BASIC INVESTIGATION
Carbon Monoxide Has Direct Toxicity on the Myocardium Distinct from Effects of Hypoxia in an Ex Vivo Rat Heart Model Selim Suner, MD, MS, Gregory Jay, MD, PhD
Abstract Objectives: Carbon monoxide (CO) toxicity causes significant central nervous system and cardiac injury. Although the neurological damage caused by CO toxicity is extensively described, the mechanisms underlying myocardial insult are unclear. The authors used an externally perfused isolated rat heart model to examine the effects of a physiological saline solution (Krebs Henseleit HEPES, KHH) aerated with CO on cardiac function. Methods: Fifteen rats were equally divided into three groups: the control group (KHH + 100% O2), the nitrogen control group (KHH + 70% O2, 30% N2), and the CO group (KHH + 70% oxygen, 30% CO). Left ventricular peak systolic pressure (LVPsP), end diastolic pressure (LVEdP), and coronary perfusion pressure were measured while the isolated heart was paced and perfused on a modified Langendorf apparatus. Results: Left ventricular generated pressure (LVGP = LVPsP ) LVEdP) decreased in the nitrogen control and CO groups compared to the control group. There was higher LVGP in the recovery phase between the nitrogen control group compared to the CO group. Both groups had increased lactic acid levels in the experimental phase. Conclusions: Carbon monoxide with hypoxia and hypoxemic hypoxia both result in similar depression of cardiac function. Hearts poisoned with CO with hypoxia do not recover function to the extent that hearts rendered hypoxic with nitrogen do when perfused with 100% oxygen after the insult. This suggests that CO causes direct myocardial toxicity distinct from the effects of hypoxia. ACADEMIC EMERGENCY MEDICINE 2008; 15:59–65 ª 2008 by the Society for Academic Emergency Medicine Keywords: carbon monoxide, toxicity, cardiac, myocardium, poisoning, hypoxia
From the Department of Emergency Medicine (SS, GJ) and the Department of Surgery (SS), Warren Alpert Medical School of Brown University, Providence, RI; the Division of Engineering, Brown University (SS, GJ), Providence, RI; Rhode Island Hospital, Andrew F. Anderson Emergency Center (SS, GJ), Providence, RI. Received June 14, 2007; revisions received September 7 and September 14, 2007; accepted September 16, 2007. Address for correspondence and reprints: Selim Suner MD, MS; e-mail:
[email protected]. Presented at the 3rd Annual New England Regional Society for Academic Emergency Medicine Conference, Boston, MA, April 10, 1999 (abstract published as Suner S, Jay G, Raymond R. Myocardial depression in rat hearts perfused ex-vivo with carbon monoxide. Acad Emerg Med. 1999; 6:394). Awarded the Society for Academic Emergency Medicine 1999 Annual Meeting Presentation Award for best basic science poster. Suner S, Jay G, Raymond R. Myocardial Depression in Rat Hearts Perfused Ex-Vivo with Carbon Monoxide.
ª 2008 by the Society for Academic Emergency Medicine doi: 10.1111/j.1553-2712.2007.00012.x
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arbon monoxide (CO) is an odorless, clear, nonirritating gas that is produced whenever there is incomplete combustion of carbonaceous fossil fuels. Common exogenous sources of CO include tobacco smoke, carbon fuel engine exhaust, heating equipment, and fires. CO is also routinely produced as the heme molecule is processed enzymatically, but this is not a source of toxicity. In the United States, CO is one of the leading causes of morbidity and mortality from poisoning.1 In addition to its well-described neurological toxicity, clinically, CO poisoning may lead to myocardial ischemia, manifested by chest pain, electrocardiogram changes (ST-segment depression and T-wave depression), and myocardial infarction, and is associated with long-term morbidity and mortality.2,3 Previous animal studies have suggested that CO may produce direct toxicity to the myocardium rather than indirect hypoxic damage.4 Experimentally, CO has been shown to decrease regional myocardial segment work
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and increase coronary flow with increasing concentrations; however, the site and mechanism of CO myocardial toxicity has not been elucidated.5,6 A site of CO toxicity may be cytochrome oxidase at the level of the mitochondria. Binding of CO to cytochrome oxidase may interfere with cellular respiration even though the affinity of oxygen for cytochrome oxidase is much higher than that for CO.7–9 Another candidate site for CO action on the heart is on myoglobin. CO binding to myoglobin is enhanced by hypoxia in dogs.10 CO binding to myoglobin may account for the myocardial depression seen in animal studies.11 Recent clinical studies have demonstrated that morbidity and mortality from cardiac causes are related to an episode of moderate to severe CO poisoning.3 The objective of this laboratory study is to show that CO causes depression of cardiac performance using the left ventricular generated pressure (LVGP) as an indicator of function in the ex vivo isolated rat heart model and that this toxic effect on the heart is distinct from hypoxemic hypoxia. We define ‘‘hypoxemia’’ as a lower partial pressure of oxygen in our perfusion solution and ‘‘hypoxia’’ as diminished oxygen delivery to the heart, evident by increased lactic acid in the effluent. The null hypothesis is that LVGP is not differentially affected in rats perfused with a physiological buffer solution aerated with 30% CO, and with 30% nitrogen, compared to those perfused with 100% oxygen. METHODS Study Design This was an experimental protocol to investigate the effects of CO toxicity on isolated heart using a murine model. The study was approved by the Rhode Island Hospital Institutional Animal Care and Use Committee and complied with National Institutes of Health (NIH) and United States Department of Agriculture (USDA) standards and guidelines for animal welfare and for ethical animal research. Experiments were conducted in the Emergency Medicine Laboratory at Rhode Island Hospital.
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Hills, IL) via a cannula in the aorta. A balloon-tipped catheter made in our laboratory from latex and polyethylene tubing (PE-60, Instech Laboraties Inc., Plymouth Meeting, PA) filled with 0.9% normal saline solution, devoid of air bubbles, was inserted into the left ventricle via the mitral valve through a small opening in the left atrium. This balloon catheter was used to monitor left ventricular pressure via a pressure transducer (P23, Gould, Oxnard, CA) positioned at the level of the heart. A second transducer, connected to a side arm of the aortic perfusion catheter, was used to monitor coronary perfusion pressure. Left ventricular generated pressure (LVGP = LVPsP [left ventricular peak systolic pressure] ) LVEdP [left ventricular end diastolic pressure]) was used as the index of global myocardial performance. The atrioventricular node was crushed using fine forceps and the hearts were paced at a rate of 300 beats ⁄ min with an electrical stimulator (Grass SD9 Stimulator, Astromed Inc., West Warwick, RI). Square-wave electrical impulses of 8 ms duration, 300 Hz were delivered at a magnitude of two times the ventricular capture voltage (2–5 V) via a Teflon-coated platinum-iridium wire inserted in the left ventricular wall. A photographic depiction of the ex vivo rat heart preparation is shown in Figure 1. Left ventricular peak systolic pressure (LVPsP) and left ventricular end diastolic pressure (LVEdP) were extracted from the pressure tracing by identifying peak and troughs. Left ventricular generated pressure (LVGP) was calculated as LVPsP ) LVEdP (integrated over 1 min) and used as the index of cardiac function. All data were continuously acquired by a Biopac data acquisition system and Acknowledge software (MP100A Biopac Systems Inc., Santa Barbara, CA) starting from the time the heart was connected to the system until it stopped functioning. A modified Krebs Henseleit HEPES (KHH) buffer solution was used as the perfusate. This solution contains 139 mM NaCl, 4.7 mM KCl, 21 mM HEPES, 1.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 11 mM dextrose, and 0.5 mM EDTA in deionized water. After aeration with 100% oxygen for 30 minutes, this solution has an osmolarity of 300 mosm ⁄ L and a partial pressure of O2 (pO2) of 600–650 mm Hg. Temperature was regulated at 37C,
Animal Subjects Experiments were conducted at sea level on adult, male, Sprague-Dawley albino rats (N = 15) ranging in weight from 300 to 500 g. Rats were heparinized intraperitoneally with 2500 units of heparin sodium in 0.25 mL of saline and then, after 15 minutes, they were anesthetized with 150 mg ⁄ kg intraperitoneal sodium pentobarbital. Study Protocol The heart and 5 mm of attached proximal aorta were rapidly excised through a bilateral midaxillary thoracotomy and ‘‘clam-shell’’ exposure of the mediastinum, as soon as the animal was in a surgical plane of anesthesia (no response to painful tail stimulation). The excised heart was placed in an ice-cold (1C) physiological saline solution to induce cardiac arrest and mounted on a Langendorff isolated heart perfusion apparatus by the aorta. Coronary flow was maintained at a constant 10 mL ⁄ min with a peristaltic pump (Masterflex model 7518–10, Cole Palmer Instrument Company, Vernon
Figure 1. The excised rat heart attached to the ex vivo perfusion apparatus. The attachment of the heart to the device by the aorta, the left ventricular pressure transducer, and the pacing electrode are depicted.
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and the pH was adjusted to 7.4 with HCl as necessary. HEPES (N-2-hydroxyethylpiperazine-N¢-2-ethanesulfonic acid) is an organic zwitterionic buffer used to maintaining a physiological pH. It has better buffering characteristics for maintain pH between 7.2 and 7.6 compared to bicarbonate buffer systems. A second reason for choosing this buffer is that it does not require a CO2 environment. While the hearts regained function on the ex vivo perfusion apparatus and during a period of stabilization (5–20 min), they were perfused with KHH aerated with 100% oxygen. After the period of stabilization, the hearts were perfused again with KHH aerated with 100% oxygen for a 15-minute basal period. After the basal period, the solution was switched to KHH aerated with either 100% oxygen (control group), 70% oxygen and 30% nitrogen mixture (nitrogen control group), or 70% oxygen and 30% CO mixture (CO group) for 15 minutes, during the experimental period, and then again with 100% oxygen for 30 minutes during the recovery period (see Figure 2). A systematic randomization protocol was not used, and the investigators were not blinded to the experimental group. All KHH solutions were made fresh and aerated with the gas mixture continuously for 30 minutes to reach steady
state before perfusion into the heart. Gases were mixed using a precision flowmeter (N082–03, Cole Palmer Instrument Company). The flowmeter was precisely calibrated at the factory. A linear calibration regression model was developed from the calibration data provided by the manufacturer to precisely control the gas mixture (Flowmeter scale = 18.640 + 5.4690 · Flow rate; r = 0.99). The effluent was collected via a slit in the apical pole of the heart. Effluent pH, lactic acid, and pO2 were measured using STAT Profile 9 analyzer (Nova Biomedical, Waltham, MA) at intervals throughout the experiment. The measurements were conducted immediately after sample effluent collection was completed in the laboratory.
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Data Analysis Data were transferred to the Matlab computing platform (Version 7.1, Mathworks, Natick, MA), and statistical analysis consisted of analysis of variance (ANOVA) for repeated measures followed by Bonferroni post hoc test and Student’s t-test. The repeated-measures ANOVA model was created with the three study groups and variable measurement times including the basal, experimental, and recovery periods. Significance was set at
Figure 2. Raw pressure waveforms recorded continuously by the left ventricular transducer. The black graph (top) shows the pressure variations recorded in the left ventricle over time from a rat heart in the control group; the dark gray graph (middle), from a rat in the nitrogen control group, and the light gray graph (bottom), one from the CO group. The x-axis is time in minutes (90 min total), and the y-axis, left ventricular pressure in mmHg (peak to peak 160 mm Hg). The grayscale bars above each graph show different epochs in the experiment. Black depicts the stabilization period where the rat heart initially regains function and the pressure is stabilized. Dark gray depicts the basal period during which the heart is perfused with KHH solution aerated with 100% oxygen. Light gray represents the experimental period during which the control heart is perfused with KHH aerated with 100% O2, the nitrogen control perfused with KHH aerated with 30% nitrogen and 70% oxygen, and the CO group perfused with KHH aerated with 30% CO and 70% oxygen. Dark gray following light gray is the recovery phase where all hearts are perfused with 100% O2. CO = carbon monoxide; KHH = Krebs Henseleit HEPES. (Color version of this figure available online at http://www.aemj.org.)
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p < 0.05. The p-values were adjusted to account for the multiple comparisons made in the Bonferroni post hoc analysis. All plots were created in Matlab. RESULTS The mean (±SD) weight of the rats was 435± 42 g. The baseline LVGP ranged between 85 and 110 mm Hg. There was a statistically significant decrease in LVGP between the control group (n = 5) and CO group (n = 5; p < 0.0001), between the control group and nitrogen control group (n = 5; p < 0.0001), and also between the CO group and nitrogen control group (p = 0.037; Figure 3). Analysis revealed that the difference between the control group and CO group, and between the control group and nitrogen control group, was localized to the points between 17 and 34 minutes, corresponding to the experimental period. The difference between the CO group and the nitrogen control group was significant in the recovery period between 40 and 60 minutes and not in the basal or experimental periods. Likewise, the peak systolic pressure decreased significantly in the nitrogen control and CO groups during the experimental period compared to the control group. In contrast, the end diastolic pressure increased significantly during the experimental period in the nitrogen control and CO groups compared to the control group. While there was no statistically different change in the perfusion pressure, during the experimental period there was a trend toward a decrease in the nitrogen control and
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CO groups compared with the control group, suggesting a decrease in coronary vascular resistance. The heart rate did not show any significant changes in any group throughout the experiment (Figure 4). Lactic acid measured in the effluent increased during the experimental period and then returned to baseline level in the recovery period in the CO and nitrogen groups (Figure 5). Lactic acid measured in the effluent increased during the experimental period then returned to baseline level in the recovery period in the CO and nitrogen groups (Figure 5). There was a statistical difference in lactate during the experimental period between the 100% oxygen and the nitrogen group and between the 100% oxygen and the CO group (p = 0.0086). There was no statistical difference in lactate between the nitrogen control group and the CO group, although there was a trend toward increased lactate in the CO group during the experimental and recovery periods. Lactate was not statistically different in any group during the basal and recovery periods. The pO2 decreased during the experimental period and then returned to baseline in the nitrogen control and CO groups. There was no statistically significant difference in pO2 between the CO, nitrogen control, or control groups, although a trend toward decreased pO2 was observed in the CO and nitrogen control groups compared to the control group. There was also no variation in pH between the three groups across the study period. Overall, pH remained relatively constant across all three groups (Figure 5).
Figure 3. Left ventricular generated pressure (LVGP) as a function of time. The x-axis depicts time in minutes, and the y-axis, LVGP in mm Hg. (,) Control group; (s) nitrogen control; (h) CO. Error bars show standard deviation. The vertical dotted lines show the onset and conclusion in time of the experimental period. The study epochs are shown with the gray coded bar. The gray representations are the same as in Figure 2. (Color version of this figure available online at http://www.aemj.org.)
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Figure 4. LVPsP, EDP, and PP as a function of time. (Top left) LVPsP as a function of time; (top right) LVEdP; (bottom left) perfusion pressure; and (bottom right) heart rate. x-axis is time in minutes. The y-axis is pressure in mm Hg for the first three graphs and beats per minute for heart rate. The line and symbol designations are the same as in Figure 3. LVPsP = left ventricular peak systolic pressure; EDP = end diastolic pressure; PP = perfusion pressure.
Figure 5. pH (top), pO2 (middle), and lactic acid (bottom) as a function of time for each experimental group. Error bars show standard deviation and the epochs are delineated with the bars as in previous figures. The x-axes depict time in minutes, and the y-axes, pH, pO2 in mm Hg, and lactic acid in millimoles per liter. The line and symbol designations are the same as in Figure 3.
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DISCUSSION We have demonstrated that CO depressed cardiac function distinct from hypoxemia and hypoxia in the isolated, perfused, and paced ex vivo rat heart. We used this model to test our hypothesis because systemic and humoral substances and variables that may affect cardiac function in the intact organism are eliminated. Therefore, we were able to determine the direct effects of CO on cardiac function, hemodynamics, and metabolism. Since there was no hemoglobin in our preparation, the classical mechanism of toxicity of CO mediated by hemoglobin binding (carboxyhemoglobin) is also eliminated as the cause for cardiac depression. Binding of CO to myoglobin, cytochrome oxidase, or other intracellular enzymes is more likely the mechanism underlying the effects observed in our study. We chose to use LVGP because prior studies evaluating the cardiac hemodynamics using similar models also used this or similar measures (pulse pressure, mean arterial pressure).4,6,12,13 Unlike some previous studies using this model to elucidate the effects of CO on the myocardium, in which 95% CO and 5% CO2 were used,4 we employed a mixture of CO and oxygen, which better simulates conditions of CO poisoning where the environmental conditions contain both gases. We tested various mixtures of CO and oxygen and determined that with 30% (v ⁄ v) CO in oxygen, the ex vivo rat heart preparation showed a significant depression and was able to recover function. With higher CO concentrations, while there was significant depression, the rat heart rarely recovered function for any appreciable time. In some previous experiments, conducted paced hearts did not recover function after 10 minutes of perfusion with 95% CO and 5% CO2; therefore, the delayed effects seen with CO compared to nitrogen control shown in our experiments could not have been appreciated.4 Although some investigators used very low CO concentrations in their experiments (0.01%–0.05%), these studies were designed to detect biochemical changes related to oxidative stress and did not measure hemodynamic variables other than heart rate. Although one study quotes ‘‘heart failure’’ with CO concentrations as low as 0.2%, it is not clear from the data what this represents in terms of hemodynamic variables, since this observation was from unpublished data.14,15 We used a paced heart preparation because we wanted to eliminate chronotropic effects as a variable. There has been a suggestion that CO may have a specific effect on the cardiac conduction system. Previous experiments demonstrated that CO can decrease electrical activity in the isolated rat heart16 and that CO interferes with electrical conduction in cerebellar Purkinje cells evaluated in cell culture.17 Our results show that the end diastolic pressure increased significantly during the experimental period in both the nitrogen control and the CO groups compared to control. This increase may indicate decreased myocardial compliance as a result of hypoxic injury.18 Since the coronary effluent flow was held constant at 10 mL ⁄ min, any changes in perfusion are a reflection of coronary vascular resistance. The perfusion pressure
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showed a decreasing trend during the experimental period in both the nitrogen control and the CO groups, indicating diminishing coronary vascular resistance. Studies have demonstrated that CO can inhibit vascular smooth muscle contractility, leading to decreased vascular resistance.19 This effect may be similar to nitrous oxide (NO), another small molecule that also decreases vascular smooth muscle contractility. It is not clear whether CO mediates this effect directly or indirectly through NO production. At least one study suggests that CO-related increases in coronary perfusion pressure are related to both endothelium-dependent and -independent mechanisms, suggesting more than one mechanism of action for CO.20 The trend toward decreased pO2 during the basal phase in the CO and nitrogen control groups cannot be explained. However, statistically the difference in pO2 was not significant and the pO2 decreased during the experimental phase and returned to baseline levels in the recovery in these groups (Figure 5). Blunted recovery in LVGP after correction of hypoxemia in the CO group compared to nitrogen controls support the notion that CO causes an interruption in cellular respiration that persists after hypoxemia has been corrected. LIMITATIONS Although there are many case reports and studies showing cardiac specific toxicity with CO poisoning in humans, extrapolation of similar findings obtained from animal studies to humans is limited. All rat hearts were electrically paced; therefore, we were not able to determine if CO has any direct chronotropic effects on the myocardium. Our gas mixtures did not contain any CO2, which differs from most environmental conditions causing CO toxicity, where CO, O2, and CO2 are all present. We do not believe that the lack of CO2 significantly affected the parameters measured. We used a buffering system that works independent of CO2, and measurement of effluent pH shows that this parameter was constant in all groups. CONCLUSIONS Carbon monoxide and hypoxemic hypoxia induced by nitrogen cause similar depression in myocardial function in the paced, ex vivo rat heart; however, the depression in myocardial function recovered differentially after exposure to CO compared to the depression seen with nitrogen control, suggesting a mechanism of direct myocardial CO toxicity distinct from hypoxia. References 1. McBay AJ. Carbon monoxide poisoning. N Engl J Med. 1965; 272:252–3. 2. Anderson R, Allensworth DC, De Groot WJ. Myocardial toxicity from carbon monoxide poisoning. Ann Intern Med. 1967; 67:1172–82. 3. Henry CR, Satron D, Lindgren B, et al. Myocardial injury and long-term mortality following moderate to severe carbon monoxide poisoning. JAMA. 2006; 295:398–402.
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4. Chen KC, McGrath JJ. Response of the isolated heart to carbon monoxide and nitrogen anoxia. Toxicol Appl Pharmacol. 1985; 81(3 Pt 1):363–70. 5. Zhu N, Weiss HR. Effect of hypoxic and carbon monoxide induced hypoxia on regional myocardial segment work and O2 consumption. Res Exp Med (Berl). 1994; 194:97–107. 6. McGrath JJ. The effect of carbon monoxide on the heart: an in vitro study. Pharmachol Biochem Behav. 1984; 21(Suppl 1):99–102. 7. Chance BC, Erecinska M, Wagner M. Mitochondrial responses to carbon monoxide. Ann N Y Acad Sci. 1970; 174:193–203. 8. Ball EG, Strittmatter CF, Cooper O. The reaction of cytochrome oxidase with carbon monoxide. J Biol Chem. 1951; 193:635–47. 9. Caugher WS. Carbon monoxide bonding in hemproteins. Ann N Y Acad Sci. 1970; 174:148–53. 10. Coburn RF, Mayers LB. Myoglobin oxygen tension determined from measurements of carboxyhemoglobin in skeletal muscle. Am J Physiol. 1971; 220:66–74. 11. Cramlet SH, Erickson HH, Gorman HA. Ventricular function following carbon monoxide exposure. J Appl Physiol. 1975; 39:482–6. 12. McGrath JJ, Smith DL. Response of rat coronary circulation to carbon monoxide and nitrogen hypoxia. Proc Soc Exp Biol Med. 1984; 177(1):132–6. 13. Kukoba TV, Moibenko OO, Kotsioruba AV. Cardioprotective effect of heme oxygenase-1 induc-
tion by hemin on the isolated rat heart during ischemia-reperfusion. Fiziol Zh (Russ). 2003; 49(6): 14–21. Patel AP, Moody AJ, Sneyd JR, Handy RD. Carbon monoxide exposure in rat heart: evidence for two modes of toxicity. Biochem Biophys Res Commun. 2004; 321:241–6. Patel AP, Moody AJ, Handy RD, Sneyd JR. Carbon monoxide exposure in rat heart: glutathione depletion is prevented by antioxidants. Biochem Biophys Res Commun. 2003; 302:392–6. Wenzel DG, Brenner GM. Carbon monoxide and cultured rat hearts. Toxicol Appl Pharmacol. 1973; 24:256–65. Raybourn MS, Cork C, Shimmerling W, Tobias CA. An in vitro electrophysiological assessment of the direct cellular toxicity of carbon monoxide. Toxicol Appl Pharmacol. 1978; 46:769–79. Levine FH, Copeland JG, Melvin DB, Stinson FB. Extended evaluation of effects of anoxia on ventricular performance and compliance. Ann Thorac Surg. 1980; 29(1):42–8. Duke HN, Killick EM. Pulmonary vasomotor responses of the isolated perfused cat lungs to anoxia. J Physiol. 1952; 117:303–16. Favory R, Lancel S, Tissier S, Mathieu D, Decoster B, Naviere R. Myocardial dysfunction and potential cardiac hypoxia in rats induced by carbon monoxide inhalation. Am J Respir Crit Care Med. 2006; 174(3):320–5.
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