Asian Cardiovasc Thorac Ann 2007;15:463-467
© 2007 Asia Publishing EXchange Ltd
Glutamine Improves Myocardial Function Following Ischemia-Reperfusion Injury
Gil Bolotin, MD,
Jai Raman, MD,
Ursula Williams, PhD,
Emile Bacha, MD,
Masha Kocherginsky, PhD,
Valluvan Jeevanandam, MD
Division of Cardiothoracic, Surgery Pritzker School of Medicine, University of Chicagom, USA
For reprint information contact: Gil Bolotin, MD, Tel: 31 43 387 7070, Fax: 31 43 387 5075, Email: g_bolotin{at}rambam.health.gov.il, Department of Cardiothoracic Surgery, Academic Hospital, Maastricht, P. Debyelaan 25, Postbus 5800, 6202 AZ, Maastricht, Netherlands.
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ABSTRACT
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Myocardial ischemia-reperfusion injury is common during cardiac procedures. Glutamine may protect the myocardium by preserving metabolic substrates. Glutamine (0.52 g·kg–1) or Ringer’s lactate solution (control group) was administered intraperitoneally to 63 Sprague-Dawley rats at 4 or 18 hours prior to experimental ischemia and reperfusion. The hearts were excised and perfused on an isolated working heart model, exposed to global ischemia for 15 min and reperfusion for 1 hour. Left atrial pressure, mean aortic pressure, cardiac flow, coronary flow, and aortic output were measured 15 min before ischemia and every 15 min during reperfusion. There was significantly better cardiac output in the glutamine pretreated groups. Pretreatment at 4 hours before the experiment was superior to pretreatment at 18 hours, with better maintenance of cardiac output and coronary flow. The enhanced protective effect of pretreatment at 4 hours highlights the importance of timing, and suggests a potential clinical benefit.
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INTRODUCTION
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Myocardial ischemia and reperfusion are common in cardiovascular surgery. Acute ischemia ranges from transient reversible stunning of the myocardium to myocardial infarction. These abnormalities are exacerbated by reperfusion with oxygenated blood.1,2 Several therapeutic modalities have been suggested and tested to prevent or reduce myocardial ischemia-reperfusion injury.3–6 In previous work by our group, pretreatment with glutamine was documented to induce hemodynamic advantages.7 In this study, we changed the timing of pretreatment to increase the protective effect.
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MATERIALS AND METHODS
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Sixty-three male Sprague-Dawley retired breeder rats (Harlan) weighing 400–500 g were divided into 4 groups. Group A (n = 16) was pretreated with intraperitoneal (IP) glutamine 4 hours before the experiment and compared to control group B (n = 14) that received IP Ringer’s lactate at 4 hours. Group C (n = 15) was pretreated with IP glutamine 18 hours before the experiment and compared to control group D (n = 18) that received IP Ringer’s lactate at 18 hours. For pretreatment, the animals were anesthetized with isoflurane and injected intraperitoneally with 15% (0.75 g·kg–1) alanine-glutamine in Ringer’s lactate or Ringer’s lactate only. The rats were deeply anesthetized 4 or 18 hours later by IP injection of 60 mg·kg–1 pentobarbital sodium. They were intubated with a 14G BD Angiocath (Beckton Dickinson, Franklin Lakes, NJ, USA) and ventilated with a Harvard Small Rodent Respirator (Harvard Apparatus, Holliston, MA, USA) at 7 mL per 80 rpm. The abdominal cavity was opened along the midline to facilitate injection of 500 U heparin into the inferior vena cava to prevent blood clots in the coronary circulation. The thoracic cavity was opened 5 min after the injection, the ascending aorta was freed from the overlying thymus gland, and the heart was removed and placed in a cardioplegic solution of modified Krebs-Henseleit buffer at 4°C. The ascending aorta was cannulated and connected to the Langendorff/ working heart apparatus and perfused in the retrograde Langendorff mode for 10 min. The perfusate was modified Krebs-Henseleit buffer (116.0 mM NaCl, 4.3 mM KCl, 1.17 mM Kh2PO4, 1.18 mM MgSO4, 2.4 mM CaCl2, 26.0 mM NaCO3), which was continually insufflated with 95% O2 and 5% CO2 and supplemented with 2.0 mM pyruvate, 0.5 mM EDTA, 5.38 mM fumarate, 10 U insulin, and 1,000 U heparin, and maintained at 36°C ± 5°C.
The left atrium was cannulated, and after 10 min, the circulation was switched to the antegrade working heart mode. The hearts were paced at 300–380 beats per minute, depending on the rat’s weight (Medtronic 5388 dual-chamber temporary pacemaker, Minneaspolis, MN, USA). After another 15-min period, perfusion was totally stopped for 15 min of ischemia, during which pacing was discontinued. The perfusate was replaced with fresh Krebs buffer every 30 min. At the end of the ischemic period, perfusion was restarted in Langendorff mode for 10 min, then switched to working heart mode with appropriate pacing. Every 15 min after commencement of working heart perfusion, preload and afterload (atrial and aortic pressures), cardiac and aortic flow, and blood gasses in the perfusate and effluent (coronary flow) were measured and recorded at 8 and 12 mm Hg left atrial pressure using an ABL 500 blood gas meter (Radiometer, Copenhagen, Denmark) and a Transonic System T206 dual-channel flow meter with a Hewlett Packard 78534C monitor and an Abbott Labs disposable pressure transducer. Hemodynamic measurements were taken 15 min prior to ischemia (baseline), and at 15, 30, 45, and 60 min after ischemia. At the end of the experiment, the hearts were removed from the cannulas and snap frozen for further analyses.
Initially, the hemodynamic parameters of interest were compared at each time point between the glutamine and control groups using Student’s t test. Plots of outcome measurements over time revealed a sharper drop in hemodynamic function from baseline to 15 min post-ischemia, and a more gradual deterioration afterwards. To accommodate these differences, further analyses of changes in the 3 parameters of interest (aortic mean pressure, coronary flow, and cardiac output) over time were performed in 2 stages. First, we compared changes in each of the parameters from baseline to 15 min post-ischemia between each group using t tests. In the second stage, we used repeated measures of analysis of variance models to explore longitudinal changes in the hemodynamic parameters, using all of the post-ischemia measurements, and treating the baseline measurement as a fixed continuous covariate to account for the initial differences between the animals. These models compared the 2 treatment groups (control and glutamine injection), as well as the time of infusion (4 and 18 hours before the experiment). Analyses were performed separately for preloads of 8 and 12 mm Hg. Correlation between multiple longitudinal measurements on the same animal (within-animal correlation) was incorporated into the model using auto regressive (1) covariance structure. Linear and quadratic time effects were also included in the model and treated as fixed effects, and interactions between treatment or infusion-time groups and time were also examined. Models were selected using backward elimination based on F-tests. Oxygen inflow minus O2 outflow) was calculated at 15 and 60 min, and differences in consumption relative to pre-ischemia O2 consumption (O2 consumption between the glutamine and control groups were compared using t tests. All p values are 2-tailed, and a value of p less than 0.05 is considered statistically significant. Data are presented as mean ± standard deviation
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RESULTS
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All groups showed significant gradual deterioration in most hemodynamic parameters at 15, 30, 45, and 60 min post-ischemia. Hemodynamic dysfunction was significantly less profound in the glutamine-treated groups. There were trends toward gradual reductions in mean aortic pressure after ischemia and reperfusion in all groups, at preloads of both 8 and 12 mm Hg (Table 1
), and time was found to have a significant effect on the repeated-measures models ( p < 0.01 for both preloads). However, the changes from baseline were generally small and reached statistical significance compared to the control group only at 45 and 60 min in the glutamine 18 hours pretreatment at 12 mm Hg preload (Table 1). There were no changes in consumption at 15 and O2 60 min post-ischemia in either the control or glutamine groups compared to pre-ischemia measurements (Table 2). There was a gradual significant reduction in cardiac output in all groups (at preloads of 8 and 12 mm Hg) at 15, 30, 45, and 60 min post-ischemia compared to baseline (Figure 1). The reduction was less profound in the glutamine group, and reached statistical significance at all time points in group A compared to group B. The trends in group C were similar; however, significant differences compared to group D were achieved only in the late measurements at 45 and 60 min post-ischemia (Figure 1). Coronary blood flow remained stable in the glutamine pre-treated groups (A and C) compared to a gradual reduction in control groups B and D (Figure 2). However, in group C, the differences did not achieve statistical significance; in group A, there were significant differences at 15, 30, 45, and 60 min compared to group B at a preload of 8 mm Hg (Figure 2). At a preload of 12 mm Hg, the coronary flow differences between groups A and B were significant at 60 min (Figure 2). Analysis of hemodynamic parameters over time, using repeated-measures models, revealed that deterioration continued to the end of the experiment (60 min post-ischemia). Generally, animals treated with glutamine performed better than those injected with Ringer’s lactate solution. We found significant differences between control and glutamine pretreatment groups regardless of time of pre-treatment for cardiac output ( p < 0.0001 and 0.0008, at preloads 8 and 12 mm Hg, respectively). Coronary flow remained stable in the glutamine pretreated group and decreased significantly faster in the control group ( p = 0.0128 and 0.0238, at preloads 8 and 12 mm Hg, respectively). Time had a significant negative effect on all 4 parameters, and the significant negative quadratic time terms in all models suggests that the rate of deterioration increased over time.
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Figure 1. Cardiac output in the control group (solid) vs the glutamine group (dashed) throughout the experiments. Groups A and B on the left side, groups C and D on the right side. *p < 0.05, **p < 0.01.
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Figure 2. Coronary flow in the control group (solid) vs glutamine group (dashed) throughout the experiments. Groups A and B on the left side, groups C and D on the right side. *p < 0.05, **p < 0.01.
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DISCUSSION
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Our results in the isolated working rat heart model suggest a protective effect of glutamine administered both at 4 and 18 hours before ischemia-reperfusion. These results are in agreement with our previous study that documented hemodynamic advantages in an isolated working rat heart model with glutamine administered 18 hours before ischemia-reperfusion.7 The protective effect in the current study was clear and significant in almost all cardiac output measurements. These results agree with other reports of a possible pretreatment protective effect.5,6 The differences from the current work are the timing of the treatment (15 min compared to 4 and 18 hours) and the components of the pretreatment solution.5,6
Glutamine may cause a more systemic cardiovascular effect, and it may have a significant impact on other organs and systems as well.8,9 Generally, group A (glutamine 4 hours before ischemia-reperfusion) demonstrated the best hemodynamics of all 4 groups, indicating the importance of timing in the protective effect of treatment. While previous reports focused on the protective effect of glutamine for ischemia-reperfusion damage, there are no data on the best timing for glutamine treatment.7–10 In the current study, the main advantage of group A (4 hours) over group C (18 hours) can be seen in Figures 1
and 2. The protective effect of glutamine on cardiac output was significant in all time points and at both preloads (Figure 1). The protective effect of the glutamine administrated 18 hours earlier was significant only later in the experiments, and with higher p values. These results strengthen the general conclusion of our previous study, suggesting that the protective effect may broaden with better timing of the treatment.7 The protective effect of glutamine pretreatment at 4 hours on coronary blood flow supports the same conclusion as it induces a significant protective effect at all time points with a preload of 8 mm Hg, and at 60 min with a preload of 12 mm Hg (Figure 2). This is in contrast to the low protective effect of 18 hours glutamine pretreatment on coronary blood flow (Figure 2). The advantage of 4 hours pretreatment with glutamine over 18 hours pretreatment may have clinical importance as it can be applied in more clinical situations, such as the morning before coronary interventions.
The endothelium has been shown to play a key role in the injury suffered after ischemia and reperfusion. When rendered hypoxic and then re-oxygenated, endothelial cells are activated to express pro-inflammatory properties. These changes may contribute to the noreflow phenomenon by promoting endothelial edema, neutrophil and platelet plugging, microthrombosis, and enhanced vasomotor tone.1 The potential mechanisms involved in the protective effect of glutamine against ischemia-reperfusion injury include: prevention of the reduction of glutathione and thus free-radical generation, prompt production of heat-shock proteins, and less adenosine triphosphate depletion.7,11–13 The primary energy source of the normal myocardium is free fatty acids. Metabolism of free fatty acids can cause damage to the myocardium during ischemia as a result of toxic metabolite accumulation.14,15 Several therapies have been suggested to treat or decrease cardiac damage after ischemia-reperfusion injury, including glutamate, aspartate, and glutamine.10,16 However, high concentrations of glutamic and aspartic acids are needed to exert beneficial effects on ischemic heart muscle.16 In such high plasma concentrations, glutamate was found to be a neural and cardiac toxin, and thus is less clinically appropriate.17 On the other hand, glutamine demonstrated a myocardial protective effect after ischemia-reperfusion injury in low concentrations.9
The results of this study are supported by several previous preclinical studies suggesting a possible protective effect on ischemia-reperfusion injury by metabolic pretreatment.5–7 The clinical relevance of this study is based on 2 factors: the broad clinical occurrence of ischemia-reperfusion injury, and the low toxicity of glutamine. Cardiac patients undergoing almost any intervention are exposed to ischemia-reperfusion injury. This has been documented during percutaneous angioplasty and coronary artery bypass grafting.18,19 We found pretreatment with glutamine to be protective against ischemia-reperfusion injury in a beating heart rat model. The 4-hour pretreatment should be tested in humans, and may have clinical advantages. Moreover, the low toxicity of glutamine combined with the wide range of clinical ischemia-reperfusion situations highlights the clinical relevance of this study.
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REFERENCES
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ABSTRACT
INTRODUCTION
