Objective: The present study aimed to elucidate the involvement of sodium overload and following damage to mitochondria during ischemia in the genesis of ischemia/reperfusion injury of perfused rat hearts. Methods: Isolated, perfused hearts were exposed to different durations (15–35 min) of ischemia followed by 60-min reperfusion. At the end of ischemia or reperfusion, myocardial sodium and calcium contents and myocardial high-energy phosphates were determined. The cardiac mitochondrial ability to produce ATP was measured using saponin-skinned bundles. The effects of sodium on the mitochondrial membrane potential and the oxidative phosphorylation rate were examined using isolated mitochondria from normal hearts. Results: Post-ischemic recovery of left ventricular developed pressure decreased in an ischemic duration-dependent manner. Ischemia induced an increase in myocardial sodium, but not calcium. This increase was dependent on the duration of ischemia. The oxygen consumption rate of skinned bundles from the ischemic heart decreased at the end of ischemia. Incubation of mitochondria with various concentrations of sodium chloride or sodium lactate in vitro resulted in a depolarization of mitochondrial membrane potential and a decrease in ATP-generating activity. This decrease was not restored after elimination of sodium compounds. Conclusions: The present findings suggest that ischemia induces an increase in sodium influx from the extracellular space and that accumulated sodium may induce irreversible damage to mitochondria during ischemia. This mitochondrial dysfunction may be one of the most important determinants for the genesis of ischemia/reperfusion injury in perfused rat hearts.
Time for primary review 30 days
There is accumulating evidence that sodium overload is caused by an increase in sodium flux into the intracellular space from the extracellular space under ischemic and hypoxic conditions and may play an important role in the genesis of ischemia/reperfusion injury [1,2]. The inhibition of sodium influx pathways in cardiomyocytes by pharmacological agents has been shown to attenuate sodium overload during ischemia and thus lead to better functional recovery after reperfusion [2–4]. The mechanism by which sodium overload induces ischemia/reperfusion injury was suggested to eventually induce calcium overload through the sodium/calcium exchanger during the early phase of reperfusion [1,5]. Despite such a significant role of sodium overload and subsequent calcium overload, the exact mechanism by which sodium overload may lead to the ischemia/reperfusion injury remains unclear.
Mitochondria produce significantly most of the cellular ATP in the working heart. Any perturbation in the mitochondrial ATP synthesis may have detrimental consequences for the heart [6,7]. It is well recognized that myocardial ischemia, which induces contractile failure of the reperfused heart, causes defects in the mitochondrial oxidative phosphorylation [8,9]. Since ATP is necessary for the contraction/relaxation cycle of cardiac muscle and for maintenance of myocardial energy-dependent processes, damage to the mitochondria would compromise cardiac pump function [10,11]. Several studies have shown a significant correlation of tissue ATP levels with post-ischemic functional recovery following ischemia/reperfusion [2,6,12]. However, temporal, qualitative, and quantitative aspects of multi-faced relation among mitochondrial function, myocardial energy levels, and post-ischemic cardiac function have not been well established. In the present study, we aimed to address the role of sodium overload in the mitochondrial function of the ischemic/reperfused heart.
2 Materials and methods
Male Wistar rats weighing 250–280 g (Japan Laboratory Animals Inc., Tokyo, Japan) were used in the present study. The animals were conditioned to an environment of 23±1 °C, a constant humidity of 55±5% and a cycle of 12-h light/12-h darkness, and given free access to food and tap water according to the Guide for the Care and Use of Laboratory Animals as promulgated by the National Research Council (National Academy Press, Washington D.C., 1996). The protocol of this study was approved by the Committee of Animal Care and Welfare of Tokyo University of Pharmacy and Life Science.
2.2 Perfusion of hearts
Perfusion of hearts was performed according to the method described previously . After anesthesia with diethyl ether, the hearts were isolated and perfused at 37 °C with a constant flow rate (9.0 ml/min) of Krebs–Henseleit bicarbonate 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. The perfusion buffer was equilibrated with a gaseous mixture of 95% O2 and 5% CO2 to pH 7.4. A latex balloon (diameter of 3.7 mm) connected to a pressure transducer (TP-200, Nihon Kohden, Tokyo, Japan) was inserted into the left ventricular cavity. A pressure of 5 mmHg of the initial left ventricular end–diastolic pressure (LVEDP) was loaded onto the perfused heart. Left ventricular developed pressure (LVDP) and heart rate (HR) were monitored by a pressure transducer (TP-200) and a heart rate counter (AT-601G, Nihon Kohden), respectively, throughout the experiment.
The perfusion was stopped to induce ischemia. During ischemia, the heart was immersed into the Krebs–Henseleit solution pre-gassed with a mixture of 95% N2+5% CO2 to avoid hypothermia-induced cardioprotection. After appropriate sequences of ischemia (for 15–35 min), the buffer in the organ bath was drained, and the hearts were reperfused for 60 min. The hearts were electronically paced throughout the experiment at the rate of 300 beats/min except for the first 15 min of reperfusion, to prevent contractile irregularities that might sometimes occur during this period.
2.3 Determination of myocardial sodium and calcium content
After ischemia, reperfusion, or normoxic perfusion, hearts were perfused with 8 ml of 320 mM sucrose–20 mM Tris–HCl, pH 7.4. Approximately 100 mg of the left ventricular free wall was dried at 120 °C for 48 h and then digested with 60% HNO3. After evaporation at 180 °C, the residues were dissolved in 0.75 N HNO3 and the myocardial sodium and calcium contents were determined by the atomic absorption method (AA-680, Shimazu, Kyoto, Japan).
In a previous study , we extensively characterized this method. Values for myocardial cations after washing with 8 ml of the sucrose buffer employed in the present study were quite similar to those determined theoretically by the Co2+-EDTA method on the basis that Co2+-EDTA is exclusively distributed to the extracellular space. From these findings, it is concluded that tissue cations determined by this method originated from the intracellular milieu and various organelles in cardiac cells.
2.4 Determination of high-energy phosphate (HEP)
After the appropriate sequence of perfusion, hearts were quickly freeze-clamped with aluminum tongs pre-cooled with liquid nitrogen. The frozen ventricle was extracted with 0.3 M HClO4 and 0.25 mM EDTA under cooling with liquid nitrogen. The extract was sampled for determination of ATP and creatine phosphate (CP) according to the HPLC method as described previously .
2.5 Oxygen consumption rate of myocardial skinned bundle
The mitochondrial oxygen consumption rate (OCR) was determined by the method of Sanbe et al. , a modified method of Saks et al. . After appropriate sequences of perfusion, the heart was quickly removed from the perfusion apparatus. Myocardial bundles were prepared from the left ventricular free wall and transferred into the relaxing medium A of the following composition (mM): EGTA, 10; MgSO4, 3; taurine, 20; dithiothreitol, 0.5; imidazole, 20; potassium 2-(N-morpholino)-ethanesulfonate, 160; ATP, 5; CP, 15 (pH 7.0). The bundles were incubated for 20 min in the medium A additionally containing 75 μg/ml saponin. After incubation, the myocardial bundles were washed with fresh medium B (the medium A without ATP and CP but supplemented with 0.5% bovine serum albumin). The OCR of skinned bundles was determined in the medium B at 30 °C by means of a Clark-type electrode (Central Kagaku, Tokyo). The basal OCR (V0) was measured following the addition of 5 mM glutamate, 3 mM malate, and 3 mM KH2PO4. Total (maximal) OCR (Vmax) was measured after the further addition of 1 mM ADP and 7.5 mM creatine. The ADP-stimulated OCR (VADP) of the skinned bundles was taken as the difference between Vmax and V0. After the determination of the OCR, the skinned bundles were solubilized with 1 N NaOH and then the protein concentration was determined .
2.6 Measurement of mitochondrial activity
2.6.1 Isolation of mitochondria
Myocardial mitochondria were isolated as described previously . In brief, the heart isolated from the rat anesthetized with diethyl ether was homogenized in ice-cold buffer containing 180 mM KCl, 10 mM EGTA (pH 7.4), and 0.5% fatty acid-free BSA. After centrifugation of the homogenate at 1000×g for 10 min, the resultant supernatant solution was centrifuged at 8000×g for 10 min. The crude mitochondria was again suspended in buffer and centrifuged at 8000×g for 10 min. Mitochondria were resuspended with suspension buffer (320 mM sucrose, 20 mM Tris–HCl, pH 7.4) and used for measurement of mitochondrial activity. Protein concentrations were determined by the method of Lowry et al. .
2.6.2 Measurement of mitochondrial membrane potential
Membrane potential of isolated mitochondria was measured at 30 °C by the method of Kamo et al. using tetraphenylphosphonium chloride (TPP+)-sensitive electrodes . TPP+ (3 μM) was added to the incubation medium containing 110 mM KCl, 5 mM K2HPO4, 10 mM 3-morpholinopropanesulfonic acid (pH 7.3) and then the mitochondria were added. The mitochondrial membrane potential was calculated according to the method described by others . The mitochondrial membrane potential was measured in the presence of sodium chloride or sodium lactate.
2.6.3 Measurement of mitochondrial respiration
Mitochondria were placed into the incubation medium containing (mM): sucrose, 210; K2HPO4, 5; glutamate, 10 (pH 7.4) and stirred at 30 °C . Oxygen consumption of mitochondria was measured in the chamber using a Clark-type oxygen electrode (Central Kagaku). The quality of the mitochondrial preparation was evaluated by assessing the respiratory control index determined in the presence of 240 μM ADP. Mitochondria with respiratory control index over 10 were used in this study. The effects of sodium salts (sodium chloride or sodium lactate) on state 3 respiration were examined.
To determine whether sodium salts-induced reduction in the oxidative phosphorylation activity was reversible, we at first measured the state 3 respiration of the mitochondria. The mitochondria were washed with buffer and then collected by centrifugation at 8000×g. Thereafter, the mitochondrial respiration was again determined. Values before and after the washing were compared.
2.7 Experimental protocol
In the present study the following experimental protocols (A–D) were carried out:
The perfused rat hearts were subjected to different periods of ischemia (15–35 min) and subsequent 60-min reperfusion. After assessment of hemodynamic parameters, the myocardial sodium and calcium contents were determined.
The hearts were subjected to different periods of ischemia and subsequent 60-min reperfusion as described in protocol A. After ischemia or reperfusion, the heart was freeze-clamped and then the myocardial ATP and CP were determined.
The perfused hearts were subjected to the same protocol as described in protocol A. After ischemia or reperfusion, the myocardium was used for determination of the oxygen consumption rate of saponin-skinned bundles prepared from the left ventricular free wall.
Mitochondria were isolated from normal rat hearts, and their mitochondrial membrane potentials or oxygen consumption rates were determined in the presence and absence of various concentrations of sodium chloride or sodium lactate. The oxidative phosphorylation that had been exposed to sodium chloride or sodium lactate was also determined after washing of the sodium salt.
The results were expressed as the means±S.E.M. Statistical significance was estimated by analysis of variance (ANOVA) followed by Dunnett's multiple comparison. The relationship between two parameters was calculated by the least squares method. Differences with a probability of less than 5% were considered to be statistically significant (P<0.05).
3.1 Changes in LVDP of the ischemic and ischemic/reperfused hearts
In the first set of experiments, rat hearts were subjected to different periods of ischemia (15- to 35-min) and subsequent 60-min reperfusion, and changes in LVDP were determined. The baseline (initial) value for LVDP was 81±3 mmHg (n = 25). The time courses of changes in the LVDP of perfused hearts subjected to different periods of ischemia are expressed as percentages to the initial value. After the onset of ischemia, LVDP declined to zero within 2.5 min and remained at this level during ischemia and the LVDPs of perfused hearts at the end of the different periods of ischemia ranging from 15 to 35 min were not detected. When the heart was reperfused, the LVDP was recovered toward the initial levels. The LVDP recovery of the ischemic/reperfused heart was inversely related to the duration of the ischemia loaded (Fig. 1).
The time course of changes in left ventricular developed pressure (LVDP) of perfused rat hearts. The hearts were subjected to different durations [15 (●), 20 (▴), 25 (▾), 30 (■), and 35 min (♦)] of ischemia followed by 60-min reperfusion. Each value represents the mean±S.E.M. of five experiments.
3.2 Changes in myocardial sodium and calcium contents at the end of ischemia and at the end of reperfusion
Myocardial sodium and calcium contents were determined at the end of different periods of ischemia or at the end of 60-min reperfusion (Fig. 2). The left panels in Fig. 2 show the time course of the changes in the myocardial sodium or calcium contents under ischemic conditions. The myocardial sodium content gradually increased with the duration of ischemia and reached an approximately 2-fold increase of the baseline value at 35-min ischemia (the left upper panel in Fig. 2). In contrast, the myocardial calcium content did not change regardless of the durations of ischemia loaded (the lower panel in Fig. 2).
Myocardial sodium (upper panel) and calcium contents (lower panel) of the heart prior to ischemia (Basal; open columns), at the end of different durations of ischemia ranging from 15 min (15-I) to 35 min (35-I), and at the end of reperfusion following different durations of ischemia ranging from 15 min (15-I+Re) to 35 min (35-I+Re). Basal values for myocardial sodium and calcium contents were 56.8±3.8 and 1.85±0.04 μmol/g dry tissue, respectively. Each value represents the mean±S.E.M. of five experiments. *P<0.05 vs. Basal.
The right panels in Fig. 2 show the myocardial sodium and calcium contents at the end of 60-min reperfusion following different periods of ischemia ranging from 15 to 35 min. When the heart was subjected to 30- to 35-min ischemia and then reperfused, a further increase in myocardial sodium content was observed during reperfusion (the right upper panel in Fig. 2). Myocardial calcium content of the ischemic/reperfused heart also increased with prolonged periods of ischemia (the right lower panel in Fig. 2).
3.3 Relation between post-ischemic contractile recovery and myocardial sodium or calcium content of ischemic or ischemic/reperfused heart
Post-ischemic recovery of LVDP was plotted against myocardial sodium or calcium content at the end of different durations of ischemia or at the end of ischemia/reperfusion (Fig. 3). The post-ischemic recovery of LVDP was inversely related to the myocardial sodium content at the end of ischemia (the left upper panel in Fig. 3). The post-ischemic recovery of LVDP was also inversely related to the myocardial sodium content at the end of reperfusion following different durations of ischemia (the right upper panel in Fig. 3). The post-ischemic recovery of LVDP was inversely related to myocardial calcium content at the end of reperfusion, whereas no significant relation between post-ischemic LVDP recovery and myocardial calcium content at the end of ischemia was observed (lower panels in Fig. 3).
The relation between post-ischemic recovery of left ventricular developed pressure (LVDP) and myocardial sodium (upper panels) or calcium content (lower panels) at the end of different durations of ischemia (left panels) or at the end of subsequent reperfusion (right panels). Significant relation between post-ischemic recovery of LVDP and myocardial sodium content at the end of ischemia (n = 5) or reperfusion (n = 25) were seen (P<0.05). A significant relation between post-ischemic recovery of LVDP and myocardial calcium content at the end of reperfusion, but not ischemia, was observed (P<0.05).
3.4 HEP content of ischemic/reperfused hearts
Fig. 4 shows the myocardial ATP and CP contents at the end of reperfusion following the different durations of ischemia. At the end of 60-min reperfusion, restoration of myocardial ATP was parallel to duration of ischemia loaded. Restoration of myocardial CP at the end of 60-min reperfusion, similar to that of myocardial ATP, was also parallel to the duration of the ischemia loaded.
Myocardial ATP (left upper panel) and creatine phosphate contents (right upper panel) at the end of reperfusion following the different durations of ischemia ranging from 15 min (15-I+Re) to 35 min (35-I+Re) and relation between post-ischemic recovery of left ventricular developed pressure (LVDP) and myocardial ATP (left lower panel) or creatine phosphate content (right lower panel) at the end of reperfusion. Pre-ischemic values for myocardial ATP and creatine phosphate contents were 23.3±1.5 and 35.7±2.4 μmol/g dry tissue (n = 5), respectively. Each value represents the mean±S.E.M. of five experiments. Significant relations between post-ischemic recovery of LVDP and myocardial ATP or creatine phosphate content at the end of reperfusion were observed (P<0.05).
Both ATP and CP were reduced in the ischemic heart irrespective of the periods of ischemia under the present experimental conditions (data not shown), suggesting that energy-sparing effects did not involve the post-ischemic recovery of LVDP or the post-ischemic restoration of HEP.
3.5 Effects of ischemic duration on OCR of ischemic or ischemic/reperfused hearts
Ischemic hearts subjected to different durations of ischemia and the hearts reperfused following the different durations of ischemia were removed from the apparatus and the oxygen consumption rate of their skinned bundles were determined (Fig. 5). Different periods of ischemia were employed in order to elucidate the relation between LVDP recovery and OCR activity. The OCR for pre-ischemic hearts was 63.1±1.8 n atom O/min/mg protein (n = 5). There were no significant differences in the mitochondrial OCR of perfused hearts under normoxic conditions, regardless of the time for normoxic perfusion. The OCR was decreased in an ischemic duration-dependent manner. The decrease in the OCR of the heart at 35-min ischemia reached approximately 30% of the pre-ischemic value (n = 5). The OCRs of the reperfused hearts previously subjected to 25-, 30-, and 35-min ischemia remained at the level similar to that at the end of ischemia (n = 5 each). There was a close relation between post-ischemic LVDP recovery and OCR of the myocardial bundles at the end of ischemia/reperfusion (the right panel of Fig. 6). Similarly, a significant correlation of the post-ischemic LVDP recovery with the OCR of myocardial bundles at the end of ischemia was shown in terms of the hearts loaded with the same ischemic insult (the left panel of Fig. 6). It should be noted that no necrotic area could be observed in the heart at the end of ischemia when determined by the staining with triphenyl tetrazolium chloride (data not shown).
Oxygen consumption rate of skinned bundles isolated from the heart prior to ischemia (Basal; open columns), at the end of different durations of ischemia ranging from 15 min (15-I) to 35 min (35-I), and at the end of reperfusion following different durations of ischemia ranging from 15 min (15-I+Re) to 35 min (35-I+Re). Basal value for the oxygen consumption rate of skinned bundles was 63.1±1.8 n atom O/min/mg protein (n = 5). Each value represents the mean±S.E.M. of five experiments. *P<0.05 vs. Basal.
Relations between post-ischemic recovery of left ventricular developed pressure (LVDP) and the oxygen consumption rate of skinned bundles from the hearts at the end of different durations of ischemia (left panel) (n = 5) and those at the end of reperfusion (right panel) (n = 25). Significant relations between post-ischemic recovery of LVDP and the oxygen consumption rate of skinned bundles at the end of ischemia and at the end of subsequent reperfusion were seen, respectively (P<0.05).
3.6 Mitochondrial activity and sodium
Fig. 7 shows the mitochondrial membrane potential in the presence and the absence of various concentrations of sodium chloride or sodium lactate. The mitochondrial membrane potential in the absence of these sodium salts was approximately −180 mV. When sodium chloride was added to the incubation medium, the mitochondrial membrane potential was depolarized in a concentration-dependent manner. Addition of sodium lactate into the medium induced a larger depolarization of the mitochondrial membrane potential than that of sodium chloride. To assess the effect of an increase in osmolarity of the medium, we added 50 mM choline chloride to the medium instead of 50 mM sodium chloride or sodium lactate, and found no changes in the membrane potential by the concentration of these compounds.
Effects of various concentrations of sodium chloride (striped columns) and sodium lactate (hatched columns) on the membrane potential of isolated mitochondria. The basal value for mitochondrial membrane potential was approximately −180 mV, when measured by using the membrane potential-sensitive probe of tetraphenylphosphonium chloride. Each value represents the mean±S.E.M. of four experiments. *P<0.05 vs. Cont.
To further characterize the effects of sodium on mitochondrial function, the mitochondrial ATP-generating ability was determined in the presence and absence of different concentrations of sodium chloride or sodium lactate (Table 1). The values for state 3 respiration were slightly but significantly decreased along with the increased concentrations of sodium chloride in the incubation medium. When isolated mitochondria were incubated with sodium lactate, a possible metabolite in ischemic hearts, the degree of a decrease in state 3 respiration was greater than that of mitochondria treated with sodium chloride. The increasing concentrations of sodium lactate deteriorated these parameters for mitochondrial respiration.
Effects of sodium chloride and sodium lactate on the mitochondrial state 3 respiration
State 3 respiration
(n atom O/min/mg protein)
Each value represents the mean±S.E.M. of four experiments. *P<0.05 vs. control group. The washing procedure did not affect the value of state 3 respiration of isolated mitochondria.
To elucidate whether this deterioration was reversible, the state 3 respiration of the isolated mitochondria was determined before and after washing of the isolated mitochondria with buffer. The values for the respiration of the mitochondria showed no difference before and after the washing of sodium chloride and sodium lactate, indicating that this washing procedure did not alter the mitochondrial activity. Despite removal of sodium from mitochondria by the washing procedure, the values for state 3 respiration were similar to those before washing (Table 1).
4.1 Myocardial sodium content and post-ischemic cardiac function
At first, we determined whether either sodium or calcium was pathophysiologically altered during ischemia and reperfusion in perfused rat hearts. We observed that the sodium content of the ischemic myocardium increased in an ischemic duration-dependent manner, whereas there was no change in myocardial calcium content during ischemia, as shown in Fig. 2. Although the NMR study has shown an increase in the intracellular free calcium , the present results showed that massive accumulation of calcium in cardiac cells during ischemia would be unlikely. When the heart was reperfused after 30–35 min of ischemia, a further increase in sodium content was seen, suggesting that an additional influx of sodium into the intracellular space occurred in the heart in which severe sodium overload had been evoked during ischemia. Since several investigators suggest that a sodium/hydrogen exchanger may function during the early phase of reperfusion [3,19,20], it is likely that myocardial sodium during reperfusion may be accumulated via this antiporter. Reperfusion also increased the myocardial calcium content. This calcium influx may be exerted via the sodium/calcium exchanger . As shown in Fig. 2, we also observed that the degrees of myocardial sodium accumulation during ischemia and ischemia/reperfusion were dependent on the duration of the ischemia loaded. There was no significant relation between the myocardial calcium content at the end of ischemia and post-ischemic recovery of LVDP. In contrast, the myocardial calcium accumulation of the reperfused heart was dependent on duration of ischemia. These findings suggest that sodium accumulation during ischemia may initiate the ionic disturbance, such as calcium overload, in the ischemic/reperfused heart.
There are several pathways for sodium entry into cardiac cells, including fast sodium channels, sodium/hydrogen exchanger, sodium/calcium exchanger, and passive sodium influx under physiological and pathophysiological conditions . In a previous study , we observed the improvement of ischemia/reperfusion-induced ionic disturbance and contractile failure by fast sodium channel blockers such as TTX, lidocaine, and quinidine and a sodium/hydrogen exchanger inhibitor, dimethyl amiloride [2,22–24]. These previous findings suggest that inhibition of sodium channel or sodium/hydrogen exchanger may, at least in part, play an important role in the ischemia-induced myocardial sodium accumulation and that contribution of a single pathway to this effect would be unlikely. Recently, several studies [5,25] have shown the presence of a persistent and inactivation-resistant sodium current in cardiac cells. This sodium current is observed when the cells are hypoxic, evoked by depolarization, or blocked by TTX or lidocaine. Thus, this current may be entirely different from the physiological fast sodium current. The exact route for sodium entry under the ischemic conditions remains to be elucidated.
4.2 Effects on high-energy phosphates
There are controversial findings regarding the correlation of post-ischemic recovery of contractile function with restoration of cellular ATP levels following myocardial ischemia/reperfusion. Neely and Grotyohann  postulated no relation between cellular ATP levels and post-ischemic functional recovery of ischemic/reperfused rat hearts. In contrast, Docherty et al.  reported a correlation of cellular ATP levels with post-ischemic functional recovery of ischemic/reperfused rat hearts. In our previous studies, enhanced post-ischemic contractile function was always associated with restoration of HEPs in perfused rat  and rabbit hearts . In the present study, we also showed that there was the close relation between the post-ischemic recovery of cardiac contractile recovery and restoration of myocardial HEP during reperfusion as shown in Fig. 4, suggesting that HEP levels of the heart may play an important role in the post-ischemic contractile recovery.
Since myocardial mitochondria produce most HEP to exert contractile function, we focused on the mitochondrial activity, particularly the ability to produce HEPs, of the ischemic/reperfused myocardium. We prepared skinned bundles from the left ventricular free wall of ischemic or ischemic/reperfused hearts, and then determined their oxygen consumption rate, which may reveal the mitochondrial ability to produce ATP in cardiac tissue. This method is convenient to determine the mitochondrial ATP-generating ability in cardiac muscles without isolation procedures of mitochondria . In the present study, we found that the mitochondrial activity at the end of ischemia markedly decreased despite the heart having no necrotic area at this time . As shown in Fig. 5, this decrease was dependent on the duration of ischemia. Furthermore, there was a correlation of the mitochondrial activity in the ischemic myocardium with restoration of myocardial HEPs at the end of reperfusion (Fig. 4). There was also a close relation between mitochondrial activity in the ischemic myocardium and post-ischemic recovery of the contractile function of perfused hearts (Fig. 6). The present findings suggest that the mitochondrial damage may precede the genesis of reperfusion injury, that is, this mitochondrial damage during ischemia may be substantially involved in energy production and contractile failure of the ischemic/reperfused heart.
4.3 Effect of sodium on mitochondria
To elucidate the mechanism underlying the relationship between sodium overload and mitochondrial damage, isolated mitochondria were incubated in the presence of various concentrations of sodium in vitro. As shown in Fig. 7 and Table 1, an exposure of isolated mitochondria to excess sodium, which mimicked sodium overload in the intracellular space of the ischemic myocardium, induced a depolarization of the mitochondrial membrane potential and a depression of a mitochondrial state 3 respiration. The findings indicate impairment of the abilities to induce the membrane depolarization and to generate ATP by an increased concentration of sodium. Since it is well known that myocardial lactate content markedly increases during ischemia, we have measured these parameters in the presence of sodium lactate. We also found that sodium lactate caused a larger depolarization of the mitochondrial membrane potential and a decrease in state 3 respiration than sodium chloride did. The finding suggests that metabolites produced under ischemic conditions may strengthen detrimental effects of sodium in cardiac myocytes. Furthermore, the washing procedure did not restore the mitochondrial respiration that had once been exposed to sodium, suggesting that the effect of sodium overload on the oxidative phosphorylation may be of irreversible nature.
In conclusion, we showed in the present study that accumulation of myocardial sodium, but not calcium, occurred at the end of ischemia with prolonged periods of ischemia loaded, which was associated with ischemic duration-dependent contractile dysfunction after reperfusion. These events were accompanied by a reduction in the mitochondrial ability to produce ATP at the end of ischemia as well as at the end of reperfusion. These findings suggest that impairment of mitochondrial function caused by the intracellular sodium accumulation during ischemia may be an important determinant of cardiac function during reperfusion in perfused rat hearts.
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