Vol. 179, No. 2, 1991 September
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 798-803
16, 1991
PROTECTION
BY ACIDOTIC pH AGAINST ANOXLWREOXYGENATION RAT NEONATAL CARDIAC MYOCYTESf
INJURY
TO
John M. Bond, Brian Herman and John J. Lemasters2 Laboratories for Cell Biology, Department of Cell Biology & Anatomy, and Curriculum Toxicology, University of North Carolina at Chapel Hill, NC 27599 Received
August
1,
in
1991
We assessed the effect of acidosis on cell killing during anoxia and reoxygenation in cultured rat neonatal cardiac myocytes. After 4.5 hours of anoxia and glycolytic inhibition with Z-deoxyglucose, loss of viability was >90% at pH 7.4. In contrast, at pH 6.2-7.0, viability was virtually unchanged. To model changes of pH and oxygenation during ischemia and reperfusion, myocytes were made anoxic at pH 6.2 for 4 hours, followed by reoxygenation at pH 7.4. Under these conditions, reoxygenatton precipitated loss of viability to about half the cells. When pH was increased to 7.4 without reoxy enation, similar lethal injury occurred. No cell killin occurred after reoxygenation at pH t .2. We conclude that actdosis protects against letha Iganoxic injury, and that a rapid return from acidotic to physiologic pH contributes significantly to reperfusion injury to cardiac myocytes - a ‘pH paradox’. 0 1991Academ. Press, Inc.
A key event during myocardial ischemia is tissue acidosis which results from lactic acid accumulation and hydrogen ion release during ATP hydrolysis. Previous work from this and other laboratories demonstrated that acidosis protects hepatocytes and other non-muscle cells against cell death during anoxia, metabolic inhibition, and exposure to toxic chemicals (l-4). Protection is mediated by intracellular acidification (5). Early reports suggested that acidosis may also protect during myocardial ischemia (6,7), but most workers consider acidosis harmful because it disrupts normal myocardial physiology (8). Reperfusion injury is the exacerbation of tissue damage when blood flow is restored to an ischemic organ. A marked reperfusion injury occurs to ischemic myocardium, whose mechanisms remain unclear. Generation of free radicals (9), critical depletion of ATP lev-
‘This work was supported, in art, by Grants from the Office of Naval Research, the National Institutes of Health, and t Ii e Gustavus and Louise Pfeiffer Research Foundation. Portions of this work were presented at the 74th Annual Meetin of the Federation of American Societies for Experimental Biology, Washington, DC, Apt-i B l-5,1990 (24). 2To whom correspondence should be addressed. Abbreviations used: KRH Krebs-Rin er-HEPES buffer containin 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4,1.2 n&f MgSO4, !mM CaQ, and 25 mM Na-!IEPES buffer, pH 7.4. WO6-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
798
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
els (lo), disruption of normal Ca*+ homeostasis (7), unmasking of latent injury (ll), and neutrophil infiltration (12) all may contribute to repel-fusion injury. During reperfusion of ischemic myocardium, both reoxygenation and a rapid return to physiologic pH occur. Much attention has been given to events associated with reoxygenation, but relatively little to changes of pH. Accordingly, the objective of this study was to assessthe role of changes of pH in anoxia/reoxygenation injury. To this end, we developed an in vitro model of reperfusion injury that simulates the changes of oxygenation and pH that occur during ischemia and reperfusion.
MATERL4LS
AND METHODS
Isolation and culhue of myocytes - Cardiac myocytes from 2-3 day old rat neonates were dispersed by digestion with pancreatin and collagenase (13), and purified by flowelutriation (14), as previously described (15 . Punfied myocytes were cultured on polystyrene coverslips at a density of 106 cells/3 z mm culture dish in Eagle’s MEM fortified with 5% fetal calf serum, 10% horse serum, 10 U/ml penicillin, and 10 &ml streptomycin at 37°C in humidified sir/5% carbon dioxide. Myocytes contracted spontaneously after about 3 days in culture and began to show striations after 5 days. At this time, the majority of cells exhibited vigorous, synchronous contractions at rates exceeding 60 beats/minute. Anoxia and reoaygenation - Myocytes were mounted in an airtight perfusion chamber and infused with an anoxic suspension of submitochondrial particles (1 mg of protein/ml) and succinate (5 mM) in KRH buffer, as previously described (16). Respiration by submitochondrial particles actively removed oxygen entering the chamber by back diffusion from the atmos here. Although respiration by submitochondrial particles was inhibited by approximately s 0% at pH 6.2, their concentration was sufficiently high to maintain oxygen at below limits of detectton of a Clark-type oxygen electrode (cl torr) at all values of H studied. In these experiments, Zdeoxyglucose (20 mM) was added to inhibit glycolysis. F or reoxygenation, submitochondrial partrcles were replaced with aerobic buffer not containing succinate or 2-deoxyglucose. In some experiments, anoxic submitochondrial particles were infused a second time to change pH without reoxygenation. Loss of cell viability was monitored by fluorescent labelling of nuclei with propidntm iodide (5 PM) (16,17). At the end of each experiment, digitonin (30 PM) was added to label nuclei of all cells in order to calculate percent loss of viability. Temperature was regulated at 37°C with an air curtain incubator (Laboratory Products, Boston, MA). Microscopy - Phase contrast and fluorescence images were collected with a Zeiss IM3.5 inverted microscope (Thornwood, NY). Propidium iodide fluorescence was imaged with 546~nm excitation, 580~nm dichroic, and 590-nm emission filters. Experiments were routinely recorded wrth a half-inch time-lapse video cassette recorder. h4ateriaZ.s- Eagle’s MEM, enicillin and stre tomycin were obtained from the Univer. of North Carolma Tissue 8 ulture Center ( & ape1 Hill, NC); pancreatin from Gibco 78and Island, NY); collagenase D from Boehringer Mannheim (Indianapolis, IN); and 2deoxyglucose from Sigma (St. Louis, MO). Other reagent-grade chemicals were obtained from standard commercial sources.
RESULTS Loss of viability of purified myocytes during anoxia was assessed at various values of extracellular pH. Cultured myocytes on polystyrene cover-slips were mounted into a gastight chamber. Initially, cells were pre-equilibrated in aerobic KRH, pH 7.4, for lo-20 minutes. During this period, myocytes exhibited vigorous, spontaneous contractions. Anoxia was produced by infusing an anoxic suspension of submitochondrial particles and succinate in KRH buffer at pH values ranging between 6.2 and 7.4. After l-2 minutes of anoxia, spon799
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
012345 Time
(hr)
F&J. Rote&n againstanoxickilling of culturedneonatalcardiacmyocytesby acidotic extracelhdarpH- Culturesof 6-12 day old myocyteswere mountedin a gas-tightchamber
and incubated in aerobic KRH buffer, pH 7.4, for 10-15 minutes. Anoxia was started by in-
fusionof an anoxicsuspension of submrtochondrialparticles(1 m of protein/ml succinate 5 mM , and Zdeoxyglucose(20 mM) in KRH buffer at pH va!ues betweentand74 by pro idium iodide stainin of myocyte nuclei. Ckli Loss od cell viability was assessed killing at H 6.2-7.0wassignificantlylesstKan at pH 7.4 (p V and &eun$ .Y. 21985) Am. J. Physiol. 249, C149C159. 5: Gores, G.J., ‘Ncminen, A.- , and Lemasters, J.J. (1988) Am. J. Physiol. 255, C315C322. 4. Nieminen, A-L., Dawson, T.L., Gores, G.J., Kawanishi, T., Herman, B., and Lemasters, J.J. (1990 Biochem. Biophys. Res. Commun. 167,600-606. 5. Gores, G.J., rl ieminen, A.-L., Wray, B.E., Herman, B., and Lemasters, J.J. (1989) J. Clin. Invest. 83,386-3%.
6. Bing, O.H.L., Brooks, W.W., and Messer, J.V. 1973) Science 180,1297-1298. Altschuld, R.A., Hostetler, J.R., and Brierley, d .P. (1981) Circ. Res.49,307-316. i: Williamson, J.R., Schaffer, S.W., Ford, C., and Safer, B. (1976) Circulation 53 (Suppl. I), 13-114. 9. Korthuis, R.J., and Granger, D.N. (1986) In PhysioZogyof Oxygen Radicals (Taylor, A.E., Matalon, S., and Ward, P.A., Eds. , p. 217-249. Williams & Wilkins, Baltunore. 10. Jennings, R.B., Hawkins, H.K., Lowe, II!. ., Hill, M.L., Klotman, S., and Reimer, K.A. 1978) Am. J. Pathol. 92,187-214. b anote, C.E. (1983)J. Mol. Cell. Cardiol. 15,67-73. ::: Romson, J-L., Hook, B.G., Kunkel, S.L., Abrams, G.D., Schork, M.A., and Lucchesi, B.R. (1983) Circulation 67,1016-1023. Harary, I., and Farley, B. (1963) Exp. Cell Res. 29,451-465. 4: Ulrich, R., Elli et, K.A., and Rosnick, D.K. (1988)J. T&s. Cult. Meth. 11,217-221. 15: Bond, J.M., If erman, B., and Lemasters, J.J. (1991) Res. Comm. Chem. Pathol. Phannacol, 71,195-208. 16. Herman, B., Nieminen, A.-L., Gores, G.J., and Lemasters, J.J. (1988) FASEBJ. 2, 146151. 17. grnyters, J.J., DiGuiseppi, J., Nieminen, A.-L., and Herman, B. (1987) Nature 325, - .
18. Stern, M.D., Chien, A.M., Capogrossi, MC., Pelto, D.J., and Lakatta, E.G. (1985) Cir. Res. 56,899-903.
19. Grinwald, P.M., and Naylor, W.G. (1981) J. Mol. CeZL CdioL 13,867-880. Hearse, D.J., Garlick, P.B., and Humphre , S.M. (1977) Am. J. CardioZ.39,986-993. it Currin, R.T., Gores, G.J., Thurman, R. e ., and Lemasters, J.J. (1990) FASEB J. 3, A626. 22. Lernasters, J.J., Caldwell-Kenkel, J.C., Gao, W., Nieminen, A.-L., Herman, B., and Thurman, R.G. (1991) In Puthophysiologv of Reperfusion Injury, D.K. Das, Ed., CRC Press? in press. 23. Harrtson, D.C., Lemasters, J.J., and Herman, B. (1991) Biochem. Biophys. Rex Commm 174,654-659. 24. Bond, J.M., Herman, B., and Lemasters, J.J. (1990) FASEBJ. 4, A622.
803
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 798-803
16, 1991
PROTECTION
BY ACIDOTIC pH AGAINST ANOXLWREOXYGENATION RAT NEONATAL CARDIAC MYOCYTESf
INJURY
TO
John M. Bond, Brian Herman and John J. Lemasters2 Laboratories for Cell Biology, Department of Cell Biology & Anatomy, and Curriculum Toxicology, University of North Carolina at Chapel Hill, NC 27599 Received
August
1,
in
1991
We assessed the effect of acidosis on cell killing during anoxia and reoxygenation in cultured rat neonatal cardiac myocytes. After 4.5 hours of anoxia and glycolytic inhibition with Z-deoxyglucose, loss of viability was >90% at pH 7.4. In contrast, at pH 6.2-7.0, viability was virtually unchanged. To model changes of pH and oxygenation during ischemia and reperfusion, myocytes were made anoxic at pH 6.2 for 4 hours, followed by reoxygenation at pH 7.4. Under these conditions, reoxygenatton precipitated loss of viability to about half the cells. When pH was increased to 7.4 without reoxy enation, similar lethal injury occurred. No cell killin occurred after reoxygenation at pH t .2. We conclude that actdosis protects against letha Iganoxic injury, and that a rapid return from acidotic to physiologic pH contributes significantly to reperfusion injury to cardiac myocytes - a ‘pH paradox’. 0 1991Academ. Press, Inc.
A key event during myocardial ischemia is tissue acidosis which results from lactic acid accumulation and hydrogen ion release during ATP hydrolysis. Previous work from this and other laboratories demonstrated that acidosis protects hepatocytes and other non-muscle cells against cell death during anoxia, metabolic inhibition, and exposure to toxic chemicals (l-4). Protection is mediated by intracellular acidification (5). Early reports suggested that acidosis may also protect during myocardial ischemia (6,7), but most workers consider acidosis harmful because it disrupts normal myocardial physiology (8). Reperfusion injury is the exacerbation of tissue damage when blood flow is restored to an ischemic organ. A marked reperfusion injury occurs to ischemic myocardium, whose mechanisms remain unclear. Generation of free radicals (9), critical depletion of ATP lev-
‘This work was supported, in art, by Grants from the Office of Naval Research, the National Institutes of Health, and t Ii e Gustavus and Louise Pfeiffer Research Foundation. Portions of this work were presented at the 74th Annual Meetin of the Federation of American Societies for Experimental Biology, Washington, DC, Apt-i B l-5,1990 (24). 2To whom correspondence should be addressed. Abbreviations used: KRH Krebs-Rin er-HEPES buffer containin 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4,1.2 n&f MgSO4, !mM CaQ, and 25 mM Na-!IEPES buffer, pH 7.4. WO6-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
798
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
els (lo), disruption of normal Ca*+ homeostasis (7), unmasking of latent injury (ll), and neutrophil infiltration (12) all may contribute to repel-fusion injury. During reperfusion of ischemic myocardium, both reoxygenation and a rapid return to physiologic pH occur. Much attention has been given to events associated with reoxygenation, but relatively little to changes of pH. Accordingly, the objective of this study was to assessthe role of changes of pH in anoxia/reoxygenation injury. To this end, we developed an in vitro model of reperfusion injury that simulates the changes of oxygenation and pH that occur during ischemia and reperfusion.
MATERL4LS
AND METHODS
Isolation and culhue of myocytes - Cardiac myocytes from 2-3 day old rat neonates were dispersed by digestion with pancreatin and collagenase (13), and purified by flowelutriation (14), as previously described (15 . Punfied myocytes were cultured on polystyrene coverslips at a density of 106 cells/3 z mm culture dish in Eagle’s MEM fortified with 5% fetal calf serum, 10% horse serum, 10 U/ml penicillin, and 10 &ml streptomycin at 37°C in humidified sir/5% carbon dioxide. Myocytes contracted spontaneously after about 3 days in culture and began to show striations after 5 days. At this time, the majority of cells exhibited vigorous, synchronous contractions at rates exceeding 60 beats/minute. Anoxia and reoaygenation - Myocytes were mounted in an airtight perfusion chamber and infused with an anoxic suspension of submitochondrial particles (1 mg of protein/ml) and succinate (5 mM) in KRH buffer, as previously described (16). Respiration by submitochondrial particles actively removed oxygen entering the chamber by back diffusion from the atmos here. Although respiration by submitochondrial particles was inhibited by approximately s 0% at pH 6.2, their concentration was sufficiently high to maintain oxygen at below limits of detectton of a Clark-type oxygen electrode (cl torr) at all values of H studied. In these experiments, Zdeoxyglucose (20 mM) was added to inhibit glycolysis. F or reoxygenation, submitochondrial partrcles were replaced with aerobic buffer not containing succinate or 2-deoxyglucose. In some experiments, anoxic submitochondrial particles were infused a second time to change pH without reoxygenation. Loss of cell viability was monitored by fluorescent labelling of nuclei with propidntm iodide (5 PM) (16,17). At the end of each experiment, digitonin (30 PM) was added to label nuclei of all cells in order to calculate percent loss of viability. Temperature was regulated at 37°C with an air curtain incubator (Laboratory Products, Boston, MA). Microscopy - Phase contrast and fluorescence images were collected with a Zeiss IM3.5 inverted microscope (Thornwood, NY). Propidium iodide fluorescence was imaged with 546~nm excitation, 580~nm dichroic, and 590-nm emission filters. Experiments were routinely recorded wrth a half-inch time-lapse video cassette recorder. h4ateriaZ.s- Eagle’s MEM, enicillin and stre tomycin were obtained from the Univer. of North Carolma Tissue 8 ulture Center ( & ape1 Hill, NC); pancreatin from Gibco 78and Island, NY); collagenase D from Boehringer Mannheim (Indianapolis, IN); and 2deoxyglucose from Sigma (St. Louis, MO). Other reagent-grade chemicals were obtained from standard commercial sources.
RESULTS Loss of viability of purified myocytes during anoxia was assessed at various values of extracellular pH. Cultured myocytes on polystyrene cover-slips were mounted into a gastight chamber. Initially, cells were pre-equilibrated in aerobic KRH, pH 7.4, for lo-20 minutes. During this period, myocytes exhibited vigorous, spontaneous contractions. Anoxia was produced by infusing an anoxic suspension of submitochondrial particles and succinate in KRH buffer at pH values ranging between 6.2 and 7.4. After l-2 minutes of anoxia, spon799
Vol.
179, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
012345 Time
(hr)
F&J. Rote&n againstanoxickilling of culturedneonatalcardiacmyocytesby acidotic extracelhdarpH- Culturesof 6-12 day old myocyteswere mountedin a gas-tightchamber
and incubated in aerobic KRH buffer, pH 7.4, for 10-15 minutes. Anoxia was started by in-
fusionof an anoxicsuspension of submrtochondrialparticles(1 m of protein/ml succinate 5 mM , and Zdeoxyglucose(20 mM) in KRH buffer at pH va!ues betweentand74 by pro idium iodide stainin of myocyte nuclei. Ckli Loss od cell viability was assessed killing at H 6.2-7.0wassignificantlylesstKan at pH 7.4 (p V and &eun$ .Y. 21985) Am. J. Physiol. 249, C149C159. 5: Gores, G.J., ‘Ncminen, A.- , and Lemasters, J.J. (1988) Am. J. Physiol. 255, C315C322. 4. Nieminen, A-L., Dawson, T.L., Gores, G.J., Kawanishi, T., Herman, B., and Lemasters, J.J. (1990 Biochem. Biophys. Res. Commun. 167,600-606. 5. Gores, G.J., rl ieminen, A.-L., Wray, B.E., Herman, B., and Lemasters, J.J. (1989) J. Clin. Invest. 83,386-3%.
6. Bing, O.H.L., Brooks, W.W., and Messer, J.V. 1973) Science 180,1297-1298. Altschuld, R.A., Hostetler, J.R., and Brierley, d .P. (1981) Circ. Res.49,307-316. i: Williamson, J.R., Schaffer, S.W., Ford, C., and Safer, B. (1976) Circulation 53 (Suppl. I), 13-114. 9. Korthuis, R.J., and Granger, D.N. (1986) In PhysioZogyof Oxygen Radicals (Taylor, A.E., Matalon, S., and Ward, P.A., Eds. , p. 217-249. Williams & Wilkins, Baltunore. 10. Jennings, R.B., Hawkins, H.K., Lowe, II!. ., Hill, M.L., Klotman, S., and Reimer, K.A. 1978) Am. J. Pathol. 92,187-214. b anote, C.E. (1983)J. Mol. Cell. Cardiol. 15,67-73. ::: Romson, J-L., Hook, B.G., Kunkel, S.L., Abrams, G.D., Schork, M.A., and Lucchesi, B.R. (1983) Circulation 67,1016-1023. Harary, I., and Farley, B. (1963) Exp. Cell Res. 29,451-465. 4: Ulrich, R., Elli et, K.A., and Rosnick, D.K. (1988)J. T&s. Cult. Meth. 11,217-221. 15: Bond, J.M., If erman, B., and Lemasters, J.J. (1991) Res. Comm. Chem. Pathol. Phannacol, 71,195-208. 16. Herman, B., Nieminen, A.-L., Gores, G.J., and Lemasters, J.J. (1988) FASEBJ. 2, 146151. 17. grnyters, J.J., DiGuiseppi, J., Nieminen, A.-L., and Herman, B. (1987) Nature 325, - .
18. Stern, M.D., Chien, A.M., Capogrossi, MC., Pelto, D.J., and Lakatta, E.G. (1985) Cir. Res. 56,899-903.
19. Grinwald, P.M., and Naylor, W.G. (1981) J. Mol. CeZL CdioL 13,867-880. Hearse, D.J., Garlick, P.B., and Humphre , S.M. (1977) Am. J. CardioZ.39,986-993. it Currin, R.T., Gores, G.J., Thurman, R. e ., and Lemasters, J.J. (1990) FASEB J. 3, A626. 22. Lernasters, J.J., Caldwell-Kenkel, J.C., Gao, W., Nieminen, A.-L., Herman, B., and Thurman, R.G. (1991) In Puthophysiologv of Reperfusion Injury, D.K. Das, Ed., CRC Press? in press. 23. Harrtson, D.C., Lemasters, J.J., and Herman, B. (1991) Biochem. Biophys. Rex Commm 174,654-659. 24. Bond, J.M., Herman, B., and Lemasters, J.J. (1990) FASEBJ. 4, A622.
803
