Cardiovascular Drugs and Therapy 1991;5:1021-1026 © Kluwer Academic Publishers, Boston. Printed in U.S.A.
The Leakage of Fatty Acid Binding Protein from Cultured Myocardial Cells during Hypoxia Hajime TakahashL Hideaki KawaguchL Kenji Iizuka, Hisakazu Yasuda Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo, Japan
Summary. Fatty-acid binding protein (FABP) is thought to play an i m p o r t a n t role as a carrier protein of fatty acids in cells. It may leak from damaged cells, because its molecular weight is low (mol wt 14000) and it accounts for several percent of soluble protein. In this experiment we attempted to use F A B P as a m a r k e r of cell injury under hypoxia in cultured myocytes. Newborn-rat myocytes were incubated under hypoxic treatment for 6 hours, and then the releases of F A B P and CPK were measured. The cell-death ratio during hypoxygenation increased from 4 hours and rose to 80% at 6 hours, but it was only 8% under aerobic conditions. F A B P in medium was detected at 1 hour, and rapidly increased and reached a plateau at 4 hours. On the other hand, CPK in medium was negligible during the 3 hours, then slightly increased. Ca antagonists and a lSl-adrenergic blocking agent inhibited the release of F A B P and prevented cell death. But the al-adrenergic blocking agent had little effect on preventing F A B P leakage and cell death. These results show that F A B P is of use as a m a r k e r of myocardial cell injury and revealed that the Ca antagonist and 131-adrenergic blocking agent are useful drugs for the protection of myocardial cell injury in hypoxia. Cardiovasc Drugs Ther 1021-1026
Key Words. fatty acid binding protein, hypoxia, calcium antagonist, aradrenergic receptor, [~l-adrenergic receptor
M a n y aspects of the changes associated with myocardial ischemia have been intensively studied [1,2]. Abnormal lipid metabolism alters cardiac function by changing the cardiac cell-membrane properties. These functional changes may contribute to the decreased myocardial contractility, arrhythmia, and cell death that follow coronary artery occlusion [3]. Despite early functional changes in membrane properties that are responsible for the electrophysiological manifestation of ischemia, little is known about the myocardial phospholipid metabolism. The possibility that lysophospholipids contribute to ischemic heart damage was first suggested by Hajidu et al. [4]. Lysophospholipid accumulation is caused by decreased lysophospholipase activity [5,6]. Recently, we showed that free fatty acid and prostacyclin are released from isolated heart during hypoxia [7] and confirmed that
phospholipid degradation and fatty acid release have been caused by the activation of phospholipase A2 [8]. The activation of phospholipase degrades membrane phospholipids and causes the cellular membrane to deteriorate. On the other hand, fatty acid binding protein (FABP), which is found in the cytosol and plasma membrane of many tissues, exhibits a high affinity for long-chain fatty acids and their CoA derivatives and, therefore, is considered to function in the uptake and intracellular utilization of fatty acids [9-12]. The high concentration of FABP in heart cytosol suggests that it plays a role in the storage of fatty acids and their derivatives [13]. Recently, the leakage of FABP in myocardial infarction was reported [14]. We consider this phenomenon to exist, because the molecular weight of cytosolic FABP is low (12,000-14,000 Da) and its concentration is high in heart cytosol [15]. Hence, it easily leaks from cells in the case of cell membrane damage. In this paper we attempted to use the leakage of FABP as a marker of myocardial cell injury under hypoxia in cultured cells and determined the effect of several agents for the protection of myocardial cell injury in hypoxia.
Materials and Methods
Reagents Metoprolol (CIBA-GIGY, Osaka), nifedipine (Bayer, Tokyo), and bunazosin HC1 (Eizai, Tokyo) were generous gifts from these companies.
Cell culture Neonatal rats were anesthetized in ether and the hearts were quickly removed and put into sterilized modified Krebs-Hanseleit and HEPES buffer (NaC1
Address for correspondence and reprint requests: Hideaki Kawaguchi, Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan.
1021
1022
Takahashi, Kawaguchi, Iizuka and Yasuda
8.3 g/l, KC1 0.42 g/l, CaC12 0.34 g/l, KH2P04 0.19 g/l, MgS04 0.17 g/l, and HEPES 11.92 g/l) adjusted to pH 7.4. Blood was carefully washed out and excised ventricles were cut into 1-2 mm cubes and then placed in phosphate-buffered saline (NaC1 8.0 g/l, Na2HPO4 1.5 g/l, KH2PO4 0.29 g/l, K2HPO4 0.2 g/l) containing 0.2% trypsin. The hearts were dissociated using a magnetic stir bar at a slow speed (100-150 rpm) for 8 minutes at 37°C. Cells from the first two combined treatments were discarded, and the sequence was repeated five times until almost all tissue was dissociated. Freed cells were pooled in cold Eagle's modified minimum essential medium (MEME, Flow Laboratories, Rockville, MD, USA) salts from Sigma containing 0.06% yeast extract (wt/vol), 0.25% lactalbumin hydrolysate (wt/vol), 400 U/ml penicillin g, and streptomycin (200 g/ml) with 10% fetal bovine serum (FBS, Flow Laboratories) [16] and centrifuged gently enough (8001000 rpm) to pellet the cells. The supernatant was poured off and swirled gently to disperse the cells in 10 ml MEME. Isolated cells were then plated into 100-mm tissue culture plastic dishes at 37°C in humidified air with 5% C02 and maintained at pH 7.4 [17]. In this medium, more than 90% of cells were beating. Cells were maintained in 10% FBS MEME for 1 week and then used for assays.
Cell hypaxygenation Cells were cultured at a density of 4 × 105/dish in a 60-mm dish, and MEME medium was changed to 95% N2, 5% C02 humidified Krebs-Henseleit, and HEPES buffer at 37°C in an anaerobic chamber (GasPak system, Becton Dickinson and Company, Cockeysville, MD, USA). Then culture dishes were exposed to a 95% Nae, 5% room-air atmosphere at 37°C for 1-6 hours for hypoxic treatment. After this incubation, 1 ml of each buffer was collected and used for activity assay. Under the hypoxic condition, the beating of almost all myocytes stopped within 30 minutes. Triplicate samples were used for each of six experiments.
Cell viability After cell hypoxygenation for 1-6 hours, the cell death ratio was determined from the number of myocytes per 200 cells that did not exclude 0.023% trypan blue within 2 minutes [18]. The time required for counting the numbers of cells was not long and, therefore, the cytotoxic effect of trypan blue appeared to be insignificant in this study.
Delipidation of samples and assay of fatty acid binding The delipidation of samples and the assay of fatty acid binding were carried out using Lipidex 1000 (Packard, Downers Grove, IL) according to the method of Glatz et al. [19]. Lipidex 1000, a 10% (w/w) substituted hydroxyalkoxypropyl derivative of Sephadex G-25, removes unbound fatty acids from protein-fatty acid complexes at 0°C and removes all fatty acids from aqueous solutions at 37°C according to protein-lipid interaction kinetics. For delipidation, samples (culture medium) were subjected to chromatography on a Lipidex column (1.5 × 7 cm) equilibrated with 10 mM sodium phosphate buffer (pH 7.4) at 37°C. The column was eluted with the same buffer, and all the proteins were recovered in the void volume. For the assay of fatty acid binding, the delipidated medium (0.5 ml) was incubated with [14C]palmitic acid (40,000 dpm/M, Amersham; Arlington Heights, IL) in a polyethylene tube in 10 mM sodium phosphate buffer (pH 7.4; final volume 0.9 ml) for 10 minutes at 37°C. Then, to remove unbound fatty acids, the tubes were cooled on ice and ice-cold Lipidex/buffer suspension (1:1 v/v; 0.1 ml) was added and samples were incubated for another 10 minutes at 0°C. Fatty acid binding was calculated from the amount of radioactivity present in the supernatant after centrifugation of the tubes and was expressed as pmol/dish. Purification of FABP from culture medium under hypoxia Cells (1 x 107/10 cm dish) were cultured and incubated in the same buffer described above for 6 hours under hypoxia. Then the culture medium was collected. The medium was concentrated by ultrafiltration (UK-10, Toyoroshi, Tokyo), and protein was applied to a Sephadex G-75 column (2.6 × 70 cm) equilibrated with 10 mM Tris-HC1 (pH 7.4), 10 mM KC1, 1 mM EDTA, and i mM dithiothreitol. The fractions with high binding activity for palmitate were designated as FABP [13]. They were then combined, dialyzed against 30 mM Tris-HC1 (pH 8.5), and applied to a DEAE cellulose column (2.1 x 15 cm) equilibrated with 30 mM tris-HC1 (pH 8.5). The column was initially eluted with the equilibration buffer until unbound proteins were completely eluted. Then the retained protein was eluted with a linear gradient prepared from 100 ml each of the equilibrating buffer and 0.3 M NaC1 in the same buffer. The purity of FABP was confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE was carried out according to the procedure of Meizel [20]. Purified FABP (heart type) was provided by Professor Ono, Niigata University, Japan [21].
Fatty Acid Binding Protein Leakage
(A)
pmol/dish
1023
m U Vml
150
150
>
>
The Leakage of Fatty Acid Binding Protein from Cultured Myocardial Cells during Hypoxia Hajime TakahashL Hideaki KawaguchL Kenji Iizuka, Hisakazu Yasuda Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo, Japan
Summary. Fatty-acid binding protein (FABP) is thought to play an i m p o r t a n t role as a carrier protein of fatty acids in cells. It may leak from damaged cells, because its molecular weight is low (mol wt 14000) and it accounts for several percent of soluble protein. In this experiment we attempted to use F A B P as a m a r k e r of cell injury under hypoxia in cultured myocytes. Newborn-rat myocytes were incubated under hypoxic treatment for 6 hours, and then the releases of F A B P and CPK were measured. The cell-death ratio during hypoxygenation increased from 4 hours and rose to 80% at 6 hours, but it was only 8% under aerobic conditions. F A B P in medium was detected at 1 hour, and rapidly increased and reached a plateau at 4 hours. On the other hand, CPK in medium was negligible during the 3 hours, then slightly increased. Ca antagonists and a lSl-adrenergic blocking agent inhibited the release of F A B P and prevented cell death. But the al-adrenergic blocking agent had little effect on preventing F A B P leakage and cell death. These results show that F A B P is of use as a m a r k e r of myocardial cell injury and revealed that the Ca antagonist and 131-adrenergic blocking agent are useful drugs for the protection of myocardial cell injury in hypoxia. Cardiovasc Drugs Ther 1021-1026
Key Words. fatty acid binding protein, hypoxia, calcium antagonist, aradrenergic receptor, [~l-adrenergic receptor
M a n y aspects of the changes associated with myocardial ischemia have been intensively studied [1,2]. Abnormal lipid metabolism alters cardiac function by changing the cardiac cell-membrane properties. These functional changes may contribute to the decreased myocardial contractility, arrhythmia, and cell death that follow coronary artery occlusion [3]. Despite early functional changes in membrane properties that are responsible for the electrophysiological manifestation of ischemia, little is known about the myocardial phospholipid metabolism. The possibility that lysophospholipids contribute to ischemic heart damage was first suggested by Hajidu et al. [4]. Lysophospholipid accumulation is caused by decreased lysophospholipase activity [5,6]. Recently, we showed that free fatty acid and prostacyclin are released from isolated heart during hypoxia [7] and confirmed that
phospholipid degradation and fatty acid release have been caused by the activation of phospholipase A2 [8]. The activation of phospholipase degrades membrane phospholipids and causes the cellular membrane to deteriorate. On the other hand, fatty acid binding protein (FABP), which is found in the cytosol and plasma membrane of many tissues, exhibits a high affinity for long-chain fatty acids and their CoA derivatives and, therefore, is considered to function in the uptake and intracellular utilization of fatty acids [9-12]. The high concentration of FABP in heart cytosol suggests that it plays a role in the storage of fatty acids and their derivatives [13]. Recently, the leakage of FABP in myocardial infarction was reported [14]. We consider this phenomenon to exist, because the molecular weight of cytosolic FABP is low (12,000-14,000 Da) and its concentration is high in heart cytosol [15]. Hence, it easily leaks from cells in the case of cell membrane damage. In this paper we attempted to use the leakage of FABP as a marker of myocardial cell injury under hypoxia in cultured cells and determined the effect of several agents for the protection of myocardial cell injury in hypoxia.
Materials and Methods
Reagents Metoprolol (CIBA-GIGY, Osaka), nifedipine (Bayer, Tokyo), and bunazosin HC1 (Eizai, Tokyo) were generous gifts from these companies.
Cell culture Neonatal rats were anesthetized in ether and the hearts were quickly removed and put into sterilized modified Krebs-Hanseleit and HEPES buffer (NaC1
Address for correspondence and reprint requests: Hideaki Kawaguchi, Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan.
1021
1022
Takahashi, Kawaguchi, Iizuka and Yasuda
8.3 g/l, KC1 0.42 g/l, CaC12 0.34 g/l, KH2P04 0.19 g/l, MgS04 0.17 g/l, and HEPES 11.92 g/l) adjusted to pH 7.4. Blood was carefully washed out and excised ventricles were cut into 1-2 mm cubes and then placed in phosphate-buffered saline (NaC1 8.0 g/l, Na2HPO4 1.5 g/l, KH2PO4 0.29 g/l, K2HPO4 0.2 g/l) containing 0.2% trypsin. The hearts were dissociated using a magnetic stir bar at a slow speed (100-150 rpm) for 8 minutes at 37°C. Cells from the first two combined treatments were discarded, and the sequence was repeated five times until almost all tissue was dissociated. Freed cells were pooled in cold Eagle's modified minimum essential medium (MEME, Flow Laboratories, Rockville, MD, USA) salts from Sigma containing 0.06% yeast extract (wt/vol), 0.25% lactalbumin hydrolysate (wt/vol), 400 U/ml penicillin g, and streptomycin (200 g/ml) with 10% fetal bovine serum (FBS, Flow Laboratories) [16] and centrifuged gently enough (8001000 rpm) to pellet the cells. The supernatant was poured off and swirled gently to disperse the cells in 10 ml MEME. Isolated cells were then plated into 100-mm tissue culture plastic dishes at 37°C in humidified air with 5% C02 and maintained at pH 7.4 [17]. In this medium, more than 90% of cells were beating. Cells were maintained in 10% FBS MEME for 1 week and then used for assays.
Cell hypaxygenation Cells were cultured at a density of 4 × 105/dish in a 60-mm dish, and MEME medium was changed to 95% N2, 5% C02 humidified Krebs-Henseleit, and HEPES buffer at 37°C in an anaerobic chamber (GasPak system, Becton Dickinson and Company, Cockeysville, MD, USA). Then culture dishes were exposed to a 95% Nae, 5% room-air atmosphere at 37°C for 1-6 hours for hypoxic treatment. After this incubation, 1 ml of each buffer was collected and used for activity assay. Under the hypoxic condition, the beating of almost all myocytes stopped within 30 minutes. Triplicate samples were used for each of six experiments.
Cell viability After cell hypoxygenation for 1-6 hours, the cell death ratio was determined from the number of myocytes per 200 cells that did not exclude 0.023% trypan blue within 2 minutes [18]. The time required for counting the numbers of cells was not long and, therefore, the cytotoxic effect of trypan blue appeared to be insignificant in this study.
Delipidation of samples and assay of fatty acid binding The delipidation of samples and the assay of fatty acid binding were carried out using Lipidex 1000 (Packard, Downers Grove, IL) according to the method of Glatz et al. [19]. Lipidex 1000, a 10% (w/w) substituted hydroxyalkoxypropyl derivative of Sephadex G-25, removes unbound fatty acids from protein-fatty acid complexes at 0°C and removes all fatty acids from aqueous solutions at 37°C according to protein-lipid interaction kinetics. For delipidation, samples (culture medium) were subjected to chromatography on a Lipidex column (1.5 × 7 cm) equilibrated with 10 mM sodium phosphate buffer (pH 7.4) at 37°C. The column was eluted with the same buffer, and all the proteins were recovered in the void volume. For the assay of fatty acid binding, the delipidated medium (0.5 ml) was incubated with [14C]palmitic acid (40,000 dpm/M, Amersham; Arlington Heights, IL) in a polyethylene tube in 10 mM sodium phosphate buffer (pH 7.4; final volume 0.9 ml) for 10 minutes at 37°C. Then, to remove unbound fatty acids, the tubes were cooled on ice and ice-cold Lipidex/buffer suspension (1:1 v/v; 0.1 ml) was added and samples were incubated for another 10 minutes at 0°C. Fatty acid binding was calculated from the amount of radioactivity present in the supernatant after centrifugation of the tubes and was expressed as pmol/dish. Purification of FABP from culture medium under hypoxia Cells (1 x 107/10 cm dish) were cultured and incubated in the same buffer described above for 6 hours under hypoxia. Then the culture medium was collected. The medium was concentrated by ultrafiltration (UK-10, Toyoroshi, Tokyo), and protein was applied to a Sephadex G-75 column (2.6 × 70 cm) equilibrated with 10 mM Tris-HC1 (pH 7.4), 10 mM KC1, 1 mM EDTA, and i mM dithiothreitol. The fractions with high binding activity for palmitate were designated as FABP [13]. They were then combined, dialyzed against 30 mM Tris-HC1 (pH 8.5), and applied to a DEAE cellulose column (2.1 x 15 cm) equilibrated with 30 mM tris-HC1 (pH 8.5). The column was initially eluted with the equilibration buffer until unbound proteins were completely eluted. Then the retained protein was eluted with a linear gradient prepared from 100 ml each of the equilibrating buffer and 0.3 M NaC1 in the same buffer. The purity of FABP was confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). SDS-PAGE was carried out according to the procedure of Meizel [20]. Purified FABP (heart type) was provided by Professor Ono, Niigata University, Japan [21].
Fatty Acid Binding Protein Leakage
(A)
pmol/dish
1023
m U Vml
150
150
>
>
