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Carbon monoxide improves cardiac energetics and safeguards the heart during reperfusion after cardiopulmonary bypass in pigs

MARIALUISA LAVITRANO^*,1, RYSZARD T. SMOLENSKI, ANTONINO MUSUMECI, MASSIMO MACCHERINI, EWA SLOMINSKA^||, ERNESTO DI FLORIO^**, ADELE BRACCO^**, ANTONIO MANCINI^**, GIORGIO STASSI, MARIELLA PATTI, ROBERTO GIOVANNONI^*, ALBERTO FROIO^*, FELICETTA SIMEONE, MONICA FORNI, MARIA LAURA BACCI, GIUSEPPE D’ALISE, EMANUELE COZZI^*,, LEO E. OTTERBEIN^¶, MAGDI H. YACOUB, FRITZ H. BACH^¶¶ and FULVIO CALISE^**

^* Department Medicina Sperimentale Ambientale e Biotecnologie Mediche, University of Milano-Bicocca, Italy;
Heart Science Centre, Imperial College at Harefield Hospital, UK;
Department Cardiac Surgery, University of Naples Federico II, Italy;
Department of Cardiothoracic Surgery, University of Siena, Italy;
^|| Department of Biochemistry, Medical University of Gdansk, Poland;
^** A.O.R.N. Cardarelli Hospital, Naples, Italy;
Department of Surgical and Oncological Sciences, School of Medicine, University of Palermo, Italy;
Department Morfofisiologia e Produzioni Animali, University of Bologna, Bologna, Italy;
Azienda Ospedaliera di Padova, Padua, Italy;
^¶ Department of Pulmonary and Critical Care Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania, USA; and
^¶¶ Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

¹Correspondence: Department Medicina Sperimentale Ambientale e Biotecnologie Mediche, University of Milano-Bicocca, Via Cadore, 48, 20052 Monza (Milano), Italy. E-mail: marialuisa.lavitrano{at}unimib.it

SPECIFIC AIMS

Ischemia-reperfusion injury (IRI) associated with cardioplegia, mimicking cardiopulmonary bypass and hypothermic cardiac arrest as may occur during cardiac surgery for aortic coronary grafting or valve replacement, leads to decreased efficiency of ATP generation, to cardiac edema and cardiomyocyte apoptosis, and to difficulty in restarting the heart beat. We tested whether pretreatment for 2 h with 250 ppm carbon monoxide (CO) would improve these parameters in an IRI model in which the pigs were put on cardiopulmonary bypass for 2 h followed by 1 h of reperfusion.

PRINCIPAL FINDINGS

1. Improvement in energetics with CO pretreatment
ATP concentrations were significantly elevated in CO-treated compared with control hearts at the end of reperfusion (P<0.05), which correlated with decreased ADP and AMP levels measured in the same samples. The ATP/ADP ratio, a parameter relating to energy status of the heart, was also elevated (P<0.05). The phosphocreatine (PCr) and GTP changes generally followed that of ATP, which is consistent with the common view that nucleotide triphosphate pools and their ratios remain in equilibrium.

2. Improvement in the redox status of pyridine nucleotides in the heart
There were no differences in concentrations of reduced or oxidized pyridine nucleotides at the end of CO pretreatment. At the end of ischemia, however, NADH increased to a larger extent in control pigs whereas NAD decreased both in control and in CO-treated pigs, leading to an elevated NADH/NAD ratio in controls compared with CO-treated hearts (P<0.05). There were no differences in NADH or NAD concentrations at the end of reperfusion.

Suppression of reduced pyridine nucleotide accumulation during cardioplegic arrest was even more pronounced in the NADP pool. NADPH accumulation was decreased by CO treatment while NADP remained unaffected during ischemia in CO-pretreated hearts. In contrast, NADPH accumulated and NADP concentrations decreased at the end of cardioplegic arrest in control pigs (P<0.05). NADPH/NADP ratio was higher in control than in CO-treated pigs at the end of ischemia (P<0.05).

3. Decrease of edema and apoptosis in the heart
Pretreatment with CO resulted in a marked decrease in cardiac edema and a highly significant decrease in apoptosis of cardiomyocytes. Biopsies from control animals showed far more interstitial edema than those from CO-treated animals (Fig. 1 ). The median percentage of edema in control tissues was 19.4% (mean 20.0%±5.6; range 13.1% to 28.6%); the median percentage of edema in tissues from CO-treated pigs was 9.0% (mean 9.7%±3.6; range 6.3% to 14.5%) (P=0.006).

View larger version (58K): [in this window] [in a new window]   Figure 1. Microscopic examination of edema. Histochemical analysis of edema in control pigs (abundant edema, A) and CO-treated pigs (reduced edema, B). Amount of edema was measured by computer-assisted analysis; yellow highlights the percentile of pixel below the value of 200. Percentage of predominantly white pixels (PWP) was calculated as 100% minus % of pixel below 200 (A: PWP=100–79.45=20.15%; B: PWP=100–98.22=1.78%). Percentage of edema in control and in CO-treated pigs based on computer-assisted analysis (C).

Similarly, the number of cardiomyocytes undergoing apoptosis was significantly greater in control pigs than in CO-treated animals (2±0.7 apoptotic nuclei/0.033 mm² vs. 0.5±0.3 apoptotic nuclei/0.033 mm², respectively, P<0.01).

4. Restarting the heart
Hearts of pigs not pretreated with CO required significantly more defibrillations to restart the heart. Considering the CO-treated group, the mean number of defibrillations required to restart the heart after cardioplegia (ischemia) was 2 (SD: 1.53, median: 1) whereas in the control group the mean was 6 (SD: 3.66, median: 7.5) (P<0.05).

5. Effect of CO on clinical parameters
All clinical parameters remained within normal ranges for the duration of the experiments: no differences were observed in acid base balance, electrolytes, cardiocirculatory function, cardiac output, and hematocrit. Blood oxygenation and carboxyhemoglobin were significantly different in CO-treated animals (P<0.05).

CONCLUSIONS AND SIGNIFICANCE

We tested whether CO had effects on ATP, ADP, AMP, and GTP levels before ischemia, after ischemia, and after reperfusion. Our results show that treatment with CO resulted in higher levels of ATP present after ischemia, consistent with higher levels of GTP as well. Thus, CO was better able to maintain efficient energy generation in hearts after ischemia, which correlates with the greater ease with which these hearts were restarted with defibrillation (Fig. 2 ).

View larger version (35K): [in this window] [in a new window]   Figure 2. Schematic diagram.

Phosphocreatine, a high-energy phosphate reserve in the cell, facilitates intracellular high-energy phosphate transport. Analyses of phosphocreatine concentration changes suggest that CO treatment had a slightly deleterious effect on energy metabolism of the heart before ischemia: the phosphocreatine concentration was decreased significantlyin the hearts of treated pigs. Yet, after reperfusion, phosphocreatine levels in CO-treated hearts were significantly higher than in untreated animals, suggesting that by the end of reperfusion CO had exerted cytoprotective effects on the energetics of the heart. It is known that early mild deterioration before ischemia may be important in triggering the later beneficial effect: transient energy depletion is a potent trigger for the eventual beneficial effects of preconditioning.

These differences in the energetic status between CO-treated and untreated hearts were seen primarily during reperfusion, a critical time when the heart recovers from the damage of ischemia and restores intra- and extracellular homeostasis to prevent further damage that can occur during reperfusion. Thus, efficient energy metabolism associated with CO treatment is critical during the reperfusion phase.

Presumably as a consequence of improved cardiac energetics, the amount of interstitial edema in hearts of CO-treated pigs was significantly less than in hearts of controls, with fewer apoptotic cells in the CO-treated hearts than in controls. The ability of CO to act in an anti-apoptotic manner has been documented in rodents in vitro and in vivo. It had not been noted before that CO can reduce interstitial edema in ischemia-reperfusion injury. In a rodent model of acute hyperoxic lung injury, CO reduces the accumulation of pleural effusion. Whether the CO effects noted here are due to direct effects on the edema formation or reflect overall tissue protection remains to be elucidated.

Another metabolic factor affecting virtually all processes in the cell is the redox status defined mainly by the NADH/NAD ratio or the NADPH/NADP ratio. CO suppressed the ischemia-induced reduction of pyridine nucleotides, with an increase in NADH/NAD and NADPH/NADP ratios observed at the end of ischemia.

Prevention by CO of reduced pyridine nucleotide accumulation may be due to a decreased rate of synthesis or increased utilization of reduced pyridine nucleotides. CO decreases NADH oxidation by inhibition of electron transport chain enzymes. It is therefore more likely that a decreased rate of formation of NADH or NADPH is responsible for this phenomenon. This may be due to decreased rates of NADH/NADPH-generating pathways in the CO-treated group. A more significant effect on NADPH/NADP than NADH/NAD ratios is consistent with the former being the primary event in non-CO-treated hearts. If CO is interfering with the electron transport chain, another possibility is that the tissues have decreased oxygen consumption requirements, resulting in a lower overall metabolic state that might explain the modulation of the ensuing inflammation and inhibition of apoptosis.

It was easier to restart the heart by defibrillation after ischemia. The mechanisms underlying these effects require further investigation.

CO in this study acted similarly to preconditioning of an organ with brief ischemia or other noxious stimuli that lead to greater resistance to subsequent prolonged periods of ischemia or hypoxia. CO and other modalities used for preconditioning induced transient dysfunction of energy metabolism as the triggering event. However, using other agents required much more profound changes in energy metabolism to induce preconditioning. If the ischemia time is too short, even if it brings the phosphocreatine levels almost to zero (a much greater reduction than seen in our experiments), it may not be enough to induce preconditioning. In some situations, the period of ischemia (and thus the "absence" of phosphocreatine) has to be continued for a critical length of time. Whether these differences reflect a greater or lesser beneficial effect of CO compared with these other modalities in terms of preconditioning requires further study.

These findings show for the first time that CO pretreatment results in improved cardiac energetics during ischemia-reperfusion injury and demonstrate the efficacy of CO pretreatment in preventing the complications of ischemia-reperfusion in a clinically relevant large animal model.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0996fje; doi: 10.1096/fj.03-0996fje

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