Carbon Monoxide Exposureenhances Arrhythmic Events During Recovery After Cardiac Stress

Carbon monoxide exposureenhances arrhythmic events during recovery after cardiac stress test: involvement of oxidative stress

Lucas Andre;Fares Gouzi, Jérome 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.

Materials and Methods

Study population and effort test

The study was approved by the local institutional review board (Comité de Protection des Personnes sud méditerrannée IV 2008-03-EESSS-V2)and subjects gave their informed consent.Healthy subjects(n=22) were recruited on the basis of the following criteria: 50 to 75 year/old (mean age = 62.4±1.3, 15 males, 7 females), non-smokers, no medical disease and spending less than 150 minutes of moderate-to-vigorous physical activity per week. These exclusion criteria allowed us to evaluatea homogeneous population, independently of factors that might modify cardiac function and occurrence of arrhythmic events. All had a complete clinical examination andsubjects with cardiologic treatment or suspicion of cardiac diseasewere excluded from the study.All participants performed a physician-supervised incrementalexercise test until exhaustion on an electrically-braked cycle ergometer (Ergoselect 200P, Ergolyne, Bitz, Germany) (1). During the exercise test, electrocardiogram (ECG) was recorded using standard 12-lead derivations, at rest, during exercise and during recovery for 5 minutes. Ectopic ventricular beats including isolated, sequential or repetitive extra-systoles, and ventricular tachycardia during recovery were then reviewed blindlyby two physicians.

Human Carboxyhaemoglobin and isoprostane blood levels

To estimate the degree of CO exposure in human subjects (n=22),we measured the concentration of Carboxyhemoglobin (HbCO). HbCO is considered as a specific and sensitive biomarker of CO exposure and has been correlated with air pollution (2). Venous blood samples were collected in standard, sterile, heparinized tubesbefore theeffort test.Immediately after sampling, blood was either transferred for HbCO measurement or serum and plasma were removed by centrifugation (2500 rpm for 10 minutes at 4°C), divided in 500 µL aliquots and stored at -80°C until analysis.HbCO levels were measured (COoxymeter IL682, Instrumentation Laboratory) in the Pharmacology and Toxicology laboratory (LapeyronieHospital, Montpellier, France).

Plasma-free and esterified Isoprostanes (F2-IsoP) were evaluated as markers of lipid peroxidationas previously described(3). Lipids were first extracted from samples (1 ml of plasma containing [2H4]-15-F2t-IsoP as internal standard) with Folch solution (chloroform/methanol 2:1; v/v) and then subjected to alkaline hydrolysis. The pH was adjusted to 2.2 with HCl and samples were diluted in water to 10 ml. Samples were then extracted twice using an inverse-phase and then an aminopropyl-phase cartridge (Sep-Pak Vac RC C18 and Vac RC NH2, Waters S.A., Guyancourt, France). After the last step of purification, samples were derivatized as pentafluorobenzylester and trimethylsilyl ethers. Derivatized samples were analyzed by gas chromatography-negative ion chemical ionization mass spectrometry. Quantification was achieved by relating the peak area of F2-IsoP to the internal standards. Since we added this parameter to the protocol when recruitment and exercise testing were already under way, we could measure it only in 17 patients.

Animal model ofchronic CO exposure

Experiments complied with the guide for the 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 laboratory animals, and were approved by the French Ministry of Agriculture.

In urban areas, ambient CO usually varies between 2 and 40 ppm, but under certain circumstances, such as heavy traffic, its concentration may reach levels as high as 150-200 ppm (4)(5). In our study, male Wistar rats (13 week/old, n=36) were exposed to CO in airtight exposure containersfor 4 weeks as previouslydescribed (6). Rats were exposed to a basal CO concentration of 30 ppm during a 12 hour period that included also 5 peaks at 100 ppm (1 hour each), to reproduce environmentally relevant air quality variations. For the remaining 12 hours, they 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=32). Experiments were performed 24 hours after the last CO exposure to avoid acute effects of CO.

Holter ECG (Physiotel-CA-F40, Data Sciences International, Minnesota, USA) were recorded by telemetry(IOX2, EMKA Technologies, Paris, France)as previously described(6). Spontaneous rhythmic disorders were detected from ECGs during the recovery froma -adrenergic challenge following injection of 1 mg.kg-1 isoprenaline.Ectopic ventricular beats, including isolated, sequential or repetitive extra-systoles, and ventricular tachycardia were counted over a 20 min period starting whenthe heart rate was 15% below the maximal iso-induced heart rate to avoid rhythmic storm.

Cell isolation

Single ventricular cardiomyocytes were isolated by enzymatic digestion (7). Briefly, rat hearts were quickly removed, mounted on a Langendorff apparatus and perfused at 37°Cwith an enzymatic solution (1.25 mg.mL-1Collagenase type IV, Worthington Freehold, NJ, U.S.A) for 10-20 min. The left ventricle (LV) was dissected and cardiomyocytes were dissociated from the inner sub-endocardial layer in a Ca2+-free solution. Ca2+ concentration was gradually increased to 1 mM Ca2+. Solutions for isolating intact cardiomyocytes contained:117mMNaCl, 5.7mMKCl,4.4mMNaHCO3,1.5mMKH2PO4,1.7mMMgCl2, 21mMHEPES,11mMglucose, 20mMtaurine, pH 7.2 adjusted with NaOH.

Measurement of mitochondrial ROS production

Mitochondrial ROS production was measured using MitoSOX Red as described previously (8). Single rat cardiomyocytes were loaded with MitoSOX Red (5 µmol/l) at room temperature for 15 min. MitoSOX Red fluorescence was measured at 583 nm following excitation at 488 using a Zeiss LSM 510 inverted confocal microscope with a x40 lens. In order to reproduce the exercise test protocol, cardiomyocytes were subjected to changes of workload. Cardiomyocytes were pacedat 0.5Hz for 5 min, followed by 5 minutes at high pacing (4Hz, 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 at rest was set at 100% and background noise was subtracted.

Determination of antioxidant activities in rat cardiomyocytes

Catalase and total Superoxide Dismutase (SOD) activitieswere measured in cardiomyocytes as described elsewhere (9),(10).

Determination of mitochondrial respiratory complex activities in rat cardiomyocytes

Complex I activity was measured as described (11). Complex IV (Cytochrome-c Oxidase) activity was measured as described (12). Oxidation of Cytochrome-c was followed by spectrophotometry. Citrate Synthase (CS) activity was measured as described(13) by following the formation of 5-thio-2-nitrobenzoic acid (colorimetric reaction) from 5,5’-dithiobis-2-nitrobenzoic acid (present in the reaction of citrate synthesis)caused by the deacetylation of acetyl-CoA.

Measurement of Ca2+ transients

Rat cardiomyocytes were loaded with Indo-1 AM (10 µM Invitrogen inc., France) at room temperature for 30 min in order 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 nm and 480 nm were simultaneously recorded using the IonOptix acquisition software (IonOptix system, Hilton, USA). Diastolic Ca2+fluorescence was collected during the HP phaseand during recovery at 0.5 Hz.Ectopic Ca2+ transients were counted over a 5 min period during the post-HP phase at 0.5 Hz. In some experiments, cardiomyocytes were pre-treated with the ROS scavenger N-acetylcysteine(20mmol/L for 1 hour).

Western blot and PKA activity

Proteins were separated using 2-20 % SDS-PAGE and blotted onto a PVDF membrane (Protran, Schleichen and Schuele, Dassel, Germany). Membranes were incubated with the following primary antibodies: anti-RyR-2 (Covalab, France), -Phospho Ser2809-RyR-2 (A010-30, Badrilla, UK), -SERCA-2a (A010-20, Badrilla, UK) and -PLB antibody (A010-14, Badrilla, UK) at 4°C overnight. RyR-2, SERCA-2A and PLB levels were expressed relative to Calsequestrin content (Cat # PA1-913, ABR, USA) on the same membrane. Immunodetection was carried out using the ECL Plus System (Amersham Pharmacia, Little Chalfont Buckinghamshire, England).

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 in control and CO-exposed rats a Chi-square test was used. Data are presented as mean±SEM.

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Table1: The clinical and cardiovascular characteristics of the healthy volunteers enrolled for this study were correlated with the prevalence of ventricular ectopic beats during recovery after exercise.

HR=heart rate, HbCO= Carboxyhemoglobin. The p value is given for significance of the Pearson’s correlation coefficient. The gender effect has been test with a U Mann-Whithney test.

Figure S1: Relationship between chronic CO exposure, arrhythmic events and oxidative stress in healthy human subjects. A: Typical example of Ventricular ectopic beats (VEB) recorded in healthy volunteers during the recovery phase of a maximal exercise test on ergocyle. B: Correlation between the number of VEB measured during the recovery phase of a maximal exercise test and the blood level of Carboxyhemoglobin (HbCO) (n=22 subjects). Data were fitted with an exponential decay function (Y=5.1exp(-x/(-1.9))+5.5exp(-x/(-1.9))+8.9exp(-x/(-0.1))-17.1, Chi-squared = 0.76, p<0.05).C:Correlation between plasmatic concentration of IsoprostaneandHbCO (n=17 subjects). Data were fitted with a linear function (Y= 553x-601, Chi-squared =0.82, p<0.001). D: Correlation between Isoprostane concentration and number of VEBmeasured during the recovering phase of a maximal exercise test (n=17 subjects). Data were fitted with a linear function (Y= 7.1x+230, Chi-squared =0.28, p=0.07).

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