Abstract
Background: Results from both animal and human being studies provide evidence that inhalation of concentrations of carbon monoxide (CO) at around 100 ppm has antiinflammatory effects. These low levels of CO are incriminated in ischemic heart diseases experienced by cigarette smokers and, in some cases, from air pollution. Although neurologic mechanisms have been investigated, the effects of CO on cardiovascular function are still poorly understood.
Methods and Results: The effects of CO (250 ppm; 90 min) inhalation on myocardial function were investigated in isolated heart of rats killed immediately, and 3, 24, 48, and 96 h after CO exposure. CO exposure at 250 ppm resulted in an arterial carboxyhemoglobin (HbCO) level of approximately 11%, which was not associated with changes in mean arterial pressure and heart rate. CO exposure induced coronary perfusion pressure increases, which were associated with endothelium-dependent and -independent vascular relaxation abnormalities. CO-induced coronary vascular relaxation perturbations were observed in the presence of increased heart contractility. Spontaneous peak to maximal Ca2+-activated left ventricular pressure ratio was markedly increased in CO-exposed rats, indicating increases in myofilament calcium sensitivity. Heart cyclic guanosine monophosphate/cAMP ratio and myocardial permeabilized fiber respiration (complex intravenous activity) were reduced in CO-exposed rats, which lasted after 48 h of reoxygenation in air.
Conclusions: These findings suggest that CO deteriorates heart oxygen supply to utilization and potentially may induce myocardial hypoxia through mechanisms that include increased oxygen demand due to increased contractility, reduced coronary blood flow reserve, and cardiomyocyte respiration inhibition.
In the last few years, a wide number of studies have suggested that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. Results from both animal and human being studies provide evidence that inhalation of concentrations of CO at around hundred(s) parts per million (ppm) have antiinflammatory effects. CO exposure, even at low concentrations, is associated with organ damage and its specific toxic effects have been of interest for many years in both human and animal studies (1, 2). Neurologic manifestations that result from CO poisoning include headache and fatigue, whereas more advanced symptoms consist of dizziness with lethargy, coma, seizures, and death (3). Low-level exposure to CO also causes neuropsychologic impairments in humans (4, 5). For example, in volunteers exposed at low levels of CO (17–100 ppm), a battery of neuropsychologic tests has clearly shown deterioration in memory and cognitive functions (5).
Also, exposure to CO at low concentrations experienced by cigarette smokers and during air pollution has been recognized as an element responsible for ischemic heart diseases (6, 7). Although the mechanisms of neurotoxicity of CO have been extensively studied (8, 9), information about myocardial consequences of CO exposure in humans are limited to case reports of ECG changes, myocardial infarction, and heart failure in the context of CO poisoning (10–14). In experimental models, even though CO cardiac toxicity has been described in the past (15, 16), precise CO effects on the heart are largely unknown and conclusions of previous studies are frequently contradictory (11). Indeed, experimental studies reporting acute effects of CO on myocardial performance and metabolism have yielded contradictory results depending on animal species and protocol of CO exposure (15–19).
Thus, the major objective of this study was to better understand the mechanisms of heart dysfunction induced by CO inhalation in rats. Using an acute and moderated (250 ppm) CO-poisoning model in conscious rats, we tested whether CO would impair myocardial function evaluated by the means of isolated heart preparation, coronary vasodilator response, calcium sensitivity, mitochondrial respiration, and cyclic guanosine monophosphate (cGMP)/cAMP ratio measurements.
The investigation conforms to the “Guide for the Care and Use of Laboratory Animals” (20). This study was performed in accordance with European Institutes of Health guidelines for the use of experimental animals, with approval from Lille's Animal Research Committee. Rats (250–300 g; Harlan, Gannat, France) were housed in a controlled environment and provided with standard rodent chow and free tap water.
After intramuscular anesthesia (ketamine, 50; xylazine, 15 mg/kg body weight), rats were placed supine on a surgical board while breathing air. The internal carotid artery was catheterized under sterile conditions, and the line was tunneled subcutaneously to the back of the neck and attached to a swivel device. Arterial pressure and cardiac rate was monitored continuously (Biopac Data Acquisition System; Biopac Systems, Inc., Goleta, CA).
Instrumented and noninstrumented conscious rats were placed in a 12-L airtight metabolic container. CO in air (250 ppm) was administrated at a constant flow for 90 min and monitored with an aspirative CO analyzer (Binos I type; Leyhold Berhaus, Saint-Quentin Fallavier, France). Reoxygenation in ambient air was then allowed. Control rats were prepared using the same protocol, except no CO in air was administered. Rats were prepared for either functional or biological studies at indicated times. At the time of killing, arterial blood was withdrawn by abdominal aortic punction for carboxyhemoglobin (HbCO) measurements (ABL 520; Radiometer Medical, Copenhagen, Denmark) under deep anesthesia (pentobarbital 50 mg/kg body weight, intramuscular injection).
Briefly, after heparinization and ether anesthesia, the heart was rapidly excised and placed into ice-cold Krebs-Henseleit buffer solution (21). Then, the heart was mounted onto a Langendorff heart perfusion apparatus (IH-5; Harvard Apparatus, Les Ulis, France) and perfused in a retrograde fashion via the aorta at a constant flow rate of 10 ml/min with aerated (95% O2, 5% CO2) Krebs-Henseleit buffer. The final buffer pH was 7.37–7.42, partial pressure of O2 was 550–600 mm Hg, partial pressure of CO2 was 40 mm Hg, and temperature was maintained at 37°C. Cardiac contractile function was assessed using a water-filled latex balloon inserted into the left ventricular (LV) cavity and connected to a pressure transducer (Accuflush; Baxter-Edwards Critical Care, Irvine, CA). The heart was paced at 300 beats/min and allowed to equilibrate for 30 min. LV end-diastolic pressure was adjusted to 5 mm Hg by increasing the balloon volume with a micrometer-filled syringe. LV developed pressure (LVDP), its first derivatives (maximal change in pressure over time (dP/dtmax), and coronary perfusion pressure were recorded using a Biopack Data Acquisition System (Biopack Systems).
After 10 min of perfusion with Krebs solution (in mM: NaCl, 118; KCl, 4.5; CaCl2, 1.4; NaHCO3, 25; MgSO4, 1.2; NaH2PO4, 1.4; glucose, 11), hearts were perfused with Krebs solution containing 3.2 mM KCl to increase coronary vascular tone, thus allowing vasodilator responses to be observed, as previously described (22). Hearts were perfused at a constant 10-ml/min flow and coronary perfusion pressure monitored by a pressure transducer. Vasodilatation was expressed in percentage of maximal change in coronary perfusion pressure before and after perfusion of vasodilators. Dose responses to endothelium-dependent and -independent vasodilators were assessed by bolus perfusion of Krebs (30 μl via a side port aortic cannula) containing different concentrations of either freshly prepared acetylcholine (10−9 to 10−4 M) or sodium nitroprusside (10−13 to 10−7 M). Vasodilators were successively administered when the perfusion pressure had returned to predose value.
Briefly, isolated heart was perfused for 60 min with 30°C Krebs containing 5 μM ryanodine (23, 24). Spontaneous ventricular activity (peak LV systolic pressure) was observed and “plateau” LV systolic pressure was obtained by the mean of tetanic stimulation (50 ms, 10 Hz, for 5 s). Intact-heart maximal Ca2+-activated force was determined at 0.5, 2, 5, 10 and 15 mM extracellular Ca2+ concentration. In this setting, spontaneous peak to maximal Ca2+-activated LV pressure ratio is indicative of myofilament calcium sensitivity (23, 24).
cGMP and cAMP concentrations in heart tissue were determined by using cGMP and cAMP EIA kits (Cayman Chemical, Ann Arbor, MI). Briefly, frozen hearts were homogenized in deionized water containing 5% trichloroacetic acid. After a centrifugation at 1,500 g, trichloroacetic acid contained in the supernatant was extracted using ether-saturated water. By heating samples at 70°C for 5 min, ether was removed. The resultant solution was used to perform the assay, according to the manufacturer's instructions.
After excision, hearts were rinsed and placed in the relaxing and biopsy preservation solution BIOPS containing (in mM) the following: CaK2 ethyleneglycol-bis-(β-aminoethyl ether)-N-N′-tetraacetic acid (EGTA), 2.77; K2 EGTA, 7.2 (free calcium concentration 0.1 μM); Na2 ATP, 5.7; MgCl2 · 6 H2O, 6.6; taurine, 20; Na2 phosphocreatine, 15; imidazole, 20; dithiothreitol, 0.5; 2-[N-morpholino]ethanesulfonic acid (MES), 50; pH 7.1, as previously described (25). Isolated fibers were permeabilized for 30 min with 50 μg/ml of saponin in BIOPS. Then, fibers were washed three times in respiration medium, Mitomed2 (MitoMedium, Oroboros Instruments, Innsbruck, Austria) (see below). All procedures were performed at 4°C. Respiration was then measured at 25°C in a two-chamber respirometer (Oxygraph-2k; Oroboros Instruments). Briefly, 5 to 10 mg of fiber bundles were placed in a chamber containing 2 ml of Mitomed2 (in mM: Na2 EDTA, 0.5; MgCl2 · 6 H2O, 5; KH2PO4, 10; mannitol, 110; KCl, 60; Tris, 60; pH 7.4). The O2 solubility of this medium was taken as 11.3 μM/kPa. In the presence of ADP (1 mM) and antimycine A (5 μM), complex IV respiration was stimulated by adding ascorbate (2 mM) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (0.5 mM). Data acquisition and analysis were performed with Datlab software (Oroboros).
In instrumented, conscious rats, β-adrenergic blockade was achieved by the means of propranolol intraarterial infusion (total dose of 1 mg/kg body weight in 30 min) during 250-ppm CO exposure. In ex vivo heart, β-adrenergic blockade was achieved by the means of propranolol added to the perfusate at a final concentration of 10 μM.
For biological and cardiac function studies, we tested for differences using an analysis of variance procedure (SPSS for Windows 9.0; SPSS France, Paris, France). When a significant difference was found, we identified specific differences between groups using a sequentially rejective Bonferroni procedure. After application of a Bonferroni correction, significance was achieved, with p < 0.05 for comparisons with control. Data are presented as means ± SEM throughout.
In conscious rats (n = 12 in each group), CO exposure (250 ppm in air; 90 min) resulted in an HbCO level of approximately 11% (Figure 1). There were no changes in mean arterial pressure and heart rate during CO exposure (data not shown). Arterial HbCO then decreased during reoxygenation in air at 3, 24, 48, and 96 h (Figure 1).
Figure 1. Carboxyhemoglobin (HbCO) in arterial blood is expressed in percentages in control rats and in CO-treated rats (250 ppm; 90 min) immediately, and 3, 24, 48, and 96 h after CO exposure. Results are presented as mean ± SEM (n = 12/group). *p < 0.05 versus control rats.
[More] [Minimize]Myocardial function of 250-ppm-CO–exposed rats was evaluated immediately, and 3, 24, 48, and 96 h after CO exposure (n = 12 in each group). CO induced increases in coronary perfusion pressure (Figure 2A) and LVDP first derivative dP/dtmax (Figure 2B), which lasted after 48 h of reoxygenation in air (Figure 2).
Figure 2. Coronary perfusion pressure (A) and left ventricular developed pressure first derivative dPdtmax (B) in control rats and in CO-treated rats (250 ppm; 90 min) immediately, and 3, 24, 48, and 96 h after CO exposure are displayed. Results are presented as mean ± SEM (n = 12/group). *p < 0.05 versus control rats.
[More] [Minimize]CO induced decrease in heart cGMP/cAMP ratio (n = 8 in each group), which lasted after 48 h of reoxygenation in air (Figure 3). High-resolution respirometry analysis indicated that myocardial permeabilized fiber respiration was reduced in 250-ppm-CO–exposed rats with complex IV inhibition (in pmol · s−1 · mg−1: 3,250 ± 250 in control animals and 2,000 ± 120 [p < 0.05 vs. controls], 2,150 ± 200 [p < 0.05 vs. controls], 2,250 ± 190 [p < 0.05 vs. controls], 3,000 ± 280, 3,500 ± 155 after 3, 24, 48, and 96 h after CO exposure, respectively; n = 6 in each group).
Figure 3. Heart cGMP/cAMP ratio in control rats and in CO-treated rats (250 ppm; 90 min) immediately, and 3, 24, 48, and 96 h after CO exposure is displayed. Results are presented as mean ± SEM (n = 8/group). *p < 0.05 versus control rats.
[More] [Minimize]In rats exposed to 250 ppm CO and reoxygenated in air for 3 h, independent series of experiments were conducted to evaluate the effects of in vivo and ex vivo β-adrenergic blockade on coronary hemodynamics and LV systolic performance. Neither in vivo rat treatment with propranolol (n = 6; 1 mg/kg total infused dose) nor ex vivo propranolol treatment (n = 8; 10 μM perfusate final concentration) altered coronary perfusion pressure and LVDP first derivative dP/dtmax (Figures 4A and 4B).
Figure 4. Effects of β-adrenergic blockade on coronary hemodynamics and left ventricular (LV) function. Coronary perfusion pressure (A) and LV developed pressure first derivative dPdtmax (B) in control rats and in CO-treated rats (250 ppm; 90 min) isolated 3 h after CO exposure are displayed. Results are presented as mean ± SEM (n = 8/group). *p < 0.05 versus control rats.
[More] [Minimize]In rats exposed to 250 ppm CO and reoxygenated in air for 3 h, alterations in coronary vasodilator responses and changes in intact-heart maximal Ca2+-activated force were further studied. In these experiments (n = 8 in each group), CO exposure resulted in total inhibition of vasodilator responses to acetylcholine (Figure 5A), whereas nitroprusside vasodilator responses were significantly reduced (Figure 5B). Intact-heart maximal Ca2+-activated force was determined in isolated perfused hearts (n = 8 in each group) by measuring isovolumic LV pressure during tetani by rapid pacing after exposure to ryanodine at 0.5, 2, 5, 10, and 15 mM extracellular Ca2+ concentration (Figure 5C). In 250-ppm-CO–exposure rats, spontaneous peak to maximal Ca2+-activated LV pressure ratio was shifted upward in the direction of myofilament calcium sensitivity increase.
Figure 5. Coronary vasodilator response and heart calcium sensitivity. Percentages of change in coronary perfusion pressure with increasing doses of acetylcholine (A) and sodium nitroprussiate (B) and spontaneous peak to maximal Ca2+-activated LV developed pressure (LVDP) ratio (C) in control rats and in CO-treated rats (250 ppm; 90 min) isolated 3 h after CO exposure are displayed. Results are presented as mean ± SEM (n = 8/group). *p < 0.05 versus control rats.
[More] [Minimize]The present study shows that CO promotes major abnormalities in coronary vascular relaxation, myocardial contractility, and mitochondrial respiration. New findings of the present study are twofold. First, in conscious rats, acute CO exposure (250 ppm) induced endothelium-dependent and -independent vascular relaxation abnormalities, which were associated with decrease in cardiac cGMP/cAMP ratio. Second, perturbations in coronary vascular relaxation were observed in the presence of heart contractility increase and cardiac mitochondrial respiration inhibition. Together, these findings support the contention that CO deteriorates heart oxygen supply-to-utilization with potential myocardial hypoxia via mechanisms including increased oxygen demand due to increased contractility, reduced coronary bloodflow reserve, and cardiomyocyte respiration inhibition.
CO is commonly characterized as a vasodilatory regulator of vascular tone, inasmuch as CO has been shown to relax vascular smooth muscle and to promote dilation of different vascular beds (26, 27). Studies in vitro have shown that CO has vasodilatory effects through three possible mechanisms: increased guanylate cyclase activities and cGMP intracellular levels (26), activation of high-conductance calcium-activated K channels (28), and inhibition of the P-450 system, which may impair the formation of vasoconstrictor substances (29). Despite the popular characterization of CO as a vasodilator, evidence exists suggesting that CO may also exert a vasoconstrictive influence on vascular tone. For example, recent reports suggest that physiologic concentrations of CO can suppress endothelial NO synthase activity and exerts endothelium-dependent vasoconstrictive influence in isolated arteries (30–32). Vasoactive properties of CO and NO are also ascribed to their respective affinity and binding mechanisms to guanylate cyclase. CO affinity for cyclases is far more than that of NO but binding of NO to prosthetic heme results in a 100-fold increase in cGMP generation, whereas the potency of CO is far less than that of NO. Considering such discrepancies, in vivo studies have shown that CO, which competes with NO on soluble guanylate cyclase, may function as a partial agonist/antagonist for the enzyme (33). Such a possibility is supported by previous results showing that CO promotes increase in cardiovascular tone and reduction in cGMP contents (34). Consistently, our present study shows that CO exposure reduced endothelium-dependent and -independent vasorelaxation and heart cGMP/cAMP ratio. Together, our results suggest that perturbations in coronary tone might be deleterious in limiting coronary hyperemia in response to anemic hypoxia induced by CO.
Myocardial dysfunction from CO exposure results from tissue hypoxia as well as damage at the cellular level. In vitro, CO binds to cytochrome-c oxidase of the electron transport chain, resulting in asphyxiation at the cellular level (1–3). Moreover, heart toxicity of CO, which can occur at extremely low levels of hemoglobin binding, has been attributed to cellular deleterious effects, including cytochrome interactions and generation of oxygen and nitrogen reactive species (3, 35, 36). Consistent with numerous studies, our results suggest that CO promotes partial and reversible inhibition of mitochondrial respiration, which lasted after 48 h of reoxygenation in air. Even though numerous studies have evaluated the effects of CO on cardiovascular function, findings are frequently contradictory (11, 15–18). For example, heart rate has been reported to increase or stay the same, whereas mean arterial pressure has been found to stay the same and decrease (11, 15). On the other hand, cardiac output is generally reported to increase, although stroke volume has been found to increase, stay the same, or decrease (11, 16). Because physiologic evaluation of global cardiac function in CO exposure models has been challenging due to preload and afterload alterations, we assessed myocardial function in ex vivo isolated heart preparation. Mainly, our results suggest that CO induces increases in LV contractility, which was not prevented by in vivo and ex vivo β-adrenergic blockade. Our findings are consistent with the one other study that evaluated heart function by the mean of isolated heart preparation and that reported myocardial contractility increases after CO exposure (37).
Mechanisms of CO-induced increases in LV contractility were further investigated by measuring intact-heart maximal Ca2+-activated force during twitch and tetanus stimulation after ryanodine incubation (23). Spontaneous peak to maximal Ca2+-activated LV pressure ratio and shift to the left of maximal Ca2+-activated force curve were markedly increased in 250-ppm-CO–exposure rats when compared with control subjects. Together, these results indicate that changes in myocardial contractility may be related to myofilament calcium sensitivity increase (23, 37). The reason why CO exposure may increase myofilament calcium sensitivity is not readily apparent. Calcium sensitivity of the contractile apparatus, however, is known to be modulated under various conditions, such as acidosis, accumulation of intracellular inorganic phosphate, and cGMP/cAMP-dependent phosphorylation of cardiac sarcomeric proteins (22, 23, 38, 39). It can be thus hypothesized that, in our study, changes in cGMP/cAMP ratio induced by CO may have influenced, at least in part, calcium myofilament responsiveness.
In conclusion, the present study shows that CO promotes major abnormalities in coronary vascular relaxation, myocardial contractility, and mitochondrial respiration. Our results further suggest that CO exposure may induce heart hypoxia through mechanisms including increased heart oxygen demand, reduced coronary bloodflow reserve, and heart cell respiration inhibition.
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