arrhythmic events during recovery after cardiac

challenge. We show a link between CO exposure, oxidative

stress, diastolic Ca2? and arrhythmic events during cardiac

stress recovery. Since post-cardiac stress irregular beats are

a key factor for predicting sudden cardiac death [18], our

findings suggest that chronic CO exposure represents a

potential risk factor for triggering cardiac events and thus

should be taken into account for the prevention of cardio-

vascular risk in clinical practice.

Acknowledgments This work was supported by a French National

Research Agency grant (COMYOCARD). SR, JF, AL, and OC are

scientists from the Centre National de la Recherche Scientifique. We

thank Sandrine Gayrard for technical assistance.

Conflict of interest None declared.

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