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(Circulation Research. 1996;78:564-572.)
© 1996 American Heart Association, Inc.

Articles

Mechanism of Hydrogen Peroxide and Hydroxyl Free Radical–Induced Intracellular Acidification in Cultured Rat Cardiac Myoblasts

Mei-Lin Wu, Ke-Li Tsai, Seu-Mei Wang, Jiahn-Chun Wu, Bor-Sen Wang, Yuan-Teh Lee

From the Departments of Physiology (M.-L.W., K.-L.T.) and Anatomy (S.-M.W., J.-C.W.), the Institute of Toxicology (B.-S.W.), and the Department of Internal Medicine (Y.-T.L.), Center for Cardiovascular Research, College of Medicine, National Taiwan University, Taipei, ROC.

Correspondence to Dr Yuan-Teh Lee, Department of Internal Medicine, Medical College, National Taiwan University Hospital, 7, Chung-Shan South Rd, Taipei, Taiwan, ROC.

	Abstract

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Abstract After a transient ischemic attack of the cardiac vascular system, reactive oxygen-derived free radicals, including the superoxide (O₂^-) and hydroxyl (OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H₂O₂) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pH_i acidification (0.24 pH unit) is induced by the addition of 100 µmol/L H₂O₂ and that this dose is without effect on the intracellular free Ca²⁺ levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of OH, and not O₂^- or H₂O₂, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pH_i regulators (the Na⁺-H⁺ exchanger, the Cl^--HCO₃ exchanger, and the Na⁺-HCO₃ cotransporter), (2) a rise in intracellular Ca²⁺ levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H₂O₂-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pH_i decrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia.

Key Words: cardiac myocytes • hydroxyl free radical • reperfusion cardiac stunning • intracellular acidosis • rat H9c2 cardiac cell line

	Introduction

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It is well documented that ROS, such as the superoxide (O₂^-) and hydroxyl (OH) radicals, and hydrogen peroxide (H₂O₂) are formed during reperfusion or reoxygenation of ischemic or hypoxic myocardium.^{1 2 3} The initial burst of ROS production peaks 2 minutes after reflow, and production continues for up to 3 hours.³ These ROS are derived from a variety of sources, such as activated neutrophils,^{4 5} leakage of electrons from mitochondria,⁶ the xanthine oxidase system,¹ and the cyclooxygenase pathway of arachidonic acid metabolism.⁷ O₂^- has been implicated as a mediator of ischemia/reperfusion-induced leukocyte adhesion in postcapillary venules⁸ and of increased microvascular permeability,⁹ but it is OH that is suggested as having the most damaging effects, including cytotoxicity and cardiac stunning, during ischemic reperfusion.^{1 10 11 12} Moreover, both the oxygen-derived free radicals, O₂^- and OH, are implicated as the major factors responsible for reperfusion-induced cardiac arrhythmia,^{13 14} cardiac ultrastructural abnormalities,¹⁵ reduction of the Ca²⁺ transient and contractility,¹⁶ increased diastolic Ca²⁺ levels,¹⁶ inhibition of glycolysis and oxidative phosphorylation and intracellular ATP depletion.^{10 11 16 17} During ischemia or reperfusion, intracellular measurements show that the free Mg²⁺ level in cardiac tissues increases as a result of the depletion of intracellular ATP.^{18 19 20}

H₂O₂ is an important tool for studying the effects of oxygen-derived free radicals on the reperfused ischemic myocardium.^{16 21} It can readily cross the cell membrane²² and be converted, via the Fenton reaction, to the more toxic OH radicals in the presence of ferrous ions^{22 23} :

In the present study, we have shown, for the first time, that a profound intracellular acidification (0.22 pH units) is induced by the addition of 100 µmol/L H₂O₂ to freshly isolated single rat cardiac myocytes or to an established cardiac cell line, H9c2, which also possesses the cardiac L-type Ca²⁺ channels,^{24 25} Na⁺-H⁺ exchanger, and DIDS-sensitive pH_i regulation (present study) seen in freshly isolated cardiac myocytes. Since the mechanism of this interesting pH acidification induced by H₂O₂ perfusion is not yet understood, we used the H9c2 cell line to study the mechanism of H₂O₂-induced acidification in cardiac cells. In addition, we discuss the possible role of intracellular acidification in reperfusion cardiac stunning.

	Materials and Methods

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Chemicals and Solutions
Unless otherwise stated, all chemicals were purchased from Sigma Chemical Co. All experiments were performed at 37°C in HEPES-buffered solution consisting of (mmol/L) NaCl 118, KCl 5.0, MgCl₂ 1.0, CaCl₂ 2.0, glucose 10, and HEPES 20, with pH adjusted to 7.4 at 37°C with NaOH.

Isolation of Single Rat Cardiac Myocytes
The method for isolating rat cardiac myocytes has been described in detail in our previous work.²⁶ In brief, adult male Wistar rats (250 to 300 g) were killed by injection of 35 mg/kg IP pentobarbital. Single cardiac myocytes were prepared using a combination of enzyme digestion (1.0 mg/mL collagenase type I and 0.1 mg/mL protease type XIV) and mechanical dispersion.²⁷

Cell Culture and Cell Treatment
Rat cardiac myoblasts (H9c2, a permanent cell line derived from the embryonic rat ventricle) were obtained from the American Type Culture Collection (Rockville, Md) and grown in DMEM supplemented with 10% FCS, 100 IU/mL penicillin, and 100 mg/mL streptomycin on 24-mm-diameter cover glasses in 35-mm dishes in an atmosphere of 5% CO₂/95% humidified air at 37°C.

Immunofluorescence Staining and Characteristics of Cultured H9c2 Cells
Frozen sections of rat heart or cultured H9c2 cells were fixed in cold acetone for 5 minutes and then incubated with a 1:100 dilution of rabbit anti-pan cadherin (Sigma) for 1 hour at 37°C. After they were washed in PBS, the specimens were treated with fluorescein-conjugated second antibody (1:50 dilution) for 1 hour, washed, and mounted.

N-Cadherin is a specific component of intercalated disks in cardiac muscle, as shown by the prominent staining of the intercalated disks of frozen rat heart sections using anti-pan cadherin antibody (data not shown). The cell junctions between H9c2 cells were also found to be positive for cadherin (data not shown). These cells have been shown to have all the characteristics of cardiac L-type Ca²⁺ currents^{24 25} and to undergo amiloride-sensitive Na⁺-H⁺ exchange and DIDS-sensitive pH_i regulation (present study), all of which are found in many cardiac tissues.^{28 29 30}

Measurement of pH_i
Measurement of pH_i has been described in detail elsewhere.²⁶ In brief, single cardiac myocytes or H9c2 cells, grown on a cover glass, were loaded with 5 µmol/L BCECF-AM (Molecular Probes) for 5 to 10 minutes at room temperature in HEPES-buffered solution. The cells were then washed with the same solution and excited alternately by 490- and 440-nm wavelength light, using a filter wheel (Cairn Research) rotating at 32 Hz. The excitation light was transmitted to the cell under study using a 510-nm dichroic mirror under the microscope nosepiece, and the resulting fluorescence was collected by a x40 oil-immersion lens. The overall sampling rate was 0.5 Hz. The 490/440 emission ratio from the intracellular BCECF was calculated and converted to a linear pH scale (see below) by in situ calibration at pH 4.5 and 9.5 performed at the end of the experiment using the nigericin technique, described elsewhere.^{26 31} The following equation was used to convert the fluorescent ratio into pH:

where R is the ratio of 530-nm fluorescence at 490-nm excitation to 530-nm fluorescence at 440-nm excitation; R_max and R_min are the maximum and minimum ratio values, respectively, from the data curve; and pK is the dissociation constant for the dye, taken as 7.15.²⁶

Measurement of Intracellular Ca²⁺ Levels
Intracellular Ca²⁺ ion levels were measured in the same way as the pH (see above), except the cells were loaded with fura 2-AM (2 µmol/L, Molecular Probes) for 20 to 30 minutes at 37°C. They were then excited alternately by 340- and 380-nm wavelength light by using a filter wheel rotating at 32 Hz. The excitation light was transmitted using a 400-nm dichroic mirror under the microscope nosepiece. The total sampling rate was 0.5 Hz. In the present study, the ratio of the 510-nm emission resulting from excitation at 340 and 380 nm was plotted directly, rather than after calibration and conversion to Ca²⁺ levels.

Measurement of Intracellular Mg²⁺ Levels
Intracellular Mg²⁺ levels were measured in the same way as Ca²⁺ levels, except the cells were loaded with Mg²⁺–fura 2 (2 µmol/L, Molecular Probes) for 20 to 30 minutes at 37°C.

Intracellular ATP Measurement Using the Luciferin-Luciferase Method
Intracellular ATP was extracted from H9c2 cells by boiling for 10 minutes in 1 mL of 100 mmol/L Tris-EDTA buffer (pH 7.75). After centrifugation, the ATP content in 0.2 mL of supernatant was measured using a luciferin-luciferase bioluminescent assay in an LKB 1251 luminometer (LKB-Wallac).^{32 33} The sensitivity of the assay was 1 pmol ATP, and ATP standards were used for calibration. Total protein was measured, and the results are expressed as nanomoles per milligram of protein.

Statistics
All data are expressed as the mean±SEM of n preparations.

	Results

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Intracellular Acidification Induced by Perfusion with H₂O₂ or Xanthine Oxidase/Hypoxanthine
Fig 1A shows the effects of perfusion of a freshly isolated rat cardiac myocyte with 100 µmol/L H₂O₂ in HEPES-buffered solution at 37°C; acidification of pH_i by 0.22±0.01 pH units (n=9) was seen, which could be reversed by washout of the H₂O₂ in 40% of the cells tested. When the same dose of H₂O₂ was used, a similar effect was seen with the rat cardiac myoblast cell line, H9c2 (Fig 1B); at 37°C, pH_i was rapidly acidified after the addition of H₂O₂ (0.24±0.02 pH units, n=13) and recovered completely after washout. At room temperature, however, intracellular acidification was not induced by the addition of 100 µmol/L H₂O₂ (n=4), either in rat cardiac myocytes or in H9c2 cardiac myoblasts. To test whether acidification was due to a chemical reaction between intracellular BCECF and H₂O₂, BCECF acid (Molecular Probes) was added to the cell bath (in the absence of cells); no change in the 490/440 ratio was seen after the addition of H₂O₂, showing that H₂O₂-induced intracellular acidosis was not due to a direct chemical reaction between H₂O₂ and BCECF. At a higher dose of H₂O₂ (5 mmol/L), a much larger and irreversible pH change was seen in both H9c2 cells (Fig 1C, n=5) and cardiac myocytes (Table 1, n=3), with marked reduction of viability (data not shown).

View larger version (13K): [in this window] [in a new window]   Figure 1. Acidosis induced by H2O2 or hypoxanthine/xanthine oxidase. A, Single rat cardiac myocyte. B, C, and D, Cardiac myoblast cell line H9c2. The bar above the pHi trace indicates the period of perfusion with H2O2 or hypoxanthine plus xanthine oxidase. All experiments were performed in nominally HCO3--free HEPES-buffered medium at 37°C.

View this table: [in this window] [in a new window]   Table 1. Summarization of Results for Figs 1 Through 3

Xanthine oxidase, in the presence of hypoxanthine, is known to produce H₂O₂ and oxygen-derived free radicals, including O₂^-.^{2 12} In the presence of 1 mmol/L hypoxanthine, 10 mU/mL of xanthine oxidase induced a similar effect on the internal pH of both cardiac myocytes (Table 1, n=3) and H9c2 cells (Fig 1D, n=3). The statistical results are summarized in Table 1.

Both with H9c2 and freshly isolated rat cardiac myocytes, 5 mmol/L H₂O₂ could cause a significant increase in intracellular Ca²⁺ ion levels (see Fig 5) and a marked decrease in viability. Since 100 µmol/L of H₂O₂ has been shown to exert a similar degree of oxidative stress as postischemic myocardial perfusion^{34 35} and since H9c2 cells have similar responses to cardiac myocytes, with which they share and have many similar characteristics (see "Materials and Methods"), we decided to use the combination of H9c2 cells and 100 µmol/L H₂O₂ to study (1) which chemical species is responsible for the induction of intracellular acidosis by H₂O₂ and (2) the cellular mechanism of H₂O₂-induced acidification.

View larger version (11K): [in this window] [in a new window]   Figure 5. Effect of 100 µmol/L and 5 mmol/L H2O2 on intracellular Ca2+ levels. The fura 2 ratio (340/380) of the 510-nm emission in arbitrary units is plotted on the y axis. All experiments were performed in HEPES-buffered medium at 37°C.

Which Chemical Species (H₂O₂, O₂^-, or OH) Is Involved in H₂O₂-Induced Intracellular Acidification?
Fig 1 shows that the extracellular addition of ROS, such as H₂O₂, can produce intracellular acidosis. To test whether extracellular H₂O₂ itself can produce this effect, catalase, an enzyme that specifically reduces extracellular H₂O₂ to H₂O and O₂, was added to the bath. The addition of catalase (100 µg/mL) itself had little effect on the resting pH_i but totally abolished the H₂O₂-induced acidification (Fig 2A, n=4) and xanthine oxidase/hypoxanthine–induced acidification (Fig 2B, n=4). These results indicate that extracellular H₂O₂ itself was not responsible for H₂O₂-induced acidosis; possibly, it has to cross the cell membrane and induce the acidosis. Moreover, after the addition of 150 U/mL SOD, an efficient extracellular O₂^- scavenger,^{22 35} acidification could still be induced by H₂O₂ (Fig 2C, n=4) or xanthine oxidase/hypoxanthine (Fig 2D, n=4), ruling out the possibility that H₂O₂-induced acidification is caused by extracellular O₂^-.

View larger version (13K): [in this window] [in a new window]   Figure 2. Effect of catalase and SOD on H2O2 or hypoxanthine/xanthine oxidase–induced acidosis in H9c2 cells. A, Catalase (100 µg/mL) completely eliminates H2O2-induced acidification. B, The same dose of catalase abolishes the effect of hypoxanthine (1 mmol/L)/xanthine oxidase (10 mU/mL) on the pHi. C and D, SOD (150 U/mL) has no inhibitory effect on either H2O2-induced or hypoxanthine/xanthine oxidase–induced intracellular acidification. All experiments were performed in HEPES-buffered medium at 37°C.

Pretreatment of the cells with 1 mmol/L phenanthroline, a membrane-crossing iron chelator that prevents the production of intracellular OH,^{22 35} resulted in complete abolition of H₂O₂-induced acidification (Fig 3A, n=6); moreover, another membrane-crossing iron chelator, deferoxamine (20 mmol/L),^{12 36} also had a significant inhibitory effect (Fig 3B, n=5). It should be noted that the concentration of iron needed to catalyze the Fenton reaction is very low and that contamination of the perfusate by iron may have provided sufficient metal to catalyze the reaction in the external perfusate. We tested this possibility by adding apo-transferrin (0.1 mg/mL), which binds extracellular iron,^{12 23} and found that it had no effect on H₂O₂-induced acidosis (Fig 3C, n=4), showing that it is intracellular iron that plays a crucial role in reacting with the H₂O₂, which freely diffuses into the cells and is broken down into OH (Fenton reaction, see introductory section).²² To obtain further evidence for the role of intracellular OH in H₂O₂-induced acidosis, we used N-MPG, a highly potent intracellular OH scavenger.^{37 38} When cells were pretreated for 5 minutes with 10 mmol/L N-MPG, H₂O₂-induced acidosis was totally abolished (Fig 3D, n=3), whereas H₂O₂-induced acidosis was again seen after washout of N-MPG (0.21±0.03 pH units, n=3). Since it is known that N-MPG is a powerful intracellular OH scavenger, which has no significant effect on O₂^- or H₂O₂,³⁷ the above results are strong evidence that the oxygen species involved in pH_i acidification is intracellular OH. The statistical results are summarized in Table 1.

View larger version (15K): [in this window] [in a new window]   Figure 3. Effect of iron chelators and an OH scavenger on H2O2-induced acidification. A, Phenanthroline (1 mmol/L). B, Deferoxamine (20 mmol/L). C, Apo-transferrin (0.1 mg/mL). D, N-MPG (10 mmol/L). All experiments were performed in HEPES-buffered medium at 37°C.

Are pH_i Transporters Involved in OH-Induced Acidosis?
We first tested whether the three known pH_i regulators in mammalian cardiac cells (the Na⁺-H⁺ exchanger,²⁹ the Na⁺-HCO₃ cotransporter,³⁰ and the Cl^--HCO₃ exchanger³⁹ ) are involved in OH-induced acidosis.

In HEPES-buffered solution, all extracellular Na⁺ ions were removed by replacement with N-methyl-D-glucamine in order to block the Na⁺-H⁺ exchanger. Once the initial acidification had stabilized, the addition of 100 µmol/L H₂O₂ again resulted in acidification (Fig 4A, n=4). EIPA (1 µmol/L), a potent Na⁺-H⁺ exchanger inhibitor,⁴⁰ also had no inhibitory effect on H₂O₂-induced acidosis (n=4, Table 1), suggesting that the Na⁺-H⁺ exchanger is not involved in H₂O₂-induced acidosis.

View larger version (7K): [in this window] [in a new window]   Figure 4. Lack of involvement of pHi regulators in H2O2-induced acidification. A, Na+-free medium to block the Na+-H+ exchanger. B, DIDS (0.5 mmol/L) to block the Na+-HCO3 cotransporter and Cl--HCO3 exchanger. C, EIPA (1 µmol/L) and DIDS (0.5 mmol/L). The experiments in panels A and B were performed in HEPES-buffered solutions at 37°C; that in panel C was performed in CO2/HCO3-buffered medium at 37°C.

In sheep Purkinje fibers and guinea pig cardiac myocytes,^{26 30 39 41} DIDS (0.5 mmol/L) is known to inhibit both the Na⁺-HCO₃ cotransporter and the Cl^--HCO₃ exchanger. Since these observations of H₂O₂-induced acidosis were made in nominally HCO₃-free HEPES-buffered solution (Fig 1) and our previous work²⁶ had shown substantial HCO₃-dependent transporter activity under HEPES-buffered conditions, we tested the effect of DIDS on H9c2 cells in HEPES-buffered solutions. Fig 4B shows that 0.5 mmol/L DIDS causes an initial acidification that is probably due to inhibition of the Na⁺-HCO₃ cotransporter in HEPES-buffered solution,²⁶ whereas the subsequent addition of 100 µmol/L H₂O₂ again resulted in acidosis (n=5). In Cl^--free medium, known to block the Cl^--HCO₃ exchanger, H₂O₂-induced acidosis was not inhibited (Table 1, n=5). Since the bicarbonate-dependent pH_i regulators should be optimally activated in CO₂/HCO₃-buffered medium, we also tested the effect of 100 µmol/L H₂O₂ in CO₂/HCO₃-buffered medium and found that H₂O₂-induced acidosis was not blocked (Fig 4C, n=5) in the presence of DIDS (0.5 mmol/L) and EIPA (1 µmol/L). From these results, we conclude that none of the three known cardiac membrane pH_i transporters are involved in the H₂O₂-induced acidification.

Is OH-Induced Acidosis Caused by a Rise in Intracellular Ca²⁺ Levels?
In rat cardiac myocytes, it is known that free radicals increase the levels of diastolic intracellular Ca²⁺.^{21 42} Another possible mechanism for H₂O₂-induced acidosis could therefore be that a rise in levels of internal Ca²⁺ ions induced by the addition of H₂O₂ may cause a decrease in pH_i that is due to competition between intracellular Ca²⁺ and H⁺ ions for the common internal buffering sites.³⁴ Fig 5 shows the results of intracellular free Ca²⁺ measurement using fura 2-AM (see "Materials and Methods"). After the addition of 100 µmol/L H₂O₂, the 340/380 ratio showed little change (n=6), but when 5 mmol/L H₂O₂ was added, the intracellular Ca²⁺ ion concentration increased markedly (n=6), showing that the intracellular acidification induced by 100 µmol/L H₂O₂ is not due to an increase in intracellular Ca²⁺ ion concentration.

Does OH-Induced Acidosis Depend on Hydrolysis of Intracellular ATP?
H₂O₂ has been shown to inhibit glycolysis and oxidative phosphorylation and to activate an ATP-sensitive K⁺ current in cardiac cells.^{16 17 21} ATP hydrolysis may lead to overproduction of H⁺ ions (ie, ATP^4-+H₂OADP^3-+HPO₄^2-+H⁺), suggesting that H₂O₂ might deplete intracellular ATP via ATP hydrolysis and result in pH_i acidosis.^{43 44} This hypothesis was tested in the following experiments.

Cyanide (CN^-, 3 mmol/L) is known to inhibit oxidative phosphorylation and reduce mitochondrial ATP production.^{33 45 46 47 48} Fig 6A shows that the addition of 3 mmol/L CN^- to H9c2 cells induced rapid intracellular acidification. It has recently been shown, in the isolated ferret heart, that the small degree of acidosis produced by cyanide in glucose-containing solution is mainly due to stimulation of anaerobic glycolysis and consequent lactic acid accumulation.⁴⁷ After stabilization of the pH_i, addition of 100 µmol/L H₂O₂ caused a further increase in acidification (Fig 6A, n=4). Fig 6B shows results similar to those found with another oxidative phosphorylation inhibitor, rotenone (10 µmol/L, n=4). These results in H9c2 were further confirmed in freshly isolated single rat cardiac myocytes; 3 mmol/L CN^- did not block the effect of 100 µmol/L H₂O₂ (Fig 6C, n=4), suggesting that depletion of mitochondrial ATP is not involved in H₂O₂-induced acidosis. A similar absence of any effect of CN^- alone on the pH_i (Fig 6C) has also been noted in rat ventricular myocytes⁴⁸ ; however, these results contrast with studies in H9c2 cells (see Fig 6A) and ferret hearts,⁴⁷ in which acidosis, presumably from lactate accumulation (see above), was seen. A possible explanation for this difference is that the rat cardiac myocyte lactate carrier, identified only recently,⁴⁹ is probably more active than the corresponding carrier in H9c2 cells, resulting in a lower lactate accumulation and acidosis in rat cardiac myocytes.

View larger version (6K): [in this window] [in a new window]   Figure 6. Inhibition of oxidative phosphorylation has no effect on H2O2-induced acidification. A, Sodium cyanide (3 mmol/L). B, Rotenone (10 µmol/L). C, Sodium cyanide (3 mmol/L). Experiments in panels A and B were carried out on H9c2 cells; that in panel C was performed on single rat cardiac myocytes. All experiments were performed in HEPES-buffered medium at 37°C.

IAA (0.5 mmol/L), an irreversible and noncompetitive inhibitor of the cytoplasmic glycolytic enzyme (GAPDH),⁴⁵ induced marked intracellular acidification and completely inhibited H₂O₂-induced acidosis (Fig 7A, n=7). The initial acidosis induced by IAA (Fig 7A) was probably due to hydrolysis of glycolytic ATP and releasing protons. Two hours of pretreatment with DOG (10 mmol/L), a partial but irreversible glycolytic pathway inhibitor,^{45 50} also resulted in inhibition of the H₂O₂ effect (Fig 7B, n=6). These results in H9c2 cells were confirmed again in freshly isolated single cardiac myocytes by the absence of any additive effect of H₂O₂ in the presence of 0.5 mmol/L IAA (Fig 7C, n=4). The initial small alkalization induced by the addition of IAA (Fig 7C) has been reported in cardiac cells^{33 44} and is believed to be due to a decrease in cytoplasmic phosphocreatine; ie, net hydrolysis of phosphocreatine leads to absorption of H⁺ ions.⁴⁴ Therefore, these results strongly suggest that H₂O₂, probably acting via OH, may inhibit glycolysis and hence deplete glycolytic ATP, resulting in elevation of H⁺ levels in cardiac cells. Table 2 summarizes all the statistical results for Figs 4 through 7.

View larger version (7K): [in this window] [in a new window]   Figure 7. Inhibition of the glycolytic pathway completely blocks H2O2-induced acidification. A, IAA (0.5 mmol/L). B, DOG (10 mmol/L, 2-hour pretreatment). C, IAA (0.5 mmol/L). The experiments shown in panels A and B were carried out on H9c2 cells; that in panel C was performed with single rat cardiac myocytes. All experiments were performed in HEPES-buffered medium at 37°C.

View this table: [in this window] [in a new window]   Table 2. Summarization of Results for Figs 4 Through 7

Intracellular ATP mainly exists as Mg²⁺ salt. In the ischemic myocardium, it has been shown that the intracellular free Mg²⁺ level increases as the intracellular ATP level decreases.^{18 19 20} To determine whether ATP was depleted by H₂O₂-induced glycolytic inhibition, thus resulting in increased levels of Mg²⁺, free Mg²⁺ ion levels were measured using Mg²⁺–fura 2 (Fig 8A; see "Materials and Methods"). Fig 8A shows the changes in the Mg²⁺–fura 2 ratio; 100 µmol/L H₂O₂ caused a reversible increase in intracellular Mg²⁺ levels, which correlated well with the reversible H₂O₂-induced acidification (see Fig 1B). However, the effect of the irreversible glycolytic inhibitor IAA (0.5 mmol/L) on Mg²⁺ levels⁴⁵ was irreversible (n=5). These observations are further proof that H₂O₂-induced acidosis results from intracellular ATP hydrolysis.

View larger version (9K): [in this window] [in a new window]   Figure 8. Intracellular free Mg2+ levels rise and ATP levels decrease during H2O2 or IAA perfusion of H9c2 cells. A, Reversible increase of intracellular Mg2+ levels on addition of 100 µmol/L H2O2 and an irreversible increase on addition of 0.5 mmol/L IAA. The y axis is in arbitrary units of Mg2+ concentration (340/380 excitation with emission at 510 nm). B, Two histograms showing the percentage change of intracellular ATP content (measured by luciferin/luciferase bioluminescent assay; see "Materials and Methods") in the control (100%) and 100 µmol/L H2O2–treated cells. The vertical bars on the histograms indicate the standard error. *P<.05.

Finally, we have used the luciferin/luciferase bioluminescent assay (see "Materials and Methods") to directly compare the levels of intracellular ATP between normal Tyrode's solution–treated (control group) and H₂O₂-treated H9c2 cells. Fig 8B shows clearly that in 100 µmol/L H₂O₂–treated cells, the levels of intracellular ATP were reduced to 44.1±8.6% (23.1±0.1 nmol/mg protein, P<.05, n=4) of the levels in untreated cells (52.3±2.5 nmol/mg protein, control group, n=4). This observation further strengthens our hypothesis that the acidosis induced by H₂O₂ is mainly due to hydrolysis of intracellular ATP. A marked reduction (55%) in intracellular ATP levels was seen after H₂O₂-induced glycolytic inhibition, suggesting a high percentage of ATP production from glycolysis in H9c2 cells. This phenomenon possibly results from the fact that the rate of glycolysis in cultured myoblasts is normally much higher than the rate of glycolysis in freshly dissociated cells.⁵¹

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we have demonstrated that intracellular production of OH, after H₂O₂ influx, is the major chemical event resulting in H₂O₂-induced acidosis. The evidence is the following: First, the non–membrane-crossing enzyme, catalase, totally abolishes the acidosis induced by either H₂O₂ (Fig 2A) or xanthine oxidase/hypoxanthine (Fig 2B). Second, acidosis is not inhibited by the extracellular O₂^- scavenger, SOD (Fig 2C and 2D). Third, acidosis is completely abolished by the two lipid-soluble iron chelators, phenanthroline and deferoxamine (Fig 3A and 3B), and the potent intracellular OH scavenger, N-MPG (Fig 3D), whereas the non–membrane-crossing iron-binding protein, apo-transferrin, has no inhibitory effect (Fig 3C). Iron is normally stored in cells in the form of ferritin micelles and a number of cellular reductants, eg, GSH, NADH, and NADPH.^{52 53} H₂O₂ may react sequentially first with Fe³⁺ in ferritin to liberate Fe²⁺ and then with this Fe²⁺ to produce OH.^{52 53} In addition, a small pool of non–protein-bound iron, found in the cytoplasm and mitochondria, can also provide iron for the Fenton reaction. Since there are no specific intracellular O₂^- scavengers available, we cannot completely rule out the possibility that intracellular O₂^- may play a role in acidosis. However, the membrane-crossing iron chelators and the potent membrane-crossing OH scavenger completely inhibit H₂O₂-induced acidosis, strongly suggesting that it is intracellular OH (produced by an intracellular Fenton reaction) that causes intracellular acidification.

In the present study, we also excluded any involvement of the three known cardiac pH_i regulators (the Na⁺-H⁺ exchanger, the Cl^--HCO₃ exchanger, and the Na⁺-HCO₃ cotransporter) in the acidification induced by hydroxyl free radicals (Fig 4). In rat cardiac myocytes, it has been shown that H₂O₂ (1 to 5 mmol/L) induces a Ca²⁺ overload,²¹ which may correlate with the elevated diastolic tension seen during ischemia cardiac reperfusion.¹⁶ In the present study, we found that the levels of intracellular Ca²⁺ ions were not altered by the addition of 100 µmol/L H₂O_2, suggesting that the acidosis induced is not due to competition between Ca²⁺ and H⁺ ions for common binding sites.⁵⁴ However, the much larger changes in pH_i (Fig 1C) and intracellular Ca²⁺ (Fig 5) induced by 5 mmol/L H₂O₂ are probably due to a displacement of intracellular Ca²⁺ ions for protons.⁵⁴

Our data indicate that inhibition of glycolysis and hydrolysis of intracellular ATP could be the major cause of H₂O₂-induced acidosis in both isolated rat cardiac myocytes and cardiac myoblasts (H9c2 cells): First, inhibition of oxidative phosphorylation (by CN^- or rotenone, Fig 6) has no effect on H₂O₂-induced acidosis. Second, no additive effect of H₂O₂ was seen after the inhibition of glycolysis (Fig 7).^{33 45} Third, increased levels of free internal Mg²⁺ ions, due to ATP hydrolysis,^{18 19 20} are seen after the sequential addition of H₂O₂ and IAA (Fig 8A), which correlates with H₂O₂-induced acidosis. Fourth, in cardiac muscle, it has been demonstrated that H₂O₂ can reversibly inhibit the glycolytic enzyme, GAPDH, with the enzyme activity being decreased by 75% after treatment with 150 µmol/L of H₂O₂ and partially recovering after washout of H₂O₂.^{55 56} This phenomenon correlates with our observation of the slow recovery of pH_i, which is probably due to ATP synthesis, on returning to H₂O₂-free solution (Fig 1A and 1B). Fifth, the present study also shows that the levels of intracellular ATP were decreased by 55% in H₂O₂-treated cells (Fig 8B). This is further direct evidence that H₂O₂ treatment may partially deplete levels of intracellular ATP and result in acid production after ATP hydrolysis. However, it should be noted that the accumulation of phosphorylated glycolytic intermediates, ie, sugar phosphates, might also contribute to the acidosis.^{43 47} In the presence of 100 µmol/L H₂O₂, acid loading was stable (Fig 1A and 1B), ie, with no pH_i recovery. The mechanism of this phenomenon is not yet clear, but it is probably due to H₂O₂ (1) causing only partial inhibition of GAPDH, with the production of some ATP (see Fig 8B), and (2) inhibiting the glycolytic pathway, resulting in accumulation of sugar phosphates (see above) and hence increasing H⁺ production. The overall effect of continuous ATP production/hydrolysis and accumulation of sugar phosphates balanced against the effects of the activated acid extruders (eg, Na⁺-H⁺ exchange) is a shift of the resting pH_i to a more acidic level.

Viable myocardial cells, subjected to brief (5- to 15-minute) periods of ischemia followed by reperfusion, show depressed cardiac contractility; this is referred to as the "stunned myocardium."^{3 15} The reperfused heart shows not only reperfusion-induced arrhythmia and contractile dysfunction but also intracellular ATP depletion and metabolic inhibition.^{14 15 21} By use of electron paramagnetic resonance spectroscopy and the spin trap -phenyl N-tert-butyl nitrone, direct measurements on postischemic rabbit and dog hearts show that ROS are generated during reperfusion.^{3 57} The iron chelators, deferoxamine and phenanthroline, and the OH scavenger, N-MPG, are effective in preventing "stunned myocardium" and in increasing the contractility of postischemic hearts.^{10 11 37} In the present study, we have shown an intriguing H₂O₂-induced pH_i acidosis, which can be prevented by these same iron chelators and OH scavenger, suggesting the involvement of OH in H₂O₂-induced acidosis. Fabiato and Fabiato⁵⁸ have shown that in skinned cardiac muscle at a constant [Ca²⁺]_i, alkalosis increases the twitch tension, whereas acidosis causes the tension to fall. In sheep cardiac Purkinje fibers, an intracellular acidification of 0.1 pH unit results in a 40% reduction in twitch tension.^{59 60} Two mechanisms have been suggested for this effect of pH_i on the active tension: a rise in internal H⁺ levels may (1) markedly decrease the sensitivity of the contractile elements to [Ca²⁺]_i or (2) reduce Ca²⁺ release from the sarcoplasmic reticulum and/or interfere with Ca²⁺ movement across the sarcoplasmic reticulum.^{58 59 60} Therefore, our results strongly suggest that the reduction in pH_i induced by OH radicals may contribute, at least in part, to the contractile dysfunction of the stunned myocardium during reperfusion injury. The marked reduction in intracellular ATP levels may indeed contribute, in part, to cardiac stunning; however, it has been shown that glycolytic ATP appears to be used preferentially to fuel membrane ion pumps and channels, whereas ATP produced by mitochondrial oxidative phosphorylation is used preferentially to support myocyte contraction (ie, functional compartmentation),^{61 62} explaining the finding that H₂O₂-induced acidification via glycolytic ATP hydrolysis may still play an important role in contractile dysfunction during ischemia reperfusion.

In addition to the possibility of cardiac depression induced by OH acidification, we should also consider other possibilities that may contribute to cardiac stunning; eg, some studies have shown that NO, cytokines, and prostaglandins have either a protective^{63 64 65} or depressive effect^{66 67} on cardiac contractility during reperfusion-induced cardiac stunning. These possible relationships between pH_i, NO, prostaglandins, and cytokines during reperfusion-induced cardiac stunning require further investigation.

In summary, we have shown that H₂O₂, probably acting via intracellular OH, can induce profound intracellular acidosis both in single rat cardiac myocytes and in a cardiac myoblast cell line. This acidosis is due to the inhibition of glycolytic metabolism and results from intracellular ATP hydrolysis. Acidification induced by the hydroxyl free radical may possibly contribute to the process of contractile dysfunction during myocardial reperfusion injury.

	Selected Abbreviations and Acronyms

 

DOG = 2-deoxy-D-glucose EIPA = 5-(N-ethyl-N-isopropyl)-amiloride IAA = iodoacetate N-MPG = N-(2-mercaptopropionyl)-glycine ROS = reactive oxygen-derived free radical(s) SOD = superoxide dismutase

	Acknowledgments

 
This study was supported by the Department of Health of Taiwan (DOH 83-HR-301).

Received May 15, 1995; accepted December 29, 1995.

	References

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