Jesse H. Jones Student Research Fellow of the American Heart Associa- tion. Accepted for publication May 22, 1989. Address reprint requests to L. Maximilian ...
American Journal ofPathology Vol. 135, No. 3, September 1989 Copyright © American Association ofPathologists
Association Between Inhibition of Arachidonic Acid Release and Prevention of Calcium Loading During ATP Depletion in Cultured Rat Cardiac Myocytes
Rebecca L. Jones, Joseph C. Miller, Herbert K. Hagler, Kenneth R. Chien, James T. Willerson, and L. Maximilian Buja, with the technical contributions of Dennis Bellotto, Donna Buja, Paulette K. Williams, and Elsa Yang From the Departments ofPathology and Internal Medicine (Cardiology Division), The University of Texas Southwestern Medical Center at Dallas, Texas
The development of irreversible myocardial ischemic injury is associated with progressive degradation of membrane phospholipids, accumulation of arachidonate and other free fatty acids, and electrolyte derangements, including calcium accumulation. To study the relationship between arachidonate release and calcium loading during adenosine triphosphate (A TP) depletion in cardiac myocytes, the effects of two purported phospholipase inhibitors, mepacrine and U26,384, were evaluated. Cultured neonatal rat ventricular myocytes were pretreated for 90 minutes with 5 to 10 AM U26,384 (a steroidal diamine) or 10 to 50
.uM mepacrine (an alkyl acridine) and then treated for 3 hours with 30 ,iM of the metabolic inhibitor, iodoacetic acid (IAA), with or without an additional dose of drug. IAA treatment resulted in a marked reduction in ATP level and a several-fold increase in free fatty acid radioactivity released from myocytes prelabeled with tritiated arachidonic acid (3H-AA). U26,384 produced substantial inhibition of the increased 3H-AA release, and was effective when given as a single pretreatment dose before IAA exposure oras continuous treatment before and during IAA exposure (for example, with 5 ,uM U26,384, the percentage of 3H-AA release versus IAA alone was 8% ± 2% [SEAl] [N = 15] for pretreatment only and 13% ± 4% [N = IO] for continuous treatment). Mepacrine also resulted in significant reduction in 3H-AA release, but was more effective when given as continuous treatment (for example,
with 50 MM mepacrine, the percentage of 3H-AA release versus IAA alone was 43% ± 9% [N = 61 for pretreatment only and 22% ± 7% [N = 9]for continuous treatment). More detailed analysis showed that U26,384 and mepacrine blocked the IAA-induced redistribution of 3H-AA into free fatty acids from other lipid species. Electron probe x-ray microanalysis of freeze-dried cryosections revealed marked electrolyte derangements in myocytes exposed to IAA, including a 24-fold increase in cellular Ca, a four fold increase in cellular Na, and a seven fold decrease in cellular K, and associated changes in cytoplasm and mitochondria. U26,384 treatment markedly reduced these electrolyte abnormalities, and maintained normal Ca levels in some protocols. Mepacrine treatment was less effective, but did produce normal Ca levels in 50% of myocytes. Prevention of IAA-induced cellular hypercontraction and blebbing also was observed. These data support the hypothesis that reduction of freefatty acid accumulation by inhibition of acceleratedphospholipid degradation is associated with protection of myocytes from calcium loading and morphologic damage during inhibition of energy metabolism. (AmjPathol 1989, 135:541-556)
Factors that contribute to the development of necrosis during myocardial ischemia include inhibition of energy metabolism, with ATP depletion and metabolite accumulation; altered electrolyte homeostasis; and membrane damage.1`5 To investigate mechanisms of irreversible injury potentially operating during myocardial ischemia, we This work was supported by NIH Ischemic Heart Disease SCOR Grant 5P50-HL1 7669 and the Moss Heart Fund, Dallas, Texas. Ms. Jones was a Jesse H. Jones Student Research Fellow of the American Heart Association. Accepted for publication May 22, 1989. Address reprint requests to L. Maximilian Buja, MD, Department of Pathology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9072.
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performed studies in a cultured neonatal rat ventricular myocyte model.6-9 During metabolic inhibition in the cultured myocytes, the loss of cell viability was associated with marked ATP reduction; progressive membrane phospholipid degradation, shown experimentally by release of arachidonic acid from myocardial membrane phospholipids; and ultrastructural evidence of mitochondrial damage, myofibrillar hypercontracture, and fluid-filled blebs.6-9 These myocytes also showed electrolyte alterations characterized by increased sodium and calcium and decreased potassium and magnesium in the cytoplasm, mitochondria, and nuclei.8'9 In an ischemic rat liver model, the degradation of membrane phospholipids was related temporally to the development of a membrane calcium permeability defect.10 Similar observations were made in myocardial models.11-13 Pharmacologic inhibition of phospholipid degradation during metabolic inhibition has resulted in protection against the development of irreversible injury.14-19 Das et al17 showed that mepacrine, a phospholipase A2 inhibitor, provides significant protection against reperfusion injury in the ischemic pig heart. Initial studies indicated that another compound, U26,384, inhibits phospholipid degradation during metabolic inhibition of cultured neonatal rat ventricular myocytes.16 Previous studies postulated a continuing cycle of progressively increasing cytosolic calcium accumulation and phospholipid degradation in the evolution of irreversible myocardial injury.34'11'2 However, a direct relationship between reduced phospholipid degradation and altered homeostasis of calcium and other electrolytes has not been evaluated. Therefore, the purpose of this study was to test the hypothesis that interventions that reduce phospholipid degradation during metabolic inhibition prevent the development of electrolyte derangements, particularly calcium loading, in metabolically impaired myocytes. Preliminary results of this study have been presented in abstract form.20
Materials and Methods Pharmacological Agents
Mepacrine (quinacrine) dihydrochoride is a commercially available alkyl acridine (Sigma Chemical Company, St. Louis, MO). Published reports indicate that mepacrine inhibits phospholipase A activity in enzyme assays and intact biological systems.71-'9 U26,384 made available for these experiments by The Upjohn Company (Kalamazoo, Ml.), is a steroidal diamine (N-[3-dimethylamino)propyl]-3methoxy-N-methyl estra 2,5(1 0)-dien-1 7-beta-amine) (Fig-
ure 1, inset) that acts as a phospholipase A2 inhibitor. U26,384 was developed by The Upjohn Company, Kalamazoo, Ml. In studies performed by Dr. Stuart Bunting and colleagues at Upjohn, U26,384 was shown to inhibit porcine pancreatic phospholipase (IC50, 50 ± 5MM), prevent collagen-induced platelet aggregation (IC50, 8.2 ± 0.9
uM), and block release of arachidonic acid metabolites from neutrophils (S. Bunting, personal communication, January, 1988). We confirmed U26,384's property as an A2 inhibitor in vitro by following the production of radioactive free fatty acid from 1-palmitoyl-2-[1 -14C]-arachidonyl phosphatidylcholine (PC) in an assay also containing 400 nmol egg PC and 2 ng porcine pancreatic phospholipase A2 in 200 Ml 10 mM CaCI2 and 100 mM Tris-HCI, pH 8.0.21 The U26,384 (0 to 2 nmol) and mepacrine (0 to 20 nmol) were sonicated together with the PC substrate and preincubated 15 minutes, and the assay was started by the addition of enzyme; both solutions were prepared in the Ca/Tris buffer. After incubation for 60 minutes at 37 C, the reaction was stopped by the addition of 200 Al of 10 mM EGTA and 4 ml of chloroform:methanol (1:1) followed by 1.5 ml water.22 The extracted lipid was resolved by thin layer chromatography, and the radioactivity in the fatty acid and PC bands was determined by liquid scintillation spectrophotometry. These reaction conditions were linear up to 120 minutes. Preliminary experiments, using the protocols described below, indicated that 5 to 10 zM U26,384 and 10 to 50 MM mepacrine were optimal concentrations for the cultured, neonatal myocytes (see Experimental Protocol).
Cell Culture Model Cultures of neonatal rat cardiac myocytes were prepared as previously described.6`9 Briefly, ventricles were isolated from 2- or 3-day-old rats and minced in a nominally calcium-free, HEPES-buffered balanced salt solution with pancreatin (60 mg/100 ml) (Gibco Labs, Lawrence, MA) and collagenase Type 11(6000 to 6400 units/1 00 ml) (Cooper Biomedical, Freehold, NJ). The minced tissue was incubated with the enzymes in a shaker bath at 37 C for 30 minutes. After centrifugation, the initial supernatant, containing red blood cells and debris, was discarded. Fresh pancreatin-collagenase was added to the remaining tissue, and the mixture was placed in a shaker bath for 20 minutes at 37 C. The supernatant was removed and centrifuged for 4 minutes at 1400 rpm in a tabletop centrifuge (IEC, model CL). The cells were resuspended in newborn calf serum and kept at 37 C. These steps were repeated for four more digestions. The combined cell suspensions in calf serum were centrifuged for 6 minutes at 2100 rpm. The pelleted cells were resuspended in DME: 199 (4:1) medium containing 10% horse serum, 5% fetal calf
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90 0
Figure 1. Effect ofU26,384 (chemical structure as shown) on pancreatic phospholipase A activity. Values are given as mean + SEM of the percentage of control (no drug) activity measured in four separate
experiments. Activity was determined by the production of radioactive fatty acid from 1 -palmitoyl-2-[1-_14C] - arachidonylphosphatidylcholine by pancreatic phospholipase A2 incubated for 60 minutes at 3 7 C in an assay containing 2 mM egg PC sonicated together with varying amounts of U26,384, 10 mM CaC12, and 100 mM Tris-HCI, pH 8. 0.
0-
U IL
60
30
0
0
and antibiotics (penicillin and streptomycin). The myocytes were purified from nonmyocytic cells with a Percoil density gradient system; myocytes were collected from the interface between 1.056 and 1.080 g/ml.23 The myocytes were plated at a density of 1.6 to 2.0 X 1 o6 cells per 60 mm plate and incubated at 37 C with 7% C02/ 93% room air. The myocytes attached to the culture dish and assumed strong, synchronous beating activity at 2 or 3 days. The cultures routinely contained 90% or greater beating myocytes. On day 3, the medium was replaced with medium 199 containing 20 mM HEPES, 26.2 mM bicarbonate, 5% fetal calf serum, and 0.3 uCi/plate of [5,6,8,9,11 ,12,14,15-3H(N)]-arachidonic acid (60 to 100 Ci/mmol, New England Nuclear Co., Boston, MA). After a 16- to 24-hour labeling period, 85% to 95% of the 3Harachidonic acid was incorporated into the cells. serum,
Experimental Protocol Experiments were performed to evaluate the effects of drugs, U26,384 or mepacrine, on myocytes exposed to the glycolytic inhibitor and alkylating agent, iodoacetic acid (IAA).24 The experiments were done in two steps: 1) a 90-minute period for drug pretreatment; 2) a 3-hour period of metabolic inhibition using 30MgM IAA with or without a second equimolar dose of drug. Cultured myocytes were incubated as shown in Table 1. Serum-free medium 199 containing 20 mM HEPES and 26.2 mM bicarbonate equilibrated with 7% CO2 at 37 C was used for all experiments. Thirty MM IAA was chosen because it gave a reproducible pattern of myocyte injury and ATP depletion interval.68 Dose-response studies from 0.01 to 100 MM demonstrated that 5 or 10 ,M U26,384 gave optimal protective effects (reduced fatty acid release and morphologic integrity) with minimal drug toxicity (myocyte over a short
.1
1
10
100
U26,384 (0LM)
detachment) in this model.1620 U26,384 was added in 5 to 10 MI ethanol per 10 ml medium; no effects due to ethanol were observed in specific tests from 1 to 100 ,lM ethanol per 10 ml medium. Mepacrine was used at 10- or 50,uM concentrations and was dissolved directly in the medium. Myocytes were observed and photographed using a Nikon inverted microscope with phase contrast light.
Analytic Methods Immediately after the 3 hours of metabolic inhibition, the media, including two 1 ml 100 mM EGTA washes, were extracted by the method of Bligh and Dyer,22 and the chloroform/methanol extract dried under nitrogen. The dried lipid was resuspended in chloroform:methanol (1:1) and aliquots were removed to measure the total radioactivity. Separate aliquots were spotted on silica gel G thin layer chromatography plates with either liver lipid extract or synthetic lipid standards added as a carrier. The neutral lipids were separated with a solvent system of hexane:diethyl ether:acetic acid (80:20:1). The spots were visualized either with iodine vapor or under ultraviolet light after spraying with dichlorofluorescein. Each sample track on the silica plates was scraped into three fractions: free fatty acid Table 1. Experimental Protocol 90 minutes Control Medium Drug only: Pretreat Drug Continuous Drug IAA only Medium IAA plus drug: Pretreat Drug Continuous Drug
Treatment
3 hours Medium Medium
Drug IAA
IAA IAA + drug
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(FFA) band; neutral lipid (NL) band, incorporating everything between FFA and the solvent front including cholesterol esters and triacylglycerols; and the phospholipid (PL) band, incorporating everything below FFA to the origin, including phospholipids, monoacylglycerols, diacyglycerols, and cholesterol. In some of the initial experiments, only the FFA band was collected. The samples were counted by liquid scintillation spectrophotometry. After removal of the media, including the EGTA washes, the cells were solubilized in 1 ml 0.1 M NaOH and scraped from the culture dish.25 Three aliquots were taken from each suspension for protein analysis by the method of Lowry et al.26 In some experiments, 0.5 ml 6N HCI and 0.5 ml 6% LaCI3 were added to the remaining suspension, and the solution was assayed for total calcium by atomic
absorption spectrophotometry.25 ATP measurements used cells scraped from the culture plates with two 750 ,ul aliquots of 6% cold perchloric acid containing 50 AM bromodeoxyuridine as an internal standard. In these experiments, the cells were washed with cold phosphate-buffered saline rather than EGTA before the perchloric acid extraction. Protein was collected by centrifugation, solubilized overnight in 1 M NaOH, neutralized with HCI, and measured.26 Supernatants were neutralized with 3N KOH and filtered, and the ATP was measured by reverse phase high pressure liquid chromatography using an internal standard ratio of ATP and bromodeoxyuridine.27
freeze-dried and maintained at -80 C under vacuum in the electron microscope.28 X-ray spectra were obtained with a JEOL 1 200EX electron microscope in the scanning transmission mode and processed with a Tracor Northern 5500 multichannel analyzer.8 Whole cells were analyzed at a magnification of 12,000 to 15,000, with selected areas of cytoplasm analyzed at a magnification of 60,000 and mitochondria analyzed at 200,000, so that the entire raster was filled with the mitochondrion. The whole cell spectra included mitochondria and extramitochondrial cytoplasm, but nuclei were excluded. The x-ray spectra were collected over the range of 0 to 20 KeV for 100 seconds at a resolution of 10 eV per channel. Analysis of spectra was based on the direct relationship between peak-to-continuum ratio and elemental concentration.29 Elemental peak intensities were obtained by deconvolution of the spectra using a multiple least squares fitting technique.x The first and second derivatives of the potassium K alpha peak were included in the least squares fit of the spectra.31 The peakfree continuum used for the calculations was from 5.5 to 7.5 KeV. Continuum counts were corrected for extraneous counts from the copper grids, aluminum specimen holder, and the formvar support films. Peak-to-continuum ratios were converted to elemental concentrations using weighting factors derived from standard curves obtained from freeze-dried sections of gelatin-glycerol preparations containing different concentrations of the elements of interest.' Scanning transmission electron micrographs of the freeze-dried cryosections also were obtained.
Electron Probe Microanalysis
Statistical Evaluation Electron probe microanalysis was used to determine the content of total calcium (bound and free) and other elements in subcellular compartments as well as of the whole cell.8 Myocytes were grown on 60-mm culture dishes coated with a nominally 1.5-mm-thick layer of collagen gel (2.5 ml 80% collagen per plate) (Collagen Corporation, Palo Alto, CA). Cells in culture media were added to the plates and incubated for 3 or 4 days. The initial seeding produced a relatively sparse colonization of the gels. The medium was removed and replaced with a second seeding of myocytes, after which the myocytes established strong beating activity after an additional 3 to 4 days. The experiments were performed as described in the Experimental Protocol. Thereafter, the collagen was cut and a rectangle was floated on filter paper. The filter paper, with collagen and myocytes, was clamp-frozen with liquid nitrogen-cooled copper blocks.8 Cryosections were cut at a thickness of approximately 150 nm using a DuPont-Sorvall MT5000 ultramicrotome with a FS1000 cryosystem and then transferred to the analytical electron microscope using a Gatan 626 cryotransfer stage. The sections were
Data are reported as means ± standard error of the mean. Multiple group comparisons were performed by analysis of variance followed by Duncan's or Newman-Keul's multiple range test. Differences were considered significant when P
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Previous studies established that IAA treatment is associated with a moderate reduction in ATP level after 1 hour and marked reduction thereafter, with ATP concentrations at 2 hours approximately 25% of control levels and at 3 hours only 5% to 15% of control levels.68'6 In the present study, IAA reduced the ATP to 25% of control at 2 hours and to 6% of control at 3 hours (Table 6). Treatment with 5 MM U26,384 or 50 plus 50 MM mepacrine alone had no significant effect on the ATP levels, and treatment with 10MuM U26,384 alone was associated with a slight reduction in ATP level. After 2 hours of IAA exposure, reductions in ATP levels were virtually identical in the presence or absence of pretreatment with 10 ,uM U26,384 (25% and 26% of control, respectively). After 3 hours of IAA exposure and treatment with 5 MM U26,384, 10MM U26,384, and 50 plus 50MuM mepacrine, ATP levels were 16%, 13%, and 10% of control, respectively, compared with 6% for IAA alone. The ATP levels in the four groups were all significantly different from those of the control group but not significantly different from each other, indicating that the drugs did not have a significant effect on IAA-induced ATP reduction.
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Figure 4. Scanning transmission electron micrographs offreeze-dried cryosections of myocytes from experiments involving treatment with iodoacetic acid (IAA) and U26,384. A: Myocytes exposed to 30 itM IAA alone for 3 hours have vacuolated cytoplasm consistent withfluid accumulation, and the mitochondria contain multiple electron dense inclusions consistent with calcium phosphate deposits (original magnification X 12,900). B: Myocytes pretreated for 90 minutes with 10 AsM U26,384 followed by exposure to 30 ALM IAA alone for 3 hours have compact cytoplasm, and the mitochondria exhibit uniform density and are devoid of electron dense inclusions (original magnification X9700).
Discussion Membrane damage in myocardial ischemia has been postulated to involve progressive phospholipid degradation secondary to phospholipase activation.34'12 The mechanism of the phospholipase activation may be mediated by an increase in cytosolic Ca2+ secondary to alterations in Ca2+ transport induced by energy depletion or, possibly, by other metabolic alterations. Once activated, progressive phospholipid degradation may lead to changes in membrane permeability, marked derangements in cellular water and electrolyte homeostasis, including progressive calcium overloading, and cell death.3'4"1"2 Although studies of in vivo myocardial ischemia provided support for this hypothesis, the need for further elucidation led to work using in vitro models. In models of ATP depletion using metabolic inhibitors, marked release of labeled fatty acid from prelabeled cells is an index of phospholipid degradation and is associated with prominent structural changes, including hypercontraction and blebbing, marked electrolyte alterations, including Ca2+ accumulation, and loss of cell viability.6 9 In a previous study, we showed that treatment with U26,384 prevented the release of 3H-AA during ATP depletion induced by exposure to IAA.16 The present study showed that inhibition of accelerated phospholipid degradation
with U26,384, and to a lesser degree with mepacrine, was associated with inhibition of calcium loading during energy depletion induced by IAA. In addition, there was preservation of cellular morphology contrasting to the hypercontraction and bleb formation induced by IAA in the absence of U26,384 or mepacrine treatment. IAA produced marked ATP reduction in the presence or absence of U26,384 or mepacrine. The present and previous experiments with U26,384 and IAA indicated that the time course of IAA-induced ATP depletion was similar in the presence or absence of drug.'6 However, after 3 hours, ATP levels were slightly higher with U26,384 or mepacrine treatment than with IAA alone. Although the differences were not statistically significant, the results raised the possibility that the drugs may have exerted an ATP-sparing effect. However, an alternative explanation was that the slight differences in ATP were due to better morphologic preservation of the cells in cultures treated with IAA plus drugs versus those treated with IAA alone. The latter interpretation was supported by the higher protein content recovered from the drug plus IAA-treated plates than the plates treated with IAA alone (% control protein: 5 ,M U26,384, 95 ± 3; 10 ,uM U26,384, 88 ± 2; 50- + 50 ,uM mepacrine, 78 ± 2; IAA alone, 69 ± 4), consistent with better retention of residual ATP in less fragile cells in the drug-treated cultures. Thus, it is unlikely that U26,384 or mepacrine exerted their protective effects on
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0
a
-
o
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2
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30 sM IAA
5 iM pro
2 x 5 gM
U26,384
2 x 50 M Mepacrins
Figure 5. Electron probe microanalysis ofcardiac myocytes exposed to 30 sM iodoacetic acid (IAA) for 3 hours in conjunction with various treatmentprotocols: IAA alone;pretreatment with 5MM U26,384 (5AMpretreatment); continuous treatment with 5,uM U26,384 (2 X 5yM); and continuous treatment with 50-MM mepacrine (2 X 50 MM). The top panel shows the percentages of the total myocyte population with abnormal levels of Ca, Na, Cl, Mg, and K The bottom panel shows the magnitude ofthe alteration in each element expressed as thepercentage control concentration (see Table 5)for the entire population of cells in each treatment group. IAA treatment produced marked elemental changes involving up to 95% of the myocytes. Continuous treatment with 5 MM U26,384 resulted in substantialprotection, characterized by minimal alteration of elemental levelsfor the entire population with less than 20% of myocytes having abnormal values. Pretreatment alone with 5 MM U26,384 gave less protection both in terms ofpercentages of abnormal cells and the magnitude of elemental alterations. Continuous treatment with 50MM mepacrine also showed only partialprotection.
cell and membrane integrity by
an
ATP sparing mecha-
nism.
In control cultures, U26,384 and mepacrine both increased total lipid radioactivity twofold to threefold in the medium. This was most prominent at the higher doses of U26,384. This biochemical change was associated with observational evidence of a dose-dependent drug effect characterized by detachment of some myocytes from the plates. However, these increases were associated with preservation of a relatively normal distribution of 3H-AA radioactivity between the various lipid compartments (Tables 2 and 3). The slight redistribution of radioactivity into the PL fraction may represent drug-induced inhibition of phospholipase activity in normal cells. In contrast, IAA treatment resulted both in an increase in total lipid radioactivity as well as a marked increase in the percentage of
radioactivity in the FFA. Thus, the data indicate that IAA affected phospholipid metabolism resulting in excess FFA release, whereas U26,384 and mepacrine produced an increase in medium radioactivity primarily due to myocytes detaching from the plate. In spite of this side effect, both agents significantly blunted the IAA-induced redistribution of 3H-AA from phospholipids to free fatty acids. Although both U26,384 and mepacrine inhibited phospholipid degradation as manifested by 3H-AA release, their mechanisms of action require further elucidation. Mepacrine is an alkyl acridine known to inhibit phospholipase A activity.17-19 However, mepacrine also has been shown to inhibit reactions catalyzed by phospholipase C,19 as well as other cellular reactions, including Na+-Ca2+ exchange.32 The effect of mepacrine on phospholipase activity may not be due to a direct effect on the enzyme but instead to inhibition of the catalytic effect of calmodulin as mediated by Ca2 .33 U26,384 is a steroidal diamine that inhibits pancreatic phospholipase A2 activity in vitro, as shown in the present study, and presumably in vivo, because arachidonate is esterified primarily at the sn-2 position. However, its specificity and mechanism(s) of action have not been fully determined. Results of the present study suggest that U26,384 and mepacrine have different mechanisms of action in cultured myocytes subjected to energy depletion, as U26,384 was effective as a pretreatment and as a continuous treatment, whereas mepacrine's optimal effect was only achieved as continuous treatment during IAA exposure. Presumably, U26,384 as a steroidal diamine becomes incorporated into membranes and is not removed with the medium after pretreatment. The similarities found between continuous treatment with 5 ,uM (two doses) and pretreatment with a single 10 ,M dose suggest retention of the initial U26,384 dose during pretreatment. If this occurs, the concentration of the drug would be equivalent during IAA exposure in the 5 AM continuous and 10,M pretreatment only protocols. Furthermore, this mechanism of action of U26,384 was supported by the finding that more of the drug was required for an optimal effect in the EPMA experiments (continuous 5 IuM or 10 ,uM pretreatment) than in the 3H-AA release or atomic absorption experiments (5 ,uM pretreatment), a finding presumably related to the partial absorption of the drug into the collagen matrix used in the EPMA experiments. Control myocytes exhibited the expected pattern of electrolyte levels measured by EPMA. The control values were comparable to those obtained in other EPMA studies of striated muscle and, particularly, mammalian cardiac muscle.34-3 In these studies, Cl values were consistently higher than Na values, suggesting active transport of Cl in mammalian myocardium.35-39 The electrolyte alterations induced by IAA were characterized by increases in Na, Cl, and Ca and decreases in K and Mg. These
554 Jones et al AlP) September 1989, Vol. 135, No. 3
Table 6. Effects ofIodoacetate, U26,384, and Mepacrine on ATP Concentration in Cultured, Neonatal Rat, Cardiac Myocytes
U26,384 Treatment
Control 2 hours 3 hours lodoacetate 2 hours 3 hours
No drug
49.0 ± 2.1 12.1 ± 1.5t 2.7 ± 0.5*
Mepacrine
(5 lM)
(10 JM)
(50 + 50 AM)
44.0 ± 3.4
41.2 ± 1.9t 43.0 ± 3.8
47.6 ± 2.6
8.0 ± 1.4*
12.7 ± 15t 6.6 ± 1.5*
5.1 ± 0.5*
Values expressed as mean ± SEM (nmol/mg protein) of 10 plates in five separate experiments (N = 20 plates/ten experiments for control 3 hour and IAA 3 hour). ANOVA (P < 0.0001) and Duncan's multiple range test (P < 0.05): * control 3 hour (no drug); t $ control 3 hour (no drug) or IAA 3 hour (no drug).
changes are consistent with progressive membrane dysfunction in energy-impaired myocytes, including initial perturbations in ion transport systems followed by nonspecific changes in membrane permeability.3-5 After 3 hours of IAA treatment, generalized changes in cellular electrolytes of the rapidly frozen cells included a 24-fold increase in cellular Ca with a 766-fold increase in mitochondrial Ca. The optimal treatment protocols, 5 gM continuous treatment and 10 MM pretreatment with U26,384, resulted in maintenance of normal Ca levels as well as minimal alterations in levels of other electrolytes after IAA exposure. A continuous mepacrine treatment of 50 ,M also resulted in minimal Ca elevation. However, mepacrine-treated myocytes still developed significant alterations in Na, Cl, and K after IAA exposure. These findings are consistent with amelioration of electrolyte alterations induced by a nonspecific increase in membrane permeability associated with advanced cell injury. The protection was nearly complete in the case of U26,384 and was limited to calcium in the case of mepacrine. Thus, although it is uncertain that the effects of U26,384 and mepacrine are limited to a single mechanism of action, the present study showed that inhibition of phospholipid degradation during metabolic inhibition is associated with prevention of altered elemental concentrations, particularly calcium loading, and preservation of morphology. Furthermore, this association was observed with both compounds regardless of their specific mechanisms of action. It should be noted that these protective effects occurred in spite of ATP depletion because an 85% or greater ATP reduction was produced by IAA in the presence and absence of drug treatment. It has been postulated that an early increase in free Ca2+ is an initiating event in injury of ATP-depleted cells that leads to phospholipase activation, accelerated phospholipid degradation, increased membrane permeability, and generalized electrolyte derangements.3 4'0 12 Evidence has been obtained that phospholipid degradation can be activated by an increase in cytosolic Ca2", inde-
pendent of metabolic inhibition.40 It is possible that U26,384 and mepacrine prevent the early increase in Ca2" or that the drugs block phospholipase activation in spite of an early increase in cytosolic Ca2+. Results of the present study are related to a later stage of cell injury and do not provide insight into these early events, although neither U26,384 nor mepacrine altered total Ca levels in controls. Further studies are needed to address this important issue regarding cytosolic Ca2+. Morphologic changes associated with severe myocyte injury include hypercontraction, blebbing, and physical defects in membranes. Our observations suggest that accelerated phospholipid degradation contributes to the development of these changes, both directly as a result of intrinsic changes in the membrane and indirectly as a result of the electrolyte and water shifts. Inhibition of phospholipid degradation by U26,384 and mepacrine preserves morphologic integrity during ATP depletion. Further investigation is necessary to determine if this protection will allow the cells to recover or whether it represents a transient but significant delay in cell death. In addition, it must be determined whether these effects are broadly applicapable or are limited to models of metabolic inhibition. Models other than IAA-induced injury will be more suitable for such studies because we have observed that exposure of myocytes to IAA for more than 1 hour induces progressive ATP depletion and cellular degeneration that cannot be reversed after the cells are returned to normal medium. Other workers presented evidence that damage to the cytoskeleton also contributes to the genesis of severe cellular damage in energy-depleted myocardium by allowing unrestrained membrane blebbing when water and electrolyte shifts occur.4' 43Determination of the exact interactions resulting from changes in membrane phospholipids and cytoskeletal filaments requires further study. However, this study showed that inhibition of phospholipid degradation is associated with prevention of marked electrolyte shifts, including calcium loading, protection against cell blebbing,
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and preservation of normal morphology during energy depletion in cardiac myocytes.
References 1. Jennings RB, Hawkins HK, Lowe JE, Hill ML, Klotman S, Reimer KA: Relation between high energy phosphate and lethal injury in myocardial ischemia in the dog. Am J Pathol
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Acknowledgment We thank Ms. Rebecca Lundswick for assistance with the statistical analyses and Ms. Dian Kammeyer for secretarial work.
