Animals and carbon monoxide exposure

Adult, male Wistar rats (n=66; 367 ± 7 g; Charles River Laboratories) were randomly assigned to 2 groups, carbon monoxide exposed rats (CO rats, exposed for 4 weeks to simulated CO urban pollution, n=33) and Control rats (Ctrl rats, exposed to standard filtered Air, n=33). CO rats were housed in an airtight exposure container for 4 weeks. Exposure was performed according to a 12:12-hours CO in air-ambient air cycle, as previously described (2, 13). During CO exposure, a CO concentration of 30 ppm was maintained and completed with five 1 hour peaks at 100 ppm. At the end of the 4 week CO exposure, rats were housed for 24 hours in standard filtered air, in order to avoid the acute effects of CO on the myocardium. 

Regional myocardial ischemia and reperfusion on isolated perfused rat hearts 

On a first set of rats (n=16 per group) a regional myocardial IR was achieved as previously described (13). The IR protocol was performed with or without SMT, a specific iNOS inhibitor at the concentration of 0.5 µM (n=8 per condition). The heart was mounted on a Langendorff isolated heart system and perfused with an oxygenated (95 % O2 / 5 % CO2) Krebs solution (37°C) composed of (in mM) NaCl 118.3, NaHCO3 25, KCl 4.7, MgSO4 1.2, KH2PO4 1.2, Glucose 11.1, CaCl 2.5 (pH= 7.4). When necessary, SMT was added to the Krebs solution (SMT, 0.5 µM). The hearts were perfused at a constant pressure (80 mmHg) and paced at 300 beats ⁄min with an electrical stimulator (Low voltage stimulator, BSL MP35 SS58L, 3V). The heart was allowed to stabilize for 30 min. Then a regional ischemia (left anterior descending coronary artery occlusion) was performed during 30 min. Subsequently the heart was allowed to reperfuse for 120 min. At the end of the protocol, a staining protocol was performed in order to assess infarct size (13). 

Cardiomyocyte excitation-contraction analysis after cellular anoxia/reoxygenation 

On a second set of rats (n=4 per group), single ventricular cardiomyocytes were isolated by enzymatic digestion (14). Cardiomyocytes were transferred into a glass Petri dish and placed in an anoxic chamber (O2 level ~ 2 %) for 60 minutes, followed by a 60-minute reoxygenation in ambient air (O2 ~ 19.4 %). Unloaded cell shortening and Ca2+ concentration (Indo-1 dye) were measured using field stimulation (0.5 Hz, 22°C, 1.8 mM external Ca2+) before and after anoxia/reoxygenation (A/R). Sarcomere length (SL) and fluorescence (405 and 480 nm) were simultaneously recorded (IonOptix system, Hilton, USA). The A/R experiment was carried out in presence or not of SMT (0.5 µM) or in presence or not of a non specific antioxidant N-Acetylcystein (NAC, 20 µM). 

Biochemical assays 

Lactate dehydrogenase in coronary effluents

 Etude n°2 – 126 – Lactate dehydrogenase (LDH) activity was measured in coronary effluents from isolated hearts, by spectrophotometry using an LDH kit (LDH-P, BIOLABO SA, France). Measurements were made at 5, 10 and 15 min of reperfusion time. LDH activity was normalized to coronary blood flow. 

Nitrites/Nitrates in coronary effluents

 Nitrites/nitrates in coronary effluents from isolated hearts, used as an index of total NO production, were determined using a quantitative colorimetric assay kit based on the Griess method (QuantiChromTM Nitric oxide Assay Kit (DINO-250). Measurements were made at the end of stabilisation and at 5, 10 and 15 min of reperfusion. NO production was normalized to coronary blood flow. 

Immunohistological detection of iNOS expression 

On a third set of rats (n=7 per group), LV tissues sections of 5-µm-thick were removed and used for immunohistological study. Identification of iNOS protein was performed using a Rabbit polyclonal anti-iNOS (Santa-cruz). iNOS was quantified using colorimetric quantification (20 X objective).

Thioredoxin reductase activity

On a another set of rats (n=6 per group), hearts were freeze-clamped and the frozen ventricular tissue was homogenized in Tris–HCl buffer (Tris HCl 60 mM, diethyltriaminopentaacetic acid 1 mM, pH 7.4, 10 ml/g w.wt), using a Teflon Potter homogenizer. Tissue homogenates were then centrifuged (10 min at 20.000×g at 4°C). Activities of thioredoxin reductase (TrxR), reflecting the oxidant status, were measured using supernatants as previously described (Andre et al., 2010). Etude n°2 – 127 – 2.4.5. Immunoblotting detection of TNF-α expression TNF-α expression in LV tissues was evaluated using western immunoblotting. Proteins were separated using 15 % SDS-PAGE. Identification of TNF-α protein was performed using a goat polyclonal anti-TNF-α (Santa-cruz 1:1000) and were expressed relative to GAPDH content.