Carbon monoxide exposure enhances arrhythmia ...

Carbon monoxide exposure enhances arrhythmia after cardiac stress: involvement of oxidative stress Lucas André, Fares Gouzi, Jérôme Thireau, Gregory Meyer, Julien Boissiere, Martine Delage, Aldja Abdellaoui, Christine Feillet-Coudray, et al. Basic Research in Cardiology ISSN 0300-8428 Volume 106 Number 6 Basic Res Cardiol (2011) 106:1235-1246 DOI 10.1007/s00395-011-0211-y

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Author's personal copy Basic Res Cardiol (2011) 106:1235–1246 DOI 10.1007/s00395-011-0211-y

ORIGINAL CONTRIBUTION

Carbon monoxide exposure enhances arrhythmia after cardiac stress: involvement of oxidative stress Lucas Andre´ • Fares Gouzi • Je´roˆme Thireau • Gregory Meyer • Julien Boissiere Martine Delage • Aldja Abdellaoui • Christine Feillet-Coudray • Gilles Fouret • Jean-Paul Cristol • Alain Lacampagne • Philippe Obert • Cyril Reboul • Je´re´my Fauconnier • Maurice Hayot • Sylvain Richard • Olivier Cazorla

•

Received: 3 February 2011 / Revised: 22 July 2011 / Accepted: 24 July 2011 / Published online: 6 August 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 disease 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 involvement of related oxidative stress. Wistar rats exposed chronically (4 weeks) to sustained urban CO pollution

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

Electronic supplementary material The online version of this article (doi:10.1007/s00395-011-0211-y) contains supplementary material, 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: [email protected] 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

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 tissue 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 (measured with Indo-1) in comparison to control cells after a high pacing sequence. Acute treatment with a ROS scavenger (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 following 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 occurrence of post-cardiac stress ectopic beats are still unknown. Mitochondria could play a key role since mitochondrial dysfunction produces reactive oxygen species (ROS) that

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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 cardiac death [16, 32, 46]. Carbon monoxide (CO), an ubiquitous air pollutant that is found in many common sources (secondhand smoke, vehicular exhaust, industrial emissions…), 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 experimental 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 COexposed 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 pathway(s) that associates chronic CO exposure and post-cardiac 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 cardiomyocytes 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.

Basic Res Cardiol (2011) 106:1235–1246

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 described [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 exposure 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, sequential 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, cardiomyocytes were subjected to workload changes. Cardiomyocytes 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 quiescent cells was set at 0% and background noise was subtracted.

Animal model of chronic CO exposure Anti-oxidant and mitochondrial complex activities Experiments complied with the guide for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publications No. 85-23, revised 1996) and the European Union Council Directives for care of

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Catalase, total superoxide dismutase (SOD), Complex I and V activities were measured in rat cardiomyocytes as described elsewhere [14].

Author's personal copy Basic Res Cardiol (2011) 106:1235–1246

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Ca2? imaging in intact cardiomyocytes

Statistical analysis

Cardiomyocytes were loaded with Indo-1 AM (10 lM molecular probes, France) at room temperature for 30 min to monitor intracellular Ca2?. Cells were illuminated 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-treated 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 quiescent 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 analyzed using ImageJ 1.41 coupled with SparkMaster. Ca2?-spark frequency was calculated for each cardiomyocytes, 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).

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.

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 4C. Immunodetection was carried out using the ECL Plus System (Amersham Pharmacia, England). Malondialdehyde (MDA), an end-product of lipid peroxidation, 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 primary 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).

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 homogeneous 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 comparable in control and CO-exposed rats (367 ± 1 and 365 ± 1 bpm, respectively), CO-exposed animals presented 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 confirm 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 COassociated 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 electrically stimulated at 0.5 Hz (pre-HP) and submitted to an abrupt increase in stimulation frequency to 4 Hz (high

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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 extrasystoles is shown (left panel). In CO-exposed rats we observed more complex arrhythmic events such as salvos of extrasystoles (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

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 postHP 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 comparable at HP. After HP, the diastolic Ca2? concentration decreased to almost pre-HP levels in both groups, but

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remained significantly higher in cardiomyocytes from COexposed rats than in control cells (Fig. 2c). Calcium homeostasis was further studied by measuring spontaneous Ca2?-sparks, as an index of diastolic sarcoplasmic reticulum (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 Serca2a expression, which might result in a weaker Ca2?


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reuptake capacity during relaxation. Phospholamban, the regulatory protein of SERCA, was similarly expressed in control and CO rats, but after high pacing its phosphorylation 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 indicates 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

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Fig. 2 CO exposure induces irregular Ca2? transient after high pacing. a Typical example of irregular Ca2? transients in cardiomyocytes from COexposed rats after high pacing (HP). b Number of arrhythmic control and CO-exposed cardiomyocytes before (preHP), 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

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during high-frequency stimulation (?40%) by fourfold compared to low pacing and control cells (Fig. 4b). ROS production results from an imbalance between prooxidant activity and anti-oxidant defenses. We thus evaluated in CO-exposed (n = 4) and control (n = 4) cardiomyocytes 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 endproduct of lipid peroxidation by ROS in control and COexposed 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 significance. 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 overproduction, particularly after high-frequency pacing. Higher ROS production and associated increase in diastolic Ca2? concentration seem essential to initiate the Ca2?

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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 COexposed 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 Ser16phosphorylated PLB and CamKII-dependent threo17-phosphorylated PLB were normalized to PLB (5 hearts each). *P \ 0.05 versus control rats

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-acetylcysteine (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 COexposed cardiomyocytes. Consistently, PKA activity, which was increased in co-exposed cardiomyocytes, was normalized by NAC treatment (Fig. 5d).

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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 subendocardium 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

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Discussion Air pollution is suspected to be involved in sudden death by increasing ventricular arrhythmic events. VEB prevalence 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

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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

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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 (CONAC) (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

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 animal model allowed us to study precisely the effects of chronic CO exposure on ROS production and its consequences on heart rhythm independently of the other pollutants. One key finding of this study is that chronic CO exposure potentiates at high-frequency pacing severe mitochondrial 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

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is able to control the level of ROS production under basal conditions. Moreover, the activity of other cytosolic antioxidant 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 exposure, 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 COinduced vasoconstriction linked to ROS production [30] can also be excluded. Alternatively, CO binding to Cytochrome 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 COexposed rats (Fig. 4d). The present results contrast with the described cytoprotective effect of CO at low dose and acute treatment in


cardiovascular diseases [17] with anti-oxidant, antiinflammatory, 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-mediated Ca2? handling disorders in which Ca2?/ROS potentiate 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 posttranslational 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-phosphorylation 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/threonine 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

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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 COexposed 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 diastolic Ca2? concentration and the reduction in SR Ca2? load. However, in our study acute NAC treatment normalized 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 SERCA2a 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? permeability’’ 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 diastolic Ca2?. CO level in human and incidence on cardiovascular disease Diastolic Ca2? overload induces oscillations of the cell membrane potential that trigger delayed after-depolarizations [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

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presence of VEB in all control rats (&12 VEB/h). However, 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 volunteers who performed an incremental maximal exercise test on ergocycle (Supplemental data Figure S1A). Age, gender, VO2max, and maximal heart rate were not correlated 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 exposure (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 concentration 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 subjects. 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 considering 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

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stress. This remains to be determined in epidemiological studies.

Conclusions The present study provides evidence that sustained exposure to urban CO pollution directly influences the occurrence 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 cardiovascular 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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