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Ann Thorac Surg 2001;72:1985-1990
© 2001 The Society of Thoracic Surgeons
a Department of Cardiovascular and Thoracic Surgery, Jean Minjoz University Hospital, Besançon, France
b Department of Pharmacology, Jean Minjoz University Hospital, Besançon, France
c Department of Pathology, Jean Minjoz University Hospital, Besançon, France
Accepted for publication August 7, 2001.
* Address reprint requests to Dr Davani, Department of Pharmacology, Jean Minjoz University Hospital, 25000 Besançon, France
e-mail: davani{at}ufc-chu.univ-fcomte.fr
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Abstract |
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Methods. Two groups of Sprague-Dawley rats (control, n = 10; and L-arginine, n = 10) were studied in an isolated buffer-perfused heart model. Both groups were injected intraperitoneally 24 hours before ischemia. Before experimentation blood samples were collected for cardiac troponin I and cGMP analysis. In the coronary effluents, cardiac troponin I, adenosine, cyclic guanosine monophosphate, and nitric oxide metabolites were assayed.
Results. Before heart excision, serum cardiac troponin I concentrations were higher in the L-arginine than in the control group (0.037 ± 0.01 versus 0.02 ± 0.05 µg · L-1; p < 0.05). During reperfusion, cardiac troponin I release was lower in the L-arginine than in the control group (0.04 ± 0.01 versus 0.19 ± 0.03 ng · min-1; p < 0.05). The coronary flow as well as the left ventricular developed pressure were higher in the L-arginine than in the control group before ischemia and remained so throughout the experimentation.
Conclusions. These results indicate that L-arginine administered intraperitoneally 24 hours before cardioplegic arrest reduced myocardial cell injury and seems to protect myocardium against ischemia-reperfusion injury.
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Introduction |
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The aim of this study was to test if L-arginine administered IP 24 hours before ischemia could induce pharmacologic cardioprotection against ischemia-reperfusion injury with the three more specific objectives to determine (1) the release kinetics of NO metabolites, cyclic guanosine monophosphate (cGMP), adenosine, and cardiac troponin I (cTnI) during ischemia and reperfusion; (2) the effects of L-arginine on postischemic myocardial variables; and (3) whether these biochemical events participated in the observed effect during reperfusion. Intra-peritoneal administration of L-arginine was chosen because this route of delivery has been shown to induce citrulline production and therefore NO formation in rat heart by the increase in L-arginine concentration in the heart [6]. To the best of our knowledge, there are no similar data for intravenous administration.
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Material and methods |
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The perfusion solution was a modified Krebs-Henseleit buffer (K-H) containing (in mmol · L-1): NaC1 118; NaHCO3 25; KC1 3.2; MgSO4 1.6; KH2PO4 1.2; CaCl2 2.5, and glucose 5.5. All perfusion solutions were passed through a 0.8 µm porosity filter (Millipore, Bedford, MA) to remove contaminants, gassed with 95% O2, 5% CO2, and adjusted at pH 7.4. The hearts were not paced and temperature was kept constant at 37°C during stabilization and reperfusion and at 20°C during ischemia. A water-filled latex balloon (No. 4, Hugo Sachs Electroniks, March-Hugstetten, Germany) connected to a pressure transducer (TA 240 S, Gould, Valley View, OH) was placed through the left atrium into the left ventricle, and adjusted to a left ventricular end-diastolic pressure (LVEDP) of 10 mm Hg. The LVEDP, the left ventricular developed pressure (LVDP, mm Hg) and the heart rate (HR, beats · min-1) were continuously recorded.
Coronary flow (CF, mL · min-1) was measured by timed collection of the coronary effluents. The incidence and duration of ventricular arrhythmias were measured during the reperfusion period. Only hearts with LVDP between 60 and 130 mm Hg, CF 9 to 18 mL · min-1, and HR 240 to 360 beats · min-1 at the end of stabilization were included.
Myocardial microdialysis
The technique of cardiac microdialysis was used to sample interstitial fluid (ISF) adenosine and NO metabolites during experimentation. A single 10-mm polycarbonate membrane microdialysis probe (CMA 20, Phymep, Solna, Sweden) was implanted in the left ventricular wall. The probe was perfused with a Ringer buffer containing (in mmol · L-1): NaCl 140; KCl 4; CaCl2 1.26; MgCl2 1.15 at a constant flow rate of 1.33 mL · min-1 by a micropump (CMA 100, Phymep, Sweden). A 20-µL sample of ISF was collected every 15 minutes during the experiment by a collector (CMA 140, Phymep, Sweden) and stored at -20°C for later analysis.
Experimental protocol
Rats were randomly divided into two groups, control (n = 10) and L-arginine (n = 10), and were injected, respectively, with saline buffer (3.30 mL · kg-1) and L-arginine (500 mg · kg-1) IP 24 hours before experimentation. Before heart excision a 1 mL blood sample was collected in the descending aorta. After centrifugation the serum was stored at -20°C for later anlaysis of cTnI and cGMP. After aortic cannulation, the microdialysis probe was inserted into the left ventricle wall.
The hearts were then stabilized for 15 minutes before starting the experimentation. All hearts were submitted to the same protocol: after 15 minutes stabilization by K-H perfusate, hearts were arrested by perfusion of 10 mL St Thomas’ II cardioplegic solution (ST) at 4°C into the aortic root. All hearts were subjected to global ischemia at 20°C for 60 minutes. At the end of the global ischemia they were reperfused for 120 minutes by the K-H at 37°C at the same pressure (80 mm Hg). LVDP (LVSP-LVEDP), HR, and CF were measured in both groups before ST perfusion and 30, 60, 90, and 120 minutes after the start of reperfusion. The coronary effluents were collected at the above times and stored at -20°C for later analysis (see Biochemical Measurement).
Biochemical measurement
Nitrite, a NO metabolite, was used as an index of NO release and was assayed in the ISF samples. Microdialysis ISF samples were also assayed for interstitial adenosine release. A 10-µL ISF sample assayed for adenosine using an automated high-performance liquid chromatography (HPLC) system (717 Plus autosampler, Waters, Millford, MA). Adenosine was separated using an Ultraspher ODS column (4.6 x 150 mm-5 µm, Beckmann, Fullerton, CA), with the mobile phase consisting of 12% (volume) methanol and 100 mmol · L-1 KH2PO4. Flow rate was set at 1 mL · min-1 using a pumping system (Waters 590, Waters, Millford). Eluent absorbance was continously monitored at 254 nm using an ultraviolet detector (Waters 486, Waters, Millford). The absorbance peak of adenosine was identified and quantified by comparing retention times and peak area to a known external standard of adenosine at a concentration of 1 µmol · L-1.
A 5-µL sample of collected ISF was injected into a chemiluminescence nitric oxide analyzer (Sievers 280, Boulder, CO). Adenosine, NO metabolites, cTnI, and cGMP were assayed in coronary effluents. Cardiac TnI and cGMP were assayed in serum samples. cTnI measurement was performed by an Access automated immunoassay system (Beckman, Fullerton). cGMP was assayed using a radioimmunoassay diagnostic kit from Amersham International.
Electron microscopy
At the end of reperfusion period, tissue samples (4 per heart) were taken from the left ventricle wall in all hearts. Then samples were cut into 1 mm3, fixed in glutaraldehyde buffer for 2 hours at 4°C, embedded in epoxy resine, and examined in a double-blind study in an electron microscope (Jeol, 1213).
Chemicals
St Thomas’ II cardioplegic solution was obtained from Aguettant Laboratory (Lyon, France). L-arginine was purchased from Sigma Chemicals Ltd (Lyon, France), and dissolved in saline buffer.
Statistics
Statistical comparisons were performed by a repeated measures analysis of variance (ANOVA). When significant differents were found a Bonferroni test was employed for pairwise comparisons. Differences were considered to be statistically significant when a p value was less than 0.05.
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Results |
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Hemodynamic data
Table 1 lists the hemodynamic variables measured in both groups before ischemia and during the reperfusion period. The hemodynamic variables of the reperfusion period subsequent to 60 minutes of ischemia are expressed as percentage of before ischemia values. There were no differences between the groups for HR after stabilization and during the reperfusion period except after 2 hours of reperfusion where HR was significantly higher in the L-arginine than in the control group. The percentage of HR recovery was improved in the L-arginine group. The LVDP and the CF were significantly higher in the L-arginine group before ischemia and during reperfusion even if the percentage of recovery was the same in the both groups. Throughout the reperfusion period, no arrhythmias occurred in either group.
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NO metabolites release assay
Before experimentation, K-H and Ringer buffer were assayed for nitrite and nitrate. All solutions were nitrite and nitrate free.
In the ISF, mean NO metabolite levels were the same in both groups before ischemia (3.8 ± 0.7 versus 4.5 ± 0.9 µmol · L-1, p = not significant). When comparing NO metabolites before and immediately after ischemia, this value was significantly higher just after ischemia in the control group (8.2 ± 0.3 versus 3.8 ± 0.7 µmol · L-1, p < 0.05) and steady in the L-arginine group (2.4 ± 0.6 versus 4.5 ± 0.9 µmol · L-1, p = NS). In the control group, NO metabolites peaked 15 minutes after the onset of reperfusion at 12.0 ± 0.5 µmol · L-1 and decreased progressively to reach the same levels as in the L-arginine group (1.7 ± 0.5 µmol · L-1) which were steady throughout the experimentation (data not shown).
In the coronary effluents during reperfusion period, there were no differences for NO metabolite release between control and L-arginine groups. NO metabolite released in the coronary effluents remained steady during reperfusion (data not shown).
Adenosine release assay
Mean adenosine concentration in the ISF after 15 minutes stabilization, was 7.6 ± 1.3 µmol · L-1 in the control and 9.6 ± 2.3 µmol · L-1 in the L-arginine group (p = NS) and then decreased rapidly in both groups to reach 0.5 ± 0.02 µmol · L-1. Global ischemia as well as the reperfusion period did not modify the kinetics of adenosine levels in the ISF (data not shown).
In the coronary effluents before ischemia, the adenosine release was significantly higher in the L-arginine when compared with the control group (55.6 ± 15 versus 6.3 ± 3 nmol · min-1; p < 0.001). During global ischemia, adenosine release increased in the L-arginine group when compared with the control (92.2 ± 25 versus 11.5 ± 4 nmol · min-1; p < 0.001). During the reperfusion period, the adenosine release was almost steady in both groups but remained significantly higher in the L-arginine than in the control group (Fig 1).
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cTnI release assay
The serum concentration of cTnI drawn before heart excision was significantly higher in the L-arginine than in the control group (0.037 ± 0.01 versus 0.02 ± 0.05 µg · L-1; p < 0.05; Fig 2A). Before ischemia, cTnI release in coronary effluents was higher in the control group than in the L-arginine group (2.2 ± 0.5 versus 0.6 ± 0.2 ng · min-1, p < 0.001). During the reperfusion period, the level of cTnI release, even if decreasing, remained significantly higher in the control than in the L-arginine group (Fig 2B).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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The present study showed that the intraperitoneal administration of L-arginine before ischemia; (1) induced reversible damage to mitochondrias; (2) did not modifiy the NO metabolites and cGMP kinetic released in reperfusion; (3) increased the adenosine and decreased the cTnI release during reperfusion period; and (4) improved the hemodynamic variables during reperfusion. Ischemia affected identically the hemodynamic variables as shown by the similar percentage of recovery in both groups. As LVDP and CF were significantly higher in the L-arginine group before ischemia, they remained so throughout the experimentation.
Cardiac troponin I has been shown to be a specific marker of myocardial cell ischemia and injury [8, 9]. The pretreatment with L-arginine induced a higher release of cTnI in serum before heart excision which revealed, as well as mitochondria damage, the deleterous effects of L-arginine through NO generation on myocardium before ischemia. Abe and colleagues [11] have shown that generated endogenous NO and NO-derived species could damage mitochondria and cardiac cells during postischemic reperfusion. Conversely to Engelmann and associates [12], our study did not show an increase in cGMP induced by L-arginine. Contrary to studies using L-arginine in cardioplegic solution or during the reperfusion period, in our study the NO metabolite release level was the same during ischemia and reperfusion [13, 14]. The timing of L-arginine injection, its delivery route as well as its dose may be an explanation of this discrepancy [6, 12].
Adenosine is produced from ATP by the S-adenosylhomocysteine (SAH) catabolism or from AMP by ecto-5'-nucleotidase [15]. Adenosine is known to have a cardioprotective action in the ischemia-reperfusion myocardium when added to cardioplegic solution [16, 17] as well as a vasodilatory effect dependent of the amount released [18]. Our results showed that the pretreatment with L-arginine was associated with a higher release of adenosine in preischemia as well as during the reperfusion period. The higher CF and LVDP observed in the L-arginine group before ischemia could be explained by the effect of adenosine release on coronary flow.
In the L-arginine group swollen mitochondrias corresponding to reversible lesions were found. There was no irreversible damage to the myocyte organization in both groups. Mitochondria damage decreases ATP synthetisation and increases ATP breakdown, which increases adenosine concentration within myocytes.
It can be hypothesized that mitochondria damage is due to L-arginine and occurs during the 24 hours between L-arginine administration and preischemia period as shown by a higher release of adenosine in the L-arginine group in the samples drawn before ischemia. Ischemia by itself also increased the ATP breakdown which increased the adenosine concentration. Therefore, there was a cumulative effect on adenosine concentration due to pretreatment by L-arginine and ischemia.
During reperfusion a washout of accumulated adenosine occurred explaining the higher release of adenosine in the L-arginine group before ischemia and during the reperfusion period. These observations suggest that the pretreatment with L-arginine induced a higher adenosine release before ischemia and during reperfusion and a lower release of cTnI during the same periods. This can be explained by the role of adenosine in the decrease of myocardial cell injury. This is in agreement with studies showing the vasodilatory effect of adenosine [18] and its contribution to myocardial protection after cardioplegia ischemic [19, 20]. Conversely, as shown by Belhomme and coworkers [21] the administration of exogenous adenosine to coronary artery bypass grafting patients did not influence the cTnI release.
In the present experiment, the ISF concentrations of adenosine and NO metabolites were used to estimate the interstitial concentration of adenosine and the NO metabolite release. Caution must be exercised in the extrapolation of ISF to the interstitial concentration.
In conclusion, the cardioprotective effect of L-arginine administration in cardioplegia solution or during reperfusion was demonstrated. Our data suggest that the administration of L-arginine 24 hours before cardioplegic arrest induces reversible damage to heart mitochondrias. The myocardial injury was also assessed by the higher cTnI concentration in the blood sample drawn before heart excision. This initial aggression by L-arginine administration seems to protect the myocardium from the subsequent ischemia-reperfusion injury as assessed by the lower cTnI release in coronary effluents in the L-arginine group during reperfusion. In this way, L-arginine administered intraperitoneally 24 hours before cardioplegic arrest seems to protect myocardium against ischemia-reperfusion injury. These findings could be useful in clinical practice particularly in cardiac surgery. Before a clinical application, the specific mechanisms underlying pharmacologic protection by L-arginine must be explicit. [10]
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentReferences |
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