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Cardiovascular Research 2003 57(2):416-425; doi:10.1016/S0008-6363(02)00698-3
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Effects of N-(2-mercaptopropionyl)-glycine on mitochondrial function in ischemic–reperfused heart

Kouichi Tanonaka, Takeshi Iwai, Kanataka Motegi and Satoshi Takeo*

Department of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

takeos{at}ps.toyaku.ac.jp

* Corresponding author. Tel.: +81-426-76-4583; fax: +81-426-76-5560.

Received 1 May 2002; accepted 25 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: A possible mechanism for N-(2-mercaptopropionyl)-glycine (MPG) underlying the improvement of contractile function and mitochondrial activity of ischemic–reperfused rat hearts was examined. Methods: Isolated, perfused hearts were subjected to 35 min ischemia–60 min reperfusion. At the end of ischemia or reperfusion, myocardial Na+ content and mitochondrial oxygen consumption rate (OCR) were examined. The perfused heart was treated with 0.1–1 mM MPG for 30 min prior to ischemia or for the first 30 min of reperfusion. Results: Ischemia increased myocardial Na+ content (sodium overload) and decreased mitochondrial OCR. The left ventricular developed pressure (LVDP) of the untreated heart recovered to 19.8±3.8% of the preischemic value and the infarct area amounted to 23.3±1.7% of the left ventricle. The thiobarbiturate-reacting substance (TRS) was also increased in the reperfused, but not ischemic, myocardium. Pretreatment of the perfused heart with 0.3–1 mM MPG attenuated the ischemia-induced sodium overload and decrease in the OCR. Pretreatment with the agent also enhanced the postischemic recovery of LVDP, attenuated reperfusion-induced increase in TRS, and reduced the infarct area. Although the postischemic treatment with MPG suppressed the increase in TRS in the reperfused myocardium, a LVDP recovery of reperfused hearts was not observed. Cardiac mitochondria were isolated and examined for the direct effect of MPG on their function. Incubation with either 12.5 mM sodium lactate or 1 µM phenylarsine oxide neither altered the mitochondrial membrane potential nor induced mitochondrial swelling, whereas incubation with a combination of these agents elicited the membrane potential depolarization and swelling. Incubation of mitochondria with 1 mM MPG attenuated these events. Conclusion: These results suggest that both attenuation of sodium overload and preservation of the mitochondrial function may largely contribute to cardioprotection of MPG in the ischemic–reperfused heart.

KEYWORDS Contractile function; Energy metabolism; Intra/extracellular ions; Ischemia; Mitochondria


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Reperfusion of the heart following a certain period of ischemia generates free radicals such as superoxides and hydroxyl radicals [1,2]. Such free radicals generated in the reperfused myocardium may induce peroxidation of the intracellular components such as phospholipids, proteins, and DNAs [2,3] and thus may provoke cellular damage of the heart. These deleterious sequences may lead to functional changes of cardiac cell and tissue. In particular, it is recognized that free radical attack may be one of causes for contractile dysfunction of ischemic–reperfused hearts [1]. Several investigators reported the benefits of N-(2-mercaptopropionyl)-glycine (MPG), a hydroxyl radical scavenger, for ischemic–reperfused hearts; treatment of anesthetized animals with this agent reduced ischemia–reperfusion-induced formation of the infarct size [4,5] and improved ischemia–reperfusion-induced myocardial stunning [6–8]. Furthermore, treatment of perfused hearts with MPG enhanced the recovery of contractile function and preserved the ultrastructural integrity of the myocardium [9]. MPG is capable of scavenging generated free radicals in the intracellular space and/or in the mitochondria of the reperfused heart, and thus the mechanism of MPG underlying cardioprotection is considered to be due to prevention of free radical attack. In contrast, several reports have recorded observations against the cardioprotective effects of MPG. Treatment of rabbit hearts with MPG cancelled the ischemic preconditioning-induced cardioprotection against ischemia–reperfusion injury [10] and treatment of rabbit hearts failed to decrease the infarct size [11]. Therefore, it remains unclear whether MPG protects the heart against ischemia–reperfusion injury and, if it does, how the cardioprotection may occur.

Recently, we reported that Na+ accumulation in the ischemic myocardium relates to postischemic contractile failure [12] and that treatment of perfused rat hearts with Na+ channel blockers or Na+/H+ exchanger inhibitors attenuated ischemia-induced sodium overload and enhanced the recovery of postischemic contractile function [12–14]. From these observations, we have suggested that cytosolic sodium overload largely contributes to the inability of the mitochondria to generate ATP in the ischemic–reperfused heart. No information is, however, available concerning the effects of MPG on sodium overload and mitochondrial function in the ischemic myocardium. In the present study, we examined possible mechanisms underlying cardioprotection of MPG, in particular, protection of mitochondrial function, against ischemia–reperfusion-induced contractile dysfunction of perfused rat hearts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1 Animals
Male Wistar rats (Japan Laboratory Animals Tokyo, Japan), weighing 230–270 g, were used in the present study. The animals were conditioned at 23±1 °C with a constant humidity of 55±5%, a cycle of 12-h light and 12-h darkness, and were given free access to food and tap water according to the Guide for Care and Use of Laboratory Animals as promulgated by the National Research Council. The protocol of this study was approved by the Committee of Animal Use and Welfare of Tokyo University of Pharmacy and Life Science.

2.2 Perfusion of hearts
After anesthesia with diethyl ether, the rat hearts were rapidly isolated and perfused at 37 °C with a constant flow-rate at 9 ml/min of Krebs–Henseleit buffer of the following composition (mM): NaCl 120; KCl 4.8; KH2PO4 1.2; MgSO4 1.2; CaCl2 1.25; NaHCO3 25; and glucose 11, which was equilibrated with a gas mixture of 95% O2–5% CO2 at pH 7.4. A latex balloon, connected to a pressure transducer (TP-200, Nihonkohden, Tokyo, Japan), was inserted into the left ventricular cavity. The initial ventricular end-diastolic pressure (LVEDP) of the perfused heart was adjusted to 5 mmHg to evaluate left ventricular developed pressure (LVDP), a convenient marker of cardiac contractile function. The LVDP and LVEDP were monitored by a pressure transducer. These hemodynamic parameters were recorded on a thermal pen recorder (WT-645G, Nihonkohden) throughout the experiment.

2.3 Ischemia–reperfusion
Ischemia–reperfusion of perfused hearts was carried out as described previously [15]. After equilibration, additional 40-min perfusion was carried out (preperfusion) and then the perfusion was stopped for 35 min (ischemia) under normothermic conditions. After 35 min of ischemia, the hearts were reperfused for 60 min at 37 °C (reperfusion). The hearts were paced at the rate of 300 beats/min throughout the experiment except for the first 15 min of reperfusion, to prevent contractile irregularities, which might sometimes occur during this period. For the purpose of comparison, hearts were perfused for 95 min under normoxic conditions, the time equal to that for ischemia plus reperfusion (normoxia).

2.4 Treatment of perfused hearts with MPG
MPG was purchased from Sigma (St. Louis, MO, USA). Treatment of the perfused hearts with different concentrations of MPG ranging from 0.1 to 1 mM was carried out by perfusing the hearts with the agent in Krebs–Henseleit buffer for the last 30 min of preischemia [15] or for the first 30 min of reperfusion. The agent was dissolved in the Krebs–Henseleit buffer.

2.5 Thiobarbiturate-reactive substance and high-energy phosphate in perfused hearts
At the end of ischemia or ischemia–reperfusion, the hearts were freeze-clamped to determine myocardial thiobarbiturate-reactive substance (TRS), ATP, and creatine phosphate (CP) contents.

After appropriate sequences of perfusion, TRS, a marker of free radical formation, in the perfused heart was determined by the method of Ohkawa et al. [16]. Briefly, the frozen left ventricular free wall (approximately 300 mg wet tissue) was homogenized with 5 volumes of 1.15% KCl solution. An aliquot of 200 µl homogenate was added into the reaction medium of the following composition; 40 µl of 8.1% SDS, 300 µl of 20% acetic acid, 300 µl of 0.67% thiobarbituric acid. The reaction mixture was incubated at 95 °C for 60 min. The red pigment produced during incubation was extracted with 1 ml n-butanol and its absorbance at 532 nm was measured using a spectrophotometer (U-Best 30, Jasco, Hachioji, Japan). The myocardial content of TRS as malondialdehyde was calculated as described previously [15].

Another frozen ventricle was pulverized and mixed with 0.3 M HClO4 and 0.25 mM EDTA. The extract was centrifuged at 8000 g for 15 min at 4 °C. ATP and CP in the supernatant were determined by the HPLC method [17].

2.6 Examination of perfusate
The perfusate eluted from the heart treated with or without MPG was collected during reperfusion. Creatine kinase (CK) activity of the perfusate was determined as described previously [18].

2.7 Myocardial Na+ content
Myocardial Na+ content of the ischemic or ischemic–reperfused heart was determined to assess ionic disturbances in the heart, as described previously [18]. After ischemia, reperfusion, or normoxic perfusion, hearts were perfused for 1 min with 8.0 ml of 320 mM sucrose–20 mM Tris–HCl, pH 7.4. Approximately 100 mg left ventricular tissue were dried at 120 °C for 48 h. The dried tissues were digested with 60% HNO3 and evaporated at 180 °C. The residues were suspended in 0.75 M HNO3. Myocardial Na+ concentration was determined by means of an atomic absorption spectrometer (AA-680, Shimazdu, Kyoto, Japan). This method can detect ions that are present in the intracellular milieu and cellular organelles in the heart, that is, tissue ions devoid of those in the extracellular and vascular spaces [18].

2.8 Mitochondrial oxygen consumption rate of skinned bundles
The mitochondrial oxygen consumption rate (OCR) was determined by the method of Sanbe et al. [19]. Briefly, myocardial bundles were prepared from the left ventricle using tissue chopper (Mickle Lab. Engineering, NY, USA) and transferred into relaxing medium A of the following composition (mM): EGTA, 10; MgSO4, 3; taurine, 20; dithiothreitol, 0.5; imidazole, 20; potassium 2-(N-morpholino)-ethanesulfonate (MOPS), 160; ATP, 5; CP, 15 (pH 7.0). Skinned bundles were prepared after incubation for 20 min in 1 ml of medium A containing 75 µg/ml saponin and then washed for 10 min in fresh medium B (medium A without ATP and CP but supplemented with 0.5% bovine serum albumin; BSA) to remove the saponin. The OCR was determined by means of a Clark-type electrode (Central Kagaku, Tokyo, Japan). The basal OCR was measured following the addition of 5 mM glutamate, 3 mM malate, and 3 mM KH2PO4. Maximal OCR was measured after further addition of 1 mM ADP and 7.5 mM creatine. The velocity of ADP-stimulated OCR was taken as the difference between the maximal and basal glutamate/malate-stimulated OCRs.

2.9 TTC staining
After ischemia, ischemia–reperfusion, or normoxic perfusion, the heart was isolated and sectioned into seven slices (approximately 1 mm thick) from the base to apex in a plane parallel to the atrioventricular groove. The slices were stained at 37 °C for 5 min with 1% 2,3,5-triphenyltetrazolium chloride (TTC) in physiological saline. TTC-stained and unstained areas were estimated by the modified method of Yoshida et al. [20].

2.10 Isolation of mitochondria
Cardiac mitochondria were prepared from the rat as described previously [21]. In brief, heart tissue was homogenized in ice-cold buffer containing 160 mM KCl, 10 mM EGTA (pH7.4), and 0.5% fatty acid-free BSA. The homogenate was centrifuged at 1000 g for 10 min at 2 °C, and the supernatant solution was centrifuged at 8000 g for 10 min at 2 °C. The crude mitochondria were again suspended in buffer and centrifuged at 8000 g for 10 min at 2 °C. Mitochondria were resuspended with suspension buffer (320 mM sucrose, 10 mM Tris–HCl, pH7.4).

2.11 Mitochondrial membrane potential
Mitochondrial membrane potential was measured at 30 °C by the method of Kamo et al. using tetraphenylphosphonium chloride (TPP+)-sensitive electrodes [22]. TPP+ (3 µM; Tokyo Chem. Industries, Tokyo) was added to the incubation medium containing 110 mM KCl, 5 mM K2HPO4, and 10 mM MOPS (pH 7.3) and then the mitochondria were added. The mitochondrial membrane potential was calculated according to the following equation:

Formula
where {Delta}{psi} is mitochondrial membrane potential (mV), v is mitochondrial matrix volume (1.6 µl/mg mitochondrial protein), V is the volume of the incubation medium (1 ml), and E0 and E are electrode potentials before and after the addition of mitochondria, respectively. The membrane potential was measured in the presence and absence of sodium lactate and/or phenylarsine oxide, an oxidant that induces membrane depolarization [23].

2.12 Mitochondrial swelling
Mitochondrial swelling was determined by the method of Brierley et al. with some modifications [24]. Mitochondria (approximately 0.2 mg protein) were preincubated at 30 °C in medium containing 110 mM KCl, 20 mM MOPS, 10 mM Tris–HCl, 0.5 µM rotenone, and 0.5 µM antimycin (pH 7.4) for 10 min prior to the addition of 12.5 mM sodium lactate and/or 1 µM phenylarsine oxide. The decrease in absorbance at 540 nm was detected with a split-beam spectrophotometer (U-Best30; Jasco) as a measure of mitochondrial swelling [25].

2.13 Statistics
Each value represents the mean±S.E.M. Statistical analysis was performed with the aid of StatView® for Windows (SAS Institute, Tokyo, Japan). Statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni's or Dunnett's multiple comparisons if necessary. The relationship between two parameters was determined by the least squares method. Differences with a probability of 5% or less were considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1 Contractile function, CK release, and TTC staining of perfused hearts
The upper and lower panels in Fig. 1 show changes in LVDP and LVEDP of the perfused heart. Baseline (initial) values for LVDP of agent-untreated and treated groups ranged from 75.0±5.4 to 78.0±4.1 mmHg (n=4 or 5). MPG at 0.3 or 1 mM decreased LVDP at the end of the agent treatment; 74.7±2.2 (n=4) or 62.4±3.6% (n=5) of initial value (at –30 min, P<0.05), whereas treatment with 0.1 mM MPG tended to decrease LVDP. After the onset of ischemia, LVDP decreased to zero within 2.5 min. At the end of 60-min reperfusion, the LVDP of the heart recovered to 19.8±3.8% of the basal value (n=5). In contrast, the LVDP of the hearts treated with 1 mM MPG recovered to 70.6±4.1% of the basal value (P<0.05, n=5).


Figure 1
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  		Fig. 1 Time course of changes in left ventricular developed pressure (LVDP; the upper panel) and left ventricular end-diastolic pressure (LVEDP; the lower panel) of the ischemic–reperfused heart. The hearts were untreated (bullet; Un, n=5) or treated with 0.1 ({bigtriangleup}; n=4), 0.3 ({square}; n=4), or 1 mM MPG ({circ}; n=5) for the last 30 min of preischemia and with 1 mM MPG ({bigtriangledown}; 1R, n=4) for the first 30 min of reperfusion. The hatched bar in the upper panel represents the LVDP of the heart treated with MPG and the striped bar, that of the heart reperfused with MPG. Each value represents the mean±S.E.M. Statistical significance in the recovery of LVDP evaluated using values at the end of reperfusion. *, Significantly different from the corresponding untreated ischemic–reperfused group (Un) (P<0.05).

 
The LVEDP of the untreated heart began to rise at 5 min after the onset of ischemia and reached its peak level approximately 20 min after the onset of ischemia. It further increased upon reperfusion. Treatment with MPG attenuated the rise in LVEDP during reperfusion in a concentration-dependent manner (Fig. 1).

The amount of CK released during reperfusion was also determined (Table 1). During preischemic perfusion or normoxia with and without MPG treatment, CK activity in the perfusate was <1 nmol NADPH/min/g wet tissue (n=4 or 5). CK activity in the perfusate released from the untreated heart markedly increased during reperfusion (n=5). Treatment with MPG attenuated the increase in CK release during reperfusion in a concentration-dependent manner (n=4 or 5).


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  		Table 1 Creatine kinase activity in the perfusate eluted from normoxic or ischemic–reperfused hearts untreated or treated with different concentrations of MPG prior to ischemia (MPG; 0.1 to 1) or 1 mM MPG during first 30-min of reperfusion (1R)

 
In another set of experiments, myocardial slices from the perfused heart were stained with TTC (Table 2). TTC-unstained area of the ischemic–reperfused heart was 23.3±1.7% of the left ventricle. In contrast, TTC-unstained area was decreased by pretreatment with MPG in a concentration-dependent manner.


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  		Table 2 TTC-unstained area of ischemic–reperfused hearts treated without (Un) and with various concentrations (0.1–1 mM) of MPG prior to ischemia or 1 mM MPG during the first 30 min of reperfusion (1R)

 
Furthermore, we examined the effect of treatment of the perfused heart with 1 mM MPG for the first 30 min of reperfusion (n=4; MPG 1R in Fig. 1). This treatment did not show any sign of the improvement of LVDP recovery and CK release during reperfusion (Fig. 1 and Table 1). Treatment of the heart with 1 mM MPG during the first 30-min reperfusion did not affect the formation of TTC-unstained area (Table 2).

3.2 TRS content of ischemic and ischemic–reperfused myocardium
Fig. 2 shows myocardial TRS content at the ends of ischemia and reperfusion. The preischemic value of TRS content was approximately 0.4 nmol/mg protein (n=5). At the end of ischemia, the value for myocardial TRS content was similar to the preischemic value, regardless of treatment with MPG. When the untreated heart was reperfused, myocardial TRS content increased to approximately 225% of the preischemic value (n=5). In contrast, the TRS content of the hearts pretreated with MPG was decreased in a concentration-dependent manner (n=4 or 5). There was an inverse and significant correlation between the recovery of LVDP and TRS content of the ischemic–reperfused hearts treated with different concentrations of MPG (r = –0.912, P<0.05, n=13). Treatment of the heart for the first 30 min of reperfusion suppressed the ischemia–reperfusion-induced increase in TRS content (n=4).


Figure 2
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  		Fig. 2 Myocardial thiobarbiturate-reactive substance (TRS) content at the ends of ischemia and reperfusion. The hearts were untreated (Un, n=5) or treated with 0.1 (n=4), 0.3 (n=4), or 1 mM MPG (n=5) for the last 30 min of preischemia and with 1 mM MPG (1R, n=4) for the first 30 min of reperfusion. The control (preischemic) value of TRS content was 0.405±0.019 nmol/mg protein (Cont, n=5). Each value represents the mean±S.E.M. #, Significantly different from the normoxic heart (Cont; P<0.05). *, Significantly different from the corresponding untreated group (Un; P<0.05).

 
3.3 Myocardial Na+ content
Fig. 3 shows myocardial Na+ content determined at the end of preischemia, ischemia, or reperfusion. Na+ content of the heart before treatment with MPG was approximately 55 µmol/g dry tissue (n=5). The myocardial Na+ content showed an approximately 2-fold increase in the baseline value at the end of ischemia (n=5). Treatment with MPG partially but significantly suppressed the ischemia-induced increase in the myocardial Na+ content (n=4 or 5). When the heart was subjected to 35-min ischemia and then reperfused for 60 min, a further increase in the myocardial Na+ content was observed during reperfusion (n=5). This increase in the myocardial Na+ content was attenuated by pretreatment with MPG in a concentration-dependent manner (n=4 or 5). In contrast, treatment of the reperfused heart with 1 mM MPG during the first 30-min reperfusion did not attenuate ischemia–reperfusion-induced increase in myocardial Na+ content (n=4).


Figure 3
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  		Fig. 3 Myocardial Na+ content at the ends of ischemia and reperfusion. The hearts were untreated (Un, n=5) or treated with 0.1 (n=4), 0.3 (n=4), or 1 mM MPG (n=5) for the last 30 min of preischemia and with 1 mM MPG (1R, n=4) for the first 30 min of reperfusion. The control (preischemic) value for myocardial Na+ content was 55.24±0.70 µmol/g dry tissue (Cont, n=5). Each value represents the mean±S.E.M. #, Significantly different from the control heart (Cont; P<0.05). *, Significantly different from the untreated ischemic–reperfused group (Un; P<0.05).

 
3.4 Mitochondrial oxygen consumption rate of skinned bundles
Mitochondrial oxygen consumption rate (OCR) of the skinned bundles prepared from the left ventricular free wall was determined (Fig. 4). The OCR of the skinned bundles from the preischemic heart was approximately 65 nano-atom O/min/mg protein (n=4). There were no significant differences in the OCR of perfused hearts under normoxic conditions regardless of treatment with or without MPG. The OCR of the untreated heart under ischemic conditions was approximately 35% of the value for the normoxic heart and that of the reperfused heart, approximately 25% of the value for the normoxic heart (n=4). Treatment with 0.3 or 1 mM MPG preserved the OCR at the ends of both ischemia and reperfusion (n=4 each). In contrast, when the heart was treated with 1 mM MPG for the first 30 min of reperfusion, the OCR was similar to that of the untreated heart.


Figure 4
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  		Fig. 4 Mitochondrial oxygen consumption rate (OCR) of skinned bundles prepared from perfused hearts at the ends of ischemia and reperfusion. The hearts were untreated (Un, n=5) or treated with 0.1 (n=4), 0.3 (n=4), or 1 mM MPG (n=5) for the last 30 min of preischemia and with 1 mM MPG (1R, n=4) for the first 30 min of reperfusion. Mitochondrial OCR was measured in the presence of 1 mM ADP and 5 mM glutamate. The control (preischemic) value for mitochondrial OCR (Cont) was 62.49±1.90 nano-atom O/min/mg protein (n=4). Expressions for columns and symbols as in Fig. 3.

 
3.5 Myocardial high-energy phosphates
Myocardial ATP and CP contents at 95 min of normoxia were similar to those at the end of preischemia. Myocardial ATP and CP contents at the end of the ischemia were approximately 3 and 10% of the preischemic values, respectively (n=5; Fig. 5). After reperfusion, myocardial ATP and CP contents restored to approximately 25 and 30% of the preischemic values, respectively (n=5; Fig. 5). When hearts were pretreated with different concentrations of MPG, ATP and CP contents in the reperfused heart were restored in a concentration-dependent manner (n=4 or 5; Fig. 5). Treatment with 1 mM MPG for the first 30-min of reperfusion did not enhance the restoration of myocardial ATP and CP contents (n=4). The ATP and CP levels at the end of ischemia were similar regardless of treatment with or without MPG (n=4 or 5; Fig. 5). The ATP and CP levels of 1 mM MPG-treated heart at the end of administration (24.07±0.46 and 35.05±0.67 µmol/g dry tissue, respectively, n=5) were similar to the corresponding values for the untreated heart (24.21±0.53 and 35.00±0.89 µmol/g dry tissue, respectively, n=5).


Figure 5
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  		Fig. 5 Myocardial ATP (the upper panel) and creatine phosphate (CP) contents at the ends of ischemia and reperfusion. The hearts were untreated (Un, n=5) or treated with 0.1 (n=4), 0.3 (n=4), or 1 mM MPG (n=5) for the last 30 min of preischemia and with 1 mM MPG (1R, n=4) for the first 30 min of reperfusion. Expressions for columns and symbols as in Fig. 3.

 
3.6 Effect of sodium lactate on mitochondrial membrane potential
The left panel of Fig. 6 shows the effect of sodium lactate, an end product of glycolysis in the ischemic myocardium, on the membrane potential of isolated mitochondria. The membrane potential of isolated mitochondria was approximately –180 mV (n=5). The right panel of Fig. 6 shows that incubation of isolated mitochondria with 25 or 50 mM sodium lactate decreased membrane potential in a concentration-dependent manner, whereas incubation with 12.5 mM sodium lactate did not decrease it (n=4 each). When the isolated mitochondria were treated with 1 mM MPG, sodium lactate-induced membrane depolarization was slightly but significantly attenuated (n=4). Choline chloride at a concentration of 12.5 mM, instead of sodium lactate, did not affect sodium lactate-induced changes in the membrane potential of the isolated mitochondria.


Figure 6
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  		Fig. 6 Effects of sodium lactate (Na+; cross striped columns) on membrane potential of isolated mitochondria (the left panel) and those of N-(2-mercaptopropionyl)-glycine (MPG; hatched column) on the membrane potential of isolated mitochondria in the presence of 25 mM sodium lactate (the right panel). The open column represents the basal value for the mitochondrial membrane potential untreated with any agent (Basal). The striped column represents the value for the membrane potential of the mitochondria in the presence of 25 mM sodium lactate (Na+). Each value represents the mean±S.E.M. of four experiments. #, Significantly different from the Basal group (P<0.05). *, Significantly different from the Un group (P<0.05).

 
3.7 Effect of phenylarsine oxide on mitochondrial membrane potential
Fig. 7 shows the effects of phenylarsine oxide on mitochondrial membrane potential. Mitochondrial membrane was depolarized by incubation with 3–30 µM phenylarsine oxide (n=4 each). Incubation of isolated mitochondria with 1 mM MPG attenuated the phenylarsine oxide-induced depolarization of membrane potential (n=4 each).


Figure 7
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  		Fig. 7 Effects of various concentrations (1–30 µM) of phenylarsine oxide (P; striped columns) and those of 1 mM N-(2-mercaptopropionyl)-glycine (M) in the presence of phenylarsine oxide on the membrane potential of isolated mitochondria (hatched columns). The open column represents the basal value for the mitochondrial membrane potential untreated with any agent (Basal). Each value represents the mean±S.E.M. of four experiments. #, Significantly different from the Basal group (P<0.05). *, Significantly different from the corresponding phenylarsine oxide-treated group (P<0.05).

 
3.8 Effect of MPG on sodium lactate and phenylarsine oxide at low concentrations on membrane depolarization and swelling of isolated mitochondria
Fig. 8 shows the effects of MPG on sodium lactate-, phenylarsine oxide-, or both-induced depolarization and swelling of isolated mitochondria (n=4 each). In this experiment, sodium lactate and phenylarsine oxide were used at concentrations that did not elicit significant changes in these parameters. When isolated mitochondria were incubated in the presence of either 12.5 mM sodium lactate or 1 µM phenylarsine oxide, the membrane potential of isolated mitochondria was not altered (the upper panel in Fig. 8). However, incubation with a combination of 12.5 mM sodium lactate and 1 µM phenylarsine oxide significantly depolarized the mitochondrial membrane. Treatment of isolated mitochondria with 1 mM MPG attenuated the depolarization induced by the combination of these two agents.


Figure 8
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  		Fig. 8 Effects of 12.5 mM sodium lactate (Na+), 1 µM phenylarsine oxide (Ph), combination of these two agents (Na++Ph), and combination of these two agents and 1 mM N-(2-mercaptopropionyl)-glycine (Na++Ph+MPG) on mitochondrial membrane potential (the upper panel) and mitochondrial swelling (the lower panel). The open columns represent the basal values for the membrane potential and mitochondrial swelling of isolated mitochondria untreated without any agent (Basal). #, Significantly different from the corresponding Basal group (P<0.05). *, Significantly different from the corresponding MPG-untreated group (P<0.05).

 
Incubation of isolated mitochondria with 12.5 mM sodium lactate decreased the absorbance at 540 nm, an indicator of mitochondrial swelling, to a small but significant degree, whereas there were no changes in the absorbance of the mitochondria incubated with 1 µM phenylarsine oxide alone (the lower panel in Fig. 8). When isolated mitochondria were incubated with a combination of 12.5 mM sodium lactate and 1 µM phenylarsine oxide, a significant decrease in the absorbance at 540 nm was observed. Treatment of mitochondria with 1 mM MPG attenuated this decrease. Choline chloride at 12.5 mM, instead of sodium lactate, did not affect the membrane potential and swelling of the isolated mitochondria.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
4.1 Sodium overload
In the present study, we observed a marked increase in Na+ content during ischemia and ischemia–reperfusion and a significant prevention of the ischemia-induced increase in the Na+ content by pretreatment with MPG. MPG also concentration-dependently preserved the OCR in the ischemic as well as reperfused hearts. Furthermore, myocardial HEPs were concentration-dependently restored by treatment with MPG. These findings suggest that MPG may play an appreciable role in the attenuation of cytosolic sodium overload and preservation of the mitochondrial function during ischemia, which may lead to better postischemic contractile recovery and tissue energy level of the ischemic–reperfused heart.

We showed in a previous study that ischemia-induced sodium overload and the associated impairment of the mitochondrial function were attenuated by pretreatment with a Na+ channel blocker, tetrodotoxin, and a Na+/H+ exchange inhibitor, ethylisopropyl amiloride [12]. This suggests that there are at least two pathways for ischemia-induced Na+ influx of the perfused heart. There are, however, no reports showing that MPG may pharmacologically suppress these Na+ flux routes during ischemia. Ju et al. [26] have shown that there is a Na+ current that operates during ischemia, which can be blocked by fairly high concentrations of tetrodotoxin and lidocaine. No information is available concerning the effect of MPG on this Na+ current in ischemia. Recently, Sarbi et al. [27] showed hydrogen-peroxide-activated Na+/H+ exchange in neonatal rat cardiomyocytes. This finding suggests that attenuation of a hydroxyl-radical-induced increase in the activity of Na+/H+ exchanger by MPG might modify the sodium influx in ischemic or reperfused hearts. Furthermore, it is also considered that MPG, a reducing agent, might bind to disulfide moieties present in the molecules of Na+ channels and Na+/H+ exchangers and lead to modification of sodium influx during ischemia and reperfusion. Further studies are required to address the route of Na+ entry that MPG may affect during ischemia and reperfusion.

4.2 High-energy phosphates
We observed in previous studies that amelioration of postischemic contractile function by agents always required treatment prior to ischemia [13,14,18] and that postischemic recovery of cardiac contractile function was always associated with restoration of myocardial energy levels during reperfusion in the perfused rat heart [17,28]. Thus, we focused, in the present study, on myocardial energy-producing events that may occur during ischemia. Treatment with MPG restored HEPs at the end of reperfusion in a concentration-dependent manner. Since the the infarct area of the ischemic–reperfused heart was relatively small (approximately 23% of the left ventricle), changes in HEPs of ischemic–reperfused hearts treated with and without MPG may represent those in the noninfarct area, which substantially depend on functional status of the mitochondrial ability to produce ATP.

It has been postulated that several agents such as diltiazem and propranolol may protect the heart from ischemia–reperfusion injury through the mechanism underlying negative inotropic effects during preischemia and the following energy-sparing effect during ischemia [29]. To elucidate this possibility under the present experimental conditions, we examined the negative inotropic effects on the perfused heart during administration of MPG and myocardial energy levels prior to reperfusion. Although we observed concentration-dependent negative inotropic effect in the MPG-treated heart during preischemia, HEP levels in the MPG-treated hearts prior to ischemia (at the end of the administration) were similar to those in the untreated heart. In addition, there were no differences in myocardial HEPs at the end of ischemia between the heart treated with MPG and the untreated heart. These findings suggest that energy-sparing effect is unlikely to have occurred in the MPG-treated, ischemic–reperfused heart. Despite such a suggestion, we cannot rule out the possibility that preservation of myocardial HEPs and/or modification of myocardial energy metabolites during ischemia by MPG, if any, might contribute to the maintenance of cardiac contractility or membrane integrity of the ischemic heart.

4.3 Lipid peroxidation during reperfusion
Since free radicals generated during reperfusion are considered to induce peroxidation of membrane phospholipids [2,30], we determined TRS content in the ischemic and ischemic–reperfused myocardium. There was no increase in myocardial TRS content in the ischemic heart, whereas TRS was increased in the ischemic–reperfused heart, suggesting that lipid peroxidation of the sarcolemma and/or mitochondria may be induced during reperfusion. Treatment of preischemic hearts with MPG attenuated the ischemia–reperfusion-induced increase in TRS content. This effect suggests a substantial contribution of MPG to protection of the heart against free radical attack during reperfusion. On the other hand, the heart treated only for the first 30 min of reperfusion did not show any improvement of myocardial contractile function and release of CK during reperfusion, despite the complete abolition of the ischemia–reperfusion-induced increase in TRS content. Thus it appears that free radical formation during reperfusion is dissociated from the ischemia–reperfusion-induced contractile dysfunction under experimental conditions.

4.4 Effect of MPG on mitochondrial membrane
MPG enhanced the restoration of myocardial HEPs during reperfusion and preserved the mitochondrial OCR of both ischemic and ischemic–reperfused hearts. These findings suggest that MPG may be an effective agent for preservation of the mitochondrial ability to produce energy in the ischemic and ischemic–reperfused heart. Therefore, we focused on the direct effect of MPG on mitochondrial membrane integrity using isolated mitochondria.

In the present study, the mitochondrial membrane potential was depolarized in the presence of sodium lactate, suggesting that sodium overload in the ischemic myocardium may disturb the steady state level of the mitochondrial membrane potential. This is consistent with the suggestion that oxygen deficiency in cardiomyocytes induces changes in mitochondrial membrane potential, that is, a decrease in {Delta}{psi} [31]. MPG significantly attenuated the sodium lactate-induced decrease in the mitochondrial membrane potential. Thus, MPG is likely to prevent a decrease in membrane potential of the mitochondria in ischemic hearts.

We further examined the effects of MPG on sodium overload- and membrane oxidation-induced changes in the mitochondrial membrane potential, which may be induced during reperfusion, to explore the possible cardioprotective action of the agent. We found that the combination of sodium lactate and phenylarsine oxide, although neither agent alone changed it, markedly decreased mitochondrial membrane potential. This finding suggests that mitochondrial membrane depolarization may intensively occur in the sodium-overloaded, reperfused heart. Continuous depolarization of the mitochondrial membrane potential is considered to alter the mitochondrial membrane integrity and eventually may lead to disruption of the mitochondrial structure, the so-called mitochondrial swelling [25]. In accordance with this, we observed that the combination of 12.5 mM sodium lactate and 1 µM phenylarsine oxide immediately elicited mitochondrial swelling, although incubation of isolated mitochondria with either sodium lactate or phenylarsine oxide alone induced mitochondrial swelling only to a minor or minimum degree. This finding also suggests that sodium overload induced potentiation of the mitochondrial swelling. MPG attenuated the mitochondrial swelling induced by the combination of 12.5 mM sodium lactate and 1 µM phenylarsine oxide, suggesting that the agent may be a possible protector against ischemia–reperfusion-induced structural damage to the mitochondria of cardiac cells, in which cytosolic Na+ is overloaded and free radicals are generated. It is, however, noted that treatment with MPG only during the first 30 min of reperfusion did not improve all the functional and biochemical variables of the ischemic–reperfused heart, suggesting that the presence of MPG during ischemia is essential to the cardioprotective effect of MPG under the present experimental conditions.

4.5 Negative inotropic effect of MPG
In the present study, we observed the negative inotropic effect during the administration of MPG. There are very few reports that clearly described the negative inotropic effect of MPG in perfused hearts: MPG at concentrations of 0.3 mM [32] or 3 mM [33] elicited a negative inotropic effect in perfused rabbit hearts. Unfortunately, they did not address its mechanism. Our results cannot address the mechanism either. Since this event occurred at high concentrations of MPG, it is considered that the negative inotropic effect might be induced by a nonspecific mechanism. Typical drugs that elicit a negative inotropic effect and exert an energy sparing effect such as propranolol and diltiazem produced more profound negative inotropic effect than MPG during administration. Thus, this negative inotropic effect of MPG, though speculative, appears to be involved in the energy sparing effect to a lesser degree than that of those compounds.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
The present study has shown that MPG has a cardioprotective effect against ischemia–reperfusion-induced contractile dysfunction, as consistent with previous observations for tetrodotoxin or ethylisopropyl amiloride [12]. Several investigators have postulated the protection of the cardiac membrane against free radical attack as a possible mechanism underlying the cardioprotection of MPG against reperfusion injury in in vivo and in vitro experiments [1,3–8,15]. The present study proposed another possible mechanism for cardioprotection of MPG, which may prevent deterioration of the mitochondrial membrane integrity due to prevention of cytosolic sodium overload in the ischemic myocardium.

Time for primary review 28 days.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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