J Mol
Cell
Cardio122,
227-237
(1990)
Effects of Calcium Depletion and Loading Metabolic Inhibition of Isolated Adult Dianne
S. Rim,
Ruth
A. Altschuld*
on Injury During Rat Myocytes
and Charles
E. Ganote
Department of Pathology, James H. Q&Len College of Medicine, East Tennessee State University, PO Box 1954OA, Johnson City, TN 37614-0002, USA *Physiological Chemistry, Ohio State University, Columbus, OH, USA (Received 15 March 1989, accepted in revised form 19 October 1989) D. S. RIM, R. A. ALTSCHULD AND C. E. GANOTE. Effects of Calcium Depletion and Loading on Injury During Metabolic Inhibition of Isolated Adult Rat Myocytes. Journal of Molecular and Cellular Cardiology (1990) 22, 227-237. The hypothesis that calcium influxes from the extracellular space play an important role in the pathogenesis of irreversible anoxic injury was tested using isolated adult rat myocytes. Myocytes treated with 6 rnM amytal and 3 rnM iodoacetate and subsequently incubated in either calcium-containing (1.12 mn) or calcium-free media (with or without 1 rnM EGTA) developed rigor contracture (cell squaring) and cell death (trypan blue permeability) at the same rate. The rates ofcell death in both calcium-containing and calcium-free media were increased by incubation in hypotonic media even though the rates of contracture development remained unaltered. Cells developed osmotic fragility prior to membrane permeability increases. The calcium ionophore, A23187 (10 p(M), induced rapid rounding of rod-shaped cells subjected only to mitochondrial inhibition in calcium containing media, confirming its ability to cause an increase in cellular permeability to calcium. However, A23187 did not alter the rates of cell death of totally metabolically inhibited myocytes in either calcium-containing or calcium-free media with EGTA. The results indicate that influxes of calcium are not necessary for the development of irreversible injury in metabolically inhibited, isolated myocytes. KEY WORDS:
Calcium;
Isolated
myocyte;
Anoxic
injury;
Introduction Myocardial injury can be divided into three conceptually distinct intervals: an early phase of anoxia, extending to the time of completion of rigor contracture during which cellular ATP (energy) levels decline; a middle phase of severe energy depletion; and a late phase of cell death and necrosis. Calcium is known to play important roles in the early and late phases of irreversible myocardial ischemic/anoxic injury, but the role of elevated cytosolic free calcium during the middle phase of severe ATP depletion is less well established. Cytosolic free calcium can increase during early stages of energy depletion through sodium/calcium exchange mechanisms (Murphy et al., 1987) or release of calcium from the mitochondria or sarcoplasmic reticulum (Barry et al., 1987). It has been hypothesized that micromolar increases in intracellular free calcium during the early phases of energy depletion increase cardiac 0022-2929/90/020227
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fragility;
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energy demand (Moreno-Sanchez and Hansford, 1988) and could activate cytosolic proteases (Dayton and Schollmeyer, 1980; Zeman et al., 1985) or lipases (Chien et al., 1985) which may then cause irreversible cellular damage. This hypothesis has been difficult to test because of the complexity of calcium interactions in the myocardial cell and the fact that tissue measurements of total tissue calcium may not provide a reliable assessment of the more relevant intracellular free ionized calcium levels, Calcium is important in modulating the rate of ATP utilization and in maintaining the ingegrity of intercalated discs (Ganote and Nayler, 1985). These actions of calcium, combined with the lack of adequate techniques to measure cytosolic free calcium in whole tissue, have precluded definitive calcium depletion experiments in intact tissues which could separate the effects of calcium on the process of irreversible injury from its other actions. 0
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Isolated myocyte models of injury would seem better suited to study the effects of extracellular calcium depletion on the development of irreversible anoxic injury than intact tissue models because: ( 1) suspensions of cells can develop ATP depletion rigor contracture in calcium-free media without suffering injury to intercalated disc membranes (Rajs et al., 1980; Piper et al., 1985; Ganote and Vander Heide, 1988); (2) rigor contracture is calcium independent (Allen and Orchard, 1983) and the development of contracture can be used as a biological assay for severe ATP depletion (Haworth et al., 1981; Altschuld et al., 1985); (3) the rate of ATP depletion of metabolically inhibited myocytes is similar in calciumcontaining and calcium-free media, allowing a direct comparison of the influence of calcium on the rates of progression of cell injury; (4) calcium influxes from the extracellular space and intracellular free calcium levels can be experimentally measured using fluorescent calcium probes (Li et al., 1987) and manipulated by controlling extracellular calcium in either the presence (duBelI et al., 1988) or absence (Barry et al., 1987; Cobbold and Allshire, 1988) of a calcium ionophore. The present study was designed to investigate the direct effects of extracellular calcium on cell injury in metabolically inhibited myocytes. The results indicate that given equal rates of energy depletion, raising or lowering extracellular and cytosolic free calcium have little effect on the rate of development of cell death.
hold, NJ) was added to the per&sate. After the hearts became soft (40 to 50 min), they were removed, minced, and dispersed in perfusion buffer containing 4% w/v BSA. The suspension was incubated at 30°C for 5 to 10 min in a shaking bath, filtered through a nylon mesh and washed in Ca’+-free “low potassium” (5 mM K ‘) wash buffer containing in addition to the other ions and supplements, HEPES buffer (30 mM). Cells were resuspended in this final wash buffer and calcium added to a final concentration of 1.12 mM. The cells were rinsed and suspended in the 5 mM K buffer which was either calciumfree or contained 1.12 mM Ca’ + and which was used for the experimental incubations, conducted at 30°C. The osmolality of the “isotonic” final buffer was 340 mOsmo1. Approximately 12 x lo6 cells were obtained from each isolate. The reagents used were iodoacetic acid (reagent grade, Sigma), amobarbital (Eli Lilly & Co.) and antibiotic A23187 (free acid, CalbioChem Corp.). Initial cell morphology and viability were assessed by removing 100 ~1 aliquots of cells in final suspension and mixing these with an equal volume of 1 o/0 glutaraldehyde in modified Tyrodes solution (50 mM NaCl replaced by 100 mM glutaraldehyde) (Maunsbach, 1966) containing 3% trypan blue dye and immediately counting the number of rod-, square-, blue square-, round- and blue roundshaped cells present. A total count always exceeded 300 cells and was completed within 15 min in order to preclude non-specific staining of cells.
Methods Cell isolation Myocytes were isolated from hearts of male Sprague-Dawley rats weighing 200 to 300 g as previously described (Vander Heide et al., 1986). Hearts were perfused retrograde with a “high potassium” buffer [in mM, NaCl 125; KC1 30; NaHCOs 25; KH2P04 1.2; MgClz 1.2; dextrose 1.1; taurine 60; creatine 20; Lglutamine 0.682; bovine serum albumin (BSA Pentax Fraction V) 1 mg/ml, and supplemented with a complete amino acid mixture]. The perfusate, pH 7.4, was continuously gassed with 95% 02--5% COZ. After 5 min of perfusion at 37°C collagenase (Type II, 1.25 mg/ml, Worthington Biochemicals, Free-
Experimental design For each experiment, the 10 ml of cell suspension in 1.12 mM calcium was divided into two centrifuge tubes. Cells were centrifuged twice, the suspension divided and resuspended in the appropriate Ca’ +-containing or Ca’ +-free, 5 mM K+ buffer, in accordance with the experimental protocol used. Each suspension was then divided into 2.5-ml aliquots. An additional volume of 2.5 ml of the appropriate buffer was added to each flask, bringing the final volume of each sample to 5 ml. If the cells were to be metabolically inhibited the final volume also contained 3 mM iodoacetic acid and 6 mM amobarbital. Hypotonic solutions
Calcium
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were prepared by diluting the buffer solutions to half their normal osmolarity, but maintaining the inhibitor concentrations constant. Aliquots of 100 ~1 for cell counts were removed at predetermined intervals during the incubation. Because of variation in the potency of the amytal and the collagenase, each experimental protocol was run as an individual group of experiments using the same lots of collagenase and amytal. Each value represents the mean of at least five separate experiments. Each experiment was performed on a separate day, using a new cell isolation. Variations in isolates during each protocol were acceptably small but the variations of isolates at different time periods precluded comparisons between separate protocols. Experimental protocols The six experimental protocols used were designed for the following purposes: (1) to study the effects of Ca2+ and Ca’+-free isotonic buffers on the rate of cell death of metabolically inhibited cells; (2) to study the effects of the presence or absence of calcium on control and inhibited cells in hypotonic media; (3) to compare the rates of cell death of metabolically inhibited cells in calcium-containing media to that of metabolically inhibited cells in calcium-free media containing the calcium chelator EGTA; (4) to demonstrate that metabolically inhibited myocytes develop osmotic fragility; (5) a control study to determine the effects of A-23187 on the rates of cell contracture to demonstrate that the ionophore has a significant effect under the conditions of these experiments; (6) to compare the effects of the addition of A-23187 on the rates of cell death of metabolically inhibited cells in calciumcontaining media to that of metabolically inhibited cells in calcium-free media containing 1 mM EGTA. The data is expressed as the percentage of cells showing rod-shaped morphology (length/width ratio > 3/l), square-shaped morphology (length/width ratio < 3/l but > 1/ 1)) or being round-shaped. The cells were further sub-classified as being viable (trypan blue-excluding) or non-viable (trypan bluestaining). Results are expressed as the mean & S.D. of the mean. Statistical differ-
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ences were determined using a two unpaired Students t-test. -
tailed,
Results Calcium and cell death in isotonic media The first experimental protocol determined the rates of cell death (as represented by the rates at which cells became permeable to trypan blue) of isolated myocytes in isotonic suspension after combined inhibition of both anaerobic and aerobic metabolism with iodoacetic acid and amytal. The results in Figure l(a) show that at the onset (time 0) of the
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FIGURE 1. (a) Effect of metabolic inhibition (6 rnM Amytal and 3 rnM IAA) on the rate of contracture of isolated myocytes in calcium-containing or calcium-free media. Myocytes rapidly developed ATP depletion rigor contracture, changing from an elongated rod-shaped form into contracted square-shaped forms. There was no significant difference in the rates of contracture development in the presence or absence of calcium. (b) Effect of calcium on the rate of development of trypan blue permeability of metabolically inhibited, square myocytes following rigor contracture in isotonic media. Since the rates of ATP depletion contracture were similar in calcium-containing (closed symbols) and calcium-free (open symbols) media [see (a)], the rates of cell death (monitored by ttypan blue staining) can be directly compared and were found to be similar in the presence or absence of extracellular calcium.
D. S. Rim et al.
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experiment, the initial cell counts were similar. Metabolically inhibited cells underwent rapid rigor contracture into squareshaped forms. From 30 to 120 min of incubation there was a gradual loss of viability of the square cells [Fig. 1 (b)]. There were no significant differences in the rates of contracture or loss of viability of the inhibited cells in calcium-containing as compared to those in calcium-free media.
31.2 + 19.1, 47.0 + 5.8 and 67.2 + 9.1 (each value different from control by P < 0.01). Despite the increased rate of cell death in hypotonic media as compared to isotonic media, there was no difference in the rates of conversion to trypan blue permeability of hypotonically incubated cells in calciumcontaining as compared to those in calciumfree media (Fig. 2).
Efect of calcium and no calcium on cells in hy@otonic media Metabolically inhibited cells incubated in hypotonic media converted to square-shaped cells at the same rate and extent as did cells in isotonic media, but the rate at which the square cells died in hypotonic medium was increased, although non-inhibited cells were not significantly affected by hypotonic incubation. In experiments to document this increase, the percentage of blue square cells at 30, 60 and 90 min of incubation in control isotonic medium were 3.6 + 4.1, 19.2 + 8.3 and 46.2 ) 7.1, respectively. In hypotonic media the corresponding values were
The effects of acute swelling in hypotonic media were determined by incubating metabolically inhibited cells in isotonic media for 30 min and then transferring them to hypotonic or isotonic media. Figure 3 shows that compared to resuspension in isotonic buffer, resuspension into hypotonic media results in a more rapid conversion of viable square cells into non-viable, blue square cells. Approximately 41% of the square cells stained blue after 5 min of acute osmotic swelling whereas only 12% of the square cells stained after resuspension in isotonic media (P < 0.001). Correcting the acute swelling value for the damage caused by mechanical manipulation
Effects of acute swelling
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FIGURE 2. Effect of calcium (closed symbols) or calcium-free (open symbols) incubations on control (boxes) and metabolically inhibited cells during hypotonic incubations. Non-inhibited control rod cell counts slowly declined during incubations with conversion of the rods to round-shaped cells. In the experimental preparations the number of metabolically inhibited rod cells (Exp rods) declined rapidly and cell squaring occurred. The higher initial number of rod cells in the initial calcium-free cell preparations resulted in a slightly higher number of square cells (triangles and circles) in the calcium-free inhibited group (open symbols). There were no differences in the rates of squaring or viable square to blue square conversions.
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the buffered calcium concentration in the T
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FIGURE 3. Effect of acute hypotonic swelling on the viability of metabolically inhibited square cells. The preparations were metabolically inhibited at time 0 with iodoacetic acid and amytal. By 15 min, 87.5% of rod cells in sample (a) and 89% of sample (b) had contracted into viable square-shaped cells that excluded trypan blue dye. At 30 min (arrow) the cells were resuspended into either isotonic (b) or hypotonic (a) media. The percentage of square cells that stained blue was significantly greater in hypotonic media than in isotonic, demonstrating that the cells had become osmotically fragile. Control cells (not shown) resuspended into hypotonic media demonstrated no significan; osmotic fragility. The differences at 35 and 60 min were statistically significant at P < 0.04 and P < 0.004, respectively.
of the cells during isotonic transfer of the cells, an increase in blue cells of 29% can be attributed to osmotic swelling alone. This is similar to the 3 1.2% blue cells obtained after 30 min of continuous hypotonic incubation. To rule out the possibility that hypotonic media caused cell damage through dilution of the ionic constituents rather than through osmotic effects the experiment was repeated, but the isotonic media was diluted with a 340 mM mannitol solution, instead of distilled water, to maintain the osmolarity of the buffer. Resuspension with isotonic media with reduced ionic concentration caused no increase in the relative percentage of blue cells over that of controls transferred to normal isotonic media (data not shown). Calcium-free incubation with EG TA and injury To ensure significant reduction of media calcium levels during calcium-free incubations, the experiments were repeated but with the addition of 1 mM EGTA to the calcium-free media. Assuming an initial calcium concentration in calcium-free media of 50 ,u~ and A-EGTA of 5 x lop7 (Grynkiewicz et al., 1985),
media can be calculated at below 2.5 nM. Control cells were also incubated in the absence of added calcium with EGTA in order to rule out deleterious effects of EGTA in the absence of metabolic inhibition. The results in Figure 4 show that EGTA did not affect the rate of cell death of inhibited cells or the viability of control cells. A.23187 and calcium contracture A23 187 is a calcium ionophore and increases the permeability of cell membranes to calcium. Although an increased influx of calcium into control cells would be expected to cause a calcium-induced contracture and cell rounding, the results (Fig. 5(a), (b)] demonstrate that control cells were not significantly affected by A23187. The lack of apparent effect of A23187 was postulated to be due to mitochondrial accumulation of the excess calcium influx with resultant maintenance of a low cytosolic free calcium level. This hypothesis was tested by incubating A23 187-treated cells in the presence of a mitochondrial inhibitor. The rationale was that anaerobic glycolysis would provide sufficient ATP to support active calcium-induced contracture and prevent rigor, while mitochondrial uptake of calcium would be inhibited. These conditions would allow the increased influx of calcium caused by A23187 to raise the cytosolic free calcium level while ATP levels were still sufficiently high to support active contraction. The result would be a calcium-induced rounding of cells. The results of the experiment were that mitochondrial inhibition alone caused a delayed contracture of rod-shaped mytocytes into a mixture ofround and square forms [Fig. 5(c)]. After 20 min of mitochondrial inhibition without A23187, there was an increase of 14.8% round cells and 26% square cells. In contrast, after 20 min of incubation of cells with mitochondrial inhibition and A23187 the number of round cells increased by 37% (P < 0.001 as compared to mitochondrial inhibition alo.ne) with only a small (6.8%) increase in square cells [Fig. 5(d)]. The markedly increased rate of A23 187-induced rounding of cells with inhibited mitochondria is indirect evidence that A23187 was capable of elevating cellular calcium levels under the experimental conditions used in this study.
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Time
(mln)
FIGURE 4. Effect of addition of 1 mM EGTA to the calcium-free incubation media during isotonic incubations of control non-inhibited cells (circles) and metabolically inhibited cells, in calcium-containing (closed symbols) and calcium-free (open symbols) incubations. The rates of squaring and conversion of viable square cells (triangles) to nonviable (blue) square cells (boxes) were nearly identical in calcium-free or calcium-containing media during metabolic inhibition.
on the rates of cell death was compared to a control sample of metabolically inhibited cells in the presence of calcium alone. The results shown in Figure 6 indicate that there was no difference in the rates at which cells squared before the addition of A23187 or the rates at which cells converted to blue-staining forms after A23187 was added.
Time (mm1
FIGURE 5. Effect of the calcium ionophore A23187 and mitochondrial inhibition on contracture of rodshaped cells into round cells in calcium-containing media. Control preparations with no inhibitors present showed little rounding in the presence (b) or absence (a) of the ionophore. Cells with mitochondrial inhibition, but allowed to continue anaerobic glycolysis in the absence of A23187 (c) developed a slowly developing conversion of rod cells to a mixture of square and round cells. In contrast, similarly inhibited cells in the presence of A23187 (d) rapidly contracted into round forms, confirming a calcium-induced contracture in the presence of the ionophore.
A23187 and cell death The influence of ionophore-induced intracellular loading and depletion of calcium in calcium-containing and calcium-free media
Morphologic
changes
Light microscopy of plastic embedded sections showed that control myocytes in calciumcontaining and calcium-free media were elongated. Membrane protrusions were rarely seen on control cells [Fig. 7 (a)]. Round cells in both control and metabolically inhibited preparations had a single dense contraction band and often fragmented membranes or large membrane blebs. Myocytes from metabolically inhibited preparations were contracted into square shapes. Square cells frequently had polyploid or dome-like blebs of the lateral or the intercalated disc membranes [Fig. 7(b)]. There were no apparent differences between inhibited cells in calcium-containing and calcium-free media (Fig. 7(c)].
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60 v) 5 50 0 s 40 E i! 30 l? 20
Time FIGURE metabolically containing
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6. Effect of the calcium ionophore A23187 on the rates of squaring inhibited (IAA plus amytal) cells in calcium-free ( q ), calcium-free (A) media. There were no differences among the three experimental
Discussion It was important to the design of these experiments that the rates of ATP depletion in metabolically inhibited myocytes in calciumfree and calcium-containing media coincided. The similar rates of ATP depletion with or without extracellular calcium allowed a direct comparison of the effects of calcium on injury independent of its effects on cell energy demand. Energy consumption of quiescent calcium tolerant myocytes in the presence of calcium is low unless the cells are stimulated to beat (Haworth et al., 1983), and the major energy utilization is to maintain the calcium and ionic equilibrium of the cell. In the absence of extracellular calcium, energy is not utilized to extrude excess calcium influxes, but ATP utilization by the sodium pump is increased (Cheung et al., 1982) as the cell extrudes sodium entering via the “sodium conducting calcium channels” (Haworth et al., 1982; Hohl et al., 1983). The end result is that the energy demands of isolated myocytes in the presence or the absence of calcium are similar. This is made manifest in metabolically inhibited myocytes in calcium-containing and calcium-free media having a similar time interval to the onset of severe ATP depletion (Altschuld et al., 1981; Ganote et al., 1984;
and cell death during incubations of plus 1 rnM EGTA (A) and in calciumgroups.
Haworth et al., 1981, 1988; Haworth and Berkoff, 1985; Piper, 1988) and hence rigor contracture (squaring). In analogy to intact hearts and tissue slices (Jennings et al., 1986; Steenbergen et al., 1985, 1987a; Ganote and Vander Heide, 1987), isolated myocytes develop membrane blebbing (Buja et al., 1985) and osmotic fragil.ity (Ganote and Vander Heide, 1988) as manifestations of irreversible injury. Although loss of membrane integrity occurs during isotonic incubations, osmotic swelling of irreversibly injured cells (but not control cells) increases the rate of cell death by exposing the progressively increasing fragility of cells at an earlier time (Vander Heide and Ganote, 1987; Jennings et al., 1986). The point of cell death is considered to be the time of rupture of the plasma membrane (Steenbergen et al., 1985, 1987a), and can be determined either by measurement of enzyme release from cells (Rajs et al., 1980; Murphy et al., 1982) or (as in this study) the onset of trypan blue permeability (Altschuld et al., 1980, 1981; Rajs et al., 1980). In these experiments both the rates of cell squaring of metabolically inhibited cells (as a marker of rigor contracture due to severe ATP depletion) and onset of trypan blue permea-
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FIGURE 7. Light microscopy of toluidine blue-stained, plastic embedded sections of myocytes from experimental protocol 2 after 60 min of inhibition. All cells were incubated in hypotonic media but fixed in isotonic glutaraldehyde. Non-inhibited control cells (a) were elongated and had intact cell membranes. Occasional round cells with either blebbed or ruptured membranes were also present. Inhibited cells (b, c) were contracted into rectangular or square shapes and often had polypoid of dome-like blebs (arrows) on the lateral or intercalated disc surfaces. Cells in calciumcontaining media (b) were morphologically indistinguishable from those in calcium-free media (c). x 480.
bility (as a marker of cell death) were similar in calcium-containing and calcium-free media. In hypotonic incubations the rates of cell squaring remained similar, but the rate of cell death increased. Similarly, myocytes in-
cubated in isotonic inhibiting solution and then acutely swollen with hypotonic buffer demonstrated osmotic fragility. These results demonstrate that increased fragility of myocytes occurs during metabolic inhibition and
Calcium
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that extracellular calcium depletion does not affect the process. Nominally calcium-free media has been estimated to be contaminated by up to 5 x 1o-5 M calcium. The addition of 1 mM EGTA would reduce this to a value less than about2.5 x lo-* M (Grynkiewicz et al., 1985)) an amount below the range of normal cytosolit calcium ( 10 - 5 to 10 - ’ M). Since addition of EGTA to calcium-free media did not alter the rate of cell death it can be concluded that calcium influxes from the extracellular space are not necessary for development of irreversible injury in isolated myocytes under conditions of metabolic inhibition. Calcium movements and their significance in severely denergy depleted cells are not fully understood. It has been reported that ATP depletion inhibits calcium influx in isolated myocytes, thereby questioning whether or not cytosolic free calcium actually rises during this critical phase of injury (Haworth et al., 1987). There is evidence that cytosolic free calcium can be predictably controlled in the presence of a calcium ionophore (Haworth et al., 1988; duBel1 et al., 1988; Li et al., 1987). The ionophore A23187, which selectively permeabilizes cells to calcium, was therefore tested for effects on cell injury. As a control, the drug was added to respiring cells where elevation of cytosolic calcium in the presence of ATP would be expected to produce a calciuminduced contracture of the cells into round forms (Altschuld et al., 1980, 1985; Lambert et al., 1986). However, rounding did not occur when control cells were incubated with A23187. It was reasoned that the absence of rounding was due to mitochondrial accumulation of the excess calcium influx at a rate sufficient to maintain a low cytosolic calcium level. To show that A23187 actually permeabilized cells to calcium it was necessary to inhibit mitochondrial respiration Ernster et al., 1963). Myocytes can produce ATP by either anaerobic or aerobic metabolism. When only one source is inhibited development of rigor contracture is delayed. In such preparations the relative rates of ATP production and consumption in individual cells results in a mixture of square cells and round cells (Haworth et al., 1981; Altschuld et al., 1980), rather than the predominantly square cell population seen with total metabolic inhi-
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bition (Ganote and Vander Heide, 1988). It was reasoned that if A23187 were rendering the plasma membrane permeable to calcium, then during the early period of mitochondrial inhibition (when there was still sufficient ATP being generated by anaerobic glycolysis to support calcium-induced contracture) the rapid rounding of cells would provide indirect evidence of an increased calcium influx. Such an effect would not be expected if myocytes were totally depleted of ATP (squared) prior to addition of A23 187, since no ATP would be available to support a calcium-induced contracture (rounding) even in the presence of elevated cytosolic calcium. In these experiments rapid conversion of rod cells to roun.d cells in the presence of mitochondrial inhibition suggests that the ionophore did increase under our experimental calcium flux conditions. Without continuous direct monitoring of cytosolic free calcium, incubation of myocytes in calcium-free media alone could not rule out the possibility that a rise of cytosolic calcium, due to redistribution of intracellular calcium secondary to metabolic inhibition could contribute to cell injury (Cobbold and Allshire, 1988; Haworth et al., 1981; Steenbergen et al., 1987b; Walsh and Tormey, 1988a, b). However, this seems unlikely since similar preparations of myocytes loaded with the fluorescent calcium probe Furashowed no increase in intracellular free calcium following ATP depletion with amytal and CCCP in a calcium-free medium (fig. 2 of Li et al., 1988). We have conducted Furaexperiments under conditions similar to those used in this study. In the presence of the non-fluorescence iontophore BrA23187 (Q Li and R. Altschul(d, unpubl. res.) cells developed no measurable rise of calcium from control levels in calciumfree media, while 50 min after squaring of cells in 1.12 mM calcium the cytosolic calcium had risen to 10 PM. That in the present experiments neither calcium loading nor depletion of metabolically inhibited cells in the presence of A23187 affected the rate of cell injury suggests that cytosolic free calcium levels may not directly control the intracellular processes leading to irreversible injury. These results do not rule out an influence of calcium on anoxic or ischemic injury. Even in isolated myocytes the processes causing
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irreversible injury could require only micromolar levels of cytosolic calcium in sequestered amounts that could still be present under our experimental conditions. In addition, calcium influxes into calcium depleted, sodium-loaded cells mediated through Na-Ca exchange at the time of reoxygenation could potentiate reoxygenation contracture (Shine and Douglas, 1978; Kohl et al., 1982; Piper et al., 1985; Stern et al., 1985; Lambert et al., 1986). Although results of these experiments will require confirmation using direct methods of calcium measurement in single cells and intracellular calcium chelation, these data indicate that neither increasing nor depleting
calcium during the period of severe energy depletion significantly modifies the rate of development of cell death of metabolically inhibited isolated myocytes.
Acknowledgements This study was supported by a grant AHA 86 674 from the American Heart Association. The excellent technical assistance of Walter Glogowski is appreciated. Most of the work for this study was conducted at the School of Medicine, Northwestern University, Chicago, IL, USA.
References DG, mammalian
CH (1983) Intracellular calcium concentration during hypoxia and metabolic inhibition in ventricular muscle. J Physiol339: 107-122. ALTSCHULD RA, GIBB L, ANSEL A, HOHL C, KRUGER FA, BRIERLEY GP (1980) Calcium tolerance of isolated rat heart cel1s.j Mol Cell Cardiol 12: 1383-1395. ALTSCHULD RA, HOSTELTER JR, BRIERLEY GP (1981) Response ofisolated rat heart cells to hypoxia, reoxygenation and acidosis. Circ Res 49: 307-3 16. ALTSCHULD RA, WENCER WC, LAMKA KG, KINDIG OR, CAPEN CC, MIZUHIRA V, VANDER HEIDE RS, BRIERLEY GP (1985). Structural and functional properties of adult rat heart myocytes lysed with digitonin. J Biol Chem 26: 14325-14334. BARRY WH, PEETERS GA, RASMUSSEN CAF Jr, CUNNINGHAM MJ (1987) Role of changes in [Ca’ ‘Ii in energy deprivation contracture. Circ Res 61: 726-734. BUJA LM, HAGLER HK, PARSONS D, CHIEN K, REYNOLDS RC, WILLERSON JT (1985) Alterations of ultrastructure and elemental composition in cultured neonatal rat myocytes after metabolic inhibition with iodoacetic acid. Lab Invest 53: 397411. CHEUNC JY, THOMPSON IG, BONVENTRE JV (1982) Effects of extracellular calcium removal and anoxia on isolated rat myocytes. Amer J Physio1243 (Cell Physiol 12): Cl84-Cl90. CHIEN KR, SEN A, REYNOLDS R, CHANG A, KIM Y, GUNN MD, BUJA LM, WILLERSON JT (1985) Release of arachidonate from membrane phospholipids in cultured neonatal rat myocardial cells during adenosine &phosphate depletion. J Clin Invest 75: 1770-l 780. Effects of Anoxia and Reoxygenation. In: COBBOLD P, ALLSHIRE A (1988) Cytosolic Free Ca 2 + in Single Cardiocytes: Biology of fiokzfed Adult Cardiac Mjwc~te~, WA Clark, RS Decker, TK Borg (Eds.). New York, Elsevier Science, pp 336338. DAYTON WR, SCHOLLMEYER JV (1980) Isolation from porcine cardiac muscle of a Ca*+-activated protease that partially degrades myofibrils. J Mol Cell Cardiol 12: 533-551. DUBELL WH, PHILIPS C, HOUSER SR (1988) A Technique for Measuring Cytosolic Free Gas + with INDO- 1 in Feline Myocytes. In: Biolqv of Isolated Adult Cardiac Myocytes, WA Clark, RS Decker, TK Borg (Eds.). New York, Elsevier Science, pp 187-201. ERNSTER E, DALLNER G, AZZONE GF (1963) Differential effects of rotenone and amytal on mitochondrial electron and energy transfer. J Biol Chem 238: 1124-l 131. GANOTE CE, GRINWALD PM, NAYLER WG (1984) 2,4-Dinitrophenol (DNP)-induced injury in calcium-free hearts. J Mol Cell Cardiol 16: 547-557. GANOTE CE, NAYLER WG (1985) Contracture and the calcium paradox. J Mol Cell Cardiol 17: 733-745. GANOTE CE, VANDER HEIDE RS (1987) Cytoskeletal lesions in anoxic myocardial injury: a conventional and high voltage electron microscopic and immunofluorescence study. Am J Path 129: 327-344. injury of isolated adult rat myocytes: osmotic fragility during GANOTE GE, VANDER HEIDE RS (1988) I rreversible metabolic inhibition. Am J Path 132: 212-222. GRYNKIEWICZ G, POENIE M, TSIEN RY (1985) A new generation of Ca 2 + indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450. HAWORTH RA, BERKOFF HA (1985) Contracture development in anoxia: the importance of asynchrony. Basic Res Cardiol80 [Suppl2]: 147-150.’ HAWORTH RA, GOKNUR AG, HUNTER DR, HEGGE JO, BERHOFF HA (1987) Inhibition of calcium influx in isolated adult rat heart by ATP depletion. Circ Res 60: 586-594. ALLEN
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(1981) Contracture in isolated adult rat heart cells. Role of CaZ +, ATP 128. (1982) Mechanism of calcium resistance in adult heart cells isolated with 523-530. (1983) Metabolic cost of the stimulated beating of isolated adult rat heart
HAWORTH RA, NICOLAUS A, GOKNUR AB, BERKOFF HA (1988) Synchronous depletion ofATP in isolated adult rat heart cells. J Mol Cell Cardiol20: 837-846. HOHL C, ANSEL A, ALTSCHULD R, BRIERLEY GP (1982) Contracture of isolated rat heart cells on anaerobic to aerobic transition. Am J Physio1242: H1022-H1030. Hour CM, A~rsonu~n RA, BRIERLEY GP (1983) Effects of calcium on the permeability of isolated adult rat cells to sodium and potassium. Arch Biochem Biophys 221: 197-205. JENNINGS RB, REIMER KA, STEENBERGEN C (1986) Myocardial ischemia revisited. The osmolar load, membrane damage and reperfiision. Editorial, J Mol Cell Cardiol 18: 769680. LAMBERT MR, JOHNSON JD, LAMKA KG, BRIERLEY GP, ALTSCHULD RA (1986) Intracellular free calcium and the hypercontracture of adult rat heart myocytes. Arch Biochem Biophys 245: 426-435. LI Q ALTSHULD RA, STOKES BT (1987) Quantitation of Intracellular free calcium in single adult cardiomyocytes by furaratios. Biochem Biophys Res Commun 14’1: 120-126. LI Q ALTSHULD RA, STOKES BT (1988) Myocyte deenergization and intracellular free calcium dynamics. Am J Physiol 255: C162-C168. MAUNSBACH AB (1966) The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives. J Ultrastruct Res 15: 242-282. MORENO~ANCHEZ R, HANSFORD RG (1988) Relation between cytosolic free calcium and respiratory rates in cardiac myocytes. Am J Physiol255: H347-H357. MURPHY E, LEFURCEY A, LIEBERMAN M (1987) Biochemical and structural changes in cultured heart cells induced by metabolic inhibition. Am J Physiol253: C700-C706. MURPHY MP, Iform CM, BRIERLEY GP, ALTSCHULD RA (1982) Release ofenzymes from adult rat heart myocytes. Circ Res 51: 560-568. PIPER HM (1988). Evaluation of Anoxic Injury in Isolated Adult Cardiomyocytes. In: Biology of Isolated Adult Cardiac Myocytes, WA Clark, RS Decker, TK Borg (Eds.), New York, Elsevier Science, pp 68-81. PIPER HM, SPAHR R, HUTTNER JF, SPIECKERMANN PG (1985) The calcium and the oxygen paradox: non-existent on the cellular level. Basic Res Cardiol80: 159-163. RAJS J, SUNDBERG M, HARM T, GRANDIASSON M, SODERLUND U (1980) Interrelationships between ATP levels, enzyme leakage, gluconate uptake, trypan blue exclusion and length/width ratio of isolated rat cardiac myocytes subjected to anoxia and reoxygenation. J Mol Cell Cardiol 12: 1227-1238. SHINE KI, DOUGLAS AM (1978) Low calcium reperfusion of the ischemic myocardium. J Mol Cell Cardiol 15: 251-260. STEENBERGEN C Jr, HILL ML, JENNINGS RB (1985) Volume regulation and plasma membrane injury in aerobic, anaerobic and ischemic myocardium in vitro: effect of osmotic swelling on plasma membrane integrity. Circ Rm 57: 864-875. STEENBERGEN C Jr, HILL ML, JENNINGS RB (1987a) Cytoskeletal damage during myocardial ischemia: changes in vinculin immunofluorescence staining during total in vitro ischemia in canine heart. Circ Res 61): 478-486. STEENBERGEN C, MURPHY E, LEW L, LONDON RE (1987b) Eelevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res 60: 700-707. STERN MD, CHIEN AM, CAPOGROSSI C, PELTO DJ, LAKATTA EG (1985) Direct observation ofthe “oxygen paradox” in single rat ventricular myocytes. Circ Res 56: 899-903. VANDER HEXDE RS, ANGELO JP, ALTSCHULD RA, GANOTE GE (1986) Energy dependence ofcontraction band formation in perfused hearts and isolated adult myocytes. Am J Path 125: 5568. VANDER HEIDE RS, GANOTE GE (1987) Increased myocyte fragility following anoxic injury, J Mol Cell Cardiol 19: 1085-I 103. WALSH LG, TORMEY JMcD (1988a) Cellular compartmentation in ischcmic myocardium: indirect analysis by electron probe. Am J Physio1225: H929-H936. WALSH LG, TORMEY JMcD (1988b) Subcellular electrolyte shifts during in r&o myocardial ischemia and reperfusion, Am J Physiol225: H917-H928. REMAN RJ, KAMEYAMA T, MATSUMOTO K, BERNSTEIN P, ETLINGER JD (1985) Regulation of protein degradation in muscle by calcium. J Biol Chem 260: 13619-13624.
Cell
Cardio122,
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(1990)
Effects of Calcium Depletion and Loading Metabolic Inhibition of Isolated Adult Dianne
S. Rim,
Ruth
A. Altschuld*
on Injury During Rat Myocytes
and Charles
E. Ganote
Department of Pathology, James H. Q&Len College of Medicine, East Tennessee State University, PO Box 1954OA, Johnson City, TN 37614-0002, USA *Physiological Chemistry, Ohio State University, Columbus, OH, USA (Received 15 March 1989, accepted in revised form 19 October 1989) D. S. RIM, R. A. ALTSCHULD AND C. E. GANOTE. Effects of Calcium Depletion and Loading on Injury During Metabolic Inhibition of Isolated Adult Rat Myocytes. Journal of Molecular and Cellular Cardiology (1990) 22, 227-237. The hypothesis that calcium influxes from the extracellular space play an important role in the pathogenesis of irreversible anoxic injury was tested using isolated adult rat myocytes. Myocytes treated with 6 rnM amytal and 3 rnM iodoacetate and subsequently incubated in either calcium-containing (1.12 mn) or calcium-free media (with or without 1 rnM EGTA) developed rigor contracture (cell squaring) and cell death (trypan blue permeability) at the same rate. The rates ofcell death in both calcium-containing and calcium-free media were increased by incubation in hypotonic media even though the rates of contracture development remained unaltered. Cells developed osmotic fragility prior to membrane permeability increases. The calcium ionophore, A23187 (10 p(M), induced rapid rounding of rod-shaped cells subjected only to mitochondrial inhibition in calcium containing media, confirming its ability to cause an increase in cellular permeability to calcium. However, A23187 did not alter the rates of cell death of totally metabolically inhibited myocytes in either calcium-containing or calcium-free media with EGTA. The results indicate that influxes of calcium are not necessary for the development of irreversible injury in metabolically inhibited, isolated myocytes. KEY WORDS:
Calcium;
Isolated
myocyte;
Anoxic
injury;
Introduction Myocardial injury can be divided into three conceptually distinct intervals: an early phase of anoxia, extending to the time of completion of rigor contracture during which cellular ATP (energy) levels decline; a middle phase of severe energy depletion; and a late phase of cell death and necrosis. Calcium is known to play important roles in the early and late phases of irreversible myocardial ischemic/anoxic injury, but the role of elevated cytosolic free calcium during the middle phase of severe ATP depletion is less well established. Cytosolic free calcium can increase during early stages of energy depletion through sodium/calcium exchange mechanisms (Murphy et al., 1987) or release of calcium from the mitochondria or sarcoplasmic reticulum (Barry et al., 1987). It has been hypothesized that micromolar increases in intracellular free calcium during the early phases of energy depletion increase cardiac 0022-2929/90/020227
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fragility;
Membrane
injury.
energy demand (Moreno-Sanchez and Hansford, 1988) and could activate cytosolic proteases (Dayton and Schollmeyer, 1980; Zeman et al., 1985) or lipases (Chien et al., 1985) which may then cause irreversible cellular damage. This hypothesis has been difficult to test because of the complexity of calcium interactions in the myocardial cell and the fact that tissue measurements of total tissue calcium may not provide a reliable assessment of the more relevant intracellular free ionized calcium levels, Calcium is important in modulating the rate of ATP utilization and in maintaining the ingegrity of intercalated discs (Ganote and Nayler, 1985). These actions of calcium, combined with the lack of adequate techniques to measure cytosolic free calcium in whole tissue, have precluded definitive calcium depletion experiments in intact tissues which could separate the effects of calcium on the process of irreversible injury from its other actions. 0
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Isolated myocyte models of injury would seem better suited to study the effects of extracellular calcium depletion on the development of irreversible anoxic injury than intact tissue models because: ( 1) suspensions of cells can develop ATP depletion rigor contracture in calcium-free media without suffering injury to intercalated disc membranes (Rajs et al., 1980; Piper et al., 1985; Ganote and Vander Heide, 1988); (2) rigor contracture is calcium independent (Allen and Orchard, 1983) and the development of contracture can be used as a biological assay for severe ATP depletion (Haworth et al., 1981; Altschuld et al., 1985); (3) the rate of ATP depletion of metabolically inhibited myocytes is similar in calciumcontaining and calcium-free media, allowing a direct comparison of the influence of calcium on the rates of progression of cell injury; (4) calcium influxes from the extracellular space and intracellular free calcium levels can be experimentally measured using fluorescent calcium probes (Li et al., 1987) and manipulated by controlling extracellular calcium in either the presence (duBelI et al., 1988) or absence (Barry et al., 1987; Cobbold and Allshire, 1988) of a calcium ionophore. The present study was designed to investigate the direct effects of extracellular calcium on cell injury in metabolically inhibited myocytes. The results indicate that given equal rates of energy depletion, raising or lowering extracellular and cytosolic free calcium have little effect on the rate of development of cell death.
hold, NJ) was added to the per&sate. After the hearts became soft (40 to 50 min), they were removed, minced, and dispersed in perfusion buffer containing 4% w/v BSA. The suspension was incubated at 30°C for 5 to 10 min in a shaking bath, filtered through a nylon mesh and washed in Ca’+-free “low potassium” (5 mM K ‘) wash buffer containing in addition to the other ions and supplements, HEPES buffer (30 mM). Cells were resuspended in this final wash buffer and calcium added to a final concentration of 1.12 mM. The cells were rinsed and suspended in the 5 mM K buffer which was either calciumfree or contained 1.12 mM Ca’ + and which was used for the experimental incubations, conducted at 30°C. The osmolality of the “isotonic” final buffer was 340 mOsmo1. Approximately 12 x lo6 cells were obtained from each isolate. The reagents used were iodoacetic acid (reagent grade, Sigma), amobarbital (Eli Lilly & Co.) and antibiotic A23187 (free acid, CalbioChem Corp.). Initial cell morphology and viability were assessed by removing 100 ~1 aliquots of cells in final suspension and mixing these with an equal volume of 1 o/0 glutaraldehyde in modified Tyrodes solution (50 mM NaCl replaced by 100 mM glutaraldehyde) (Maunsbach, 1966) containing 3% trypan blue dye and immediately counting the number of rod-, square-, blue square-, round- and blue roundshaped cells present. A total count always exceeded 300 cells and was completed within 15 min in order to preclude non-specific staining of cells.
Methods Cell isolation Myocytes were isolated from hearts of male Sprague-Dawley rats weighing 200 to 300 g as previously described (Vander Heide et al., 1986). Hearts were perfused retrograde with a “high potassium” buffer [in mM, NaCl 125; KC1 30; NaHCOs 25; KH2P04 1.2; MgClz 1.2; dextrose 1.1; taurine 60; creatine 20; Lglutamine 0.682; bovine serum albumin (BSA Pentax Fraction V) 1 mg/ml, and supplemented with a complete amino acid mixture]. The perfusate, pH 7.4, was continuously gassed with 95% 02--5% COZ. After 5 min of perfusion at 37°C collagenase (Type II, 1.25 mg/ml, Worthington Biochemicals, Free-
Experimental design For each experiment, the 10 ml of cell suspension in 1.12 mM calcium was divided into two centrifuge tubes. Cells were centrifuged twice, the suspension divided and resuspended in the appropriate Ca’ +-containing or Ca’ +-free, 5 mM K+ buffer, in accordance with the experimental protocol used. Each suspension was then divided into 2.5-ml aliquots. An additional volume of 2.5 ml of the appropriate buffer was added to each flask, bringing the final volume of each sample to 5 ml. If the cells were to be metabolically inhibited the final volume also contained 3 mM iodoacetic acid and 6 mM amobarbital. Hypotonic solutions
Calcium
and
were prepared by diluting the buffer solutions to half their normal osmolarity, but maintaining the inhibitor concentrations constant. Aliquots of 100 ~1 for cell counts were removed at predetermined intervals during the incubation. Because of variation in the potency of the amytal and the collagenase, each experimental protocol was run as an individual group of experiments using the same lots of collagenase and amytal. Each value represents the mean of at least five separate experiments. Each experiment was performed on a separate day, using a new cell isolation. Variations in isolates during each protocol were acceptably small but the variations of isolates at different time periods precluded comparisons between separate protocols. Experimental protocols The six experimental protocols used were designed for the following purposes: (1) to study the effects of Ca2+ and Ca’+-free isotonic buffers on the rate of cell death of metabolically inhibited cells; (2) to study the effects of the presence or absence of calcium on control and inhibited cells in hypotonic media; (3) to compare the rates of cell death of metabolically inhibited cells in calcium-containing media to that of metabolically inhibited cells in calcium-free media containing the calcium chelator EGTA; (4) to demonstrate that metabolically inhibited myocytes develop osmotic fragility; (5) a control study to determine the effects of A-23187 on the rates of cell contracture to demonstrate that the ionophore has a significant effect under the conditions of these experiments; (6) to compare the effects of the addition of A-23187 on the rates of cell death of metabolically inhibited cells in calciumcontaining media to that of metabolically inhibited cells in calcium-free media containing 1 mM EGTA. The data is expressed as the percentage of cells showing rod-shaped morphology (length/width ratio > 3/l), square-shaped morphology (length/width ratio < 3/l but > 1/ 1)) or being round-shaped. The cells were further sub-classified as being viable (trypan blue-excluding) or non-viable (trypan bluestaining). Results are expressed as the mean & S.D. of the mean. Statistical differ-
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ences were determined using a two unpaired Students t-test. -
tailed,
Results Calcium and cell death in isotonic media The first experimental protocol determined the rates of cell death (as represented by the rates at which cells became permeable to trypan blue) of isolated myocytes in isotonic suspension after combined inhibition of both anaerobic and aerobic metabolism with iodoacetic acid and amytal. The results in Figure l(a) show that at the onset (time 0) of the
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FIGURE 1. (a) Effect of metabolic inhibition (6 rnM Amytal and 3 rnM IAA) on the rate of contracture of isolated myocytes in calcium-containing or calcium-free media. Myocytes rapidly developed ATP depletion rigor contracture, changing from an elongated rod-shaped form into contracted square-shaped forms. There was no significant difference in the rates of contracture development in the presence or absence of calcium. (b) Effect of calcium on the rate of development of trypan blue permeability of metabolically inhibited, square myocytes following rigor contracture in isotonic media. Since the rates of ATP depletion contracture were similar in calcium-containing (closed symbols) and calcium-free (open symbols) media [see (a)], the rates of cell death (monitored by ttypan blue staining) can be directly compared and were found to be similar in the presence or absence of extracellular calcium.
D. S. Rim et al.
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experiment, the initial cell counts were similar. Metabolically inhibited cells underwent rapid rigor contracture into squareshaped forms. From 30 to 120 min of incubation there was a gradual loss of viability of the square cells [Fig. 1 (b)]. There were no significant differences in the rates of contracture or loss of viability of the inhibited cells in calcium-containing as compared to those in calcium-free media.
31.2 + 19.1, 47.0 + 5.8 and 67.2 + 9.1 (each value different from control by P < 0.01). Despite the increased rate of cell death in hypotonic media as compared to isotonic media, there was no difference in the rates of conversion to trypan blue permeability of hypotonically incubated cells in calciumcontaining as compared to those in calciumfree media (Fig. 2).
Efect of calcium and no calcium on cells in hy@otonic media Metabolically inhibited cells incubated in hypotonic media converted to square-shaped cells at the same rate and extent as did cells in isotonic media, but the rate at which the square cells died in hypotonic medium was increased, although non-inhibited cells were not significantly affected by hypotonic incubation. In experiments to document this increase, the percentage of blue square cells at 30, 60 and 90 min of incubation in control isotonic medium were 3.6 + 4.1, 19.2 + 8.3 and 46.2 ) 7.1, respectively. In hypotonic media the corresponding values were
The effects of acute swelling in hypotonic media were determined by incubating metabolically inhibited cells in isotonic media for 30 min and then transferring them to hypotonic or isotonic media. Figure 3 shows that compared to resuspension in isotonic buffer, resuspension into hypotonic media results in a more rapid conversion of viable square cells into non-viable, blue square cells. Approximately 41% of the square cells stained blue after 5 min of acute osmotic swelling whereas only 12% of the square cells stained after resuspension in isotonic media (P < 0.001). Correcting the acute swelling value for the damage caused by mechanical manipulation
Effects of acute swelling
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FIGURE 2. Effect of calcium (closed symbols) or calcium-free (open symbols) incubations on control (boxes) and metabolically inhibited cells during hypotonic incubations. Non-inhibited control rod cell counts slowly declined during incubations with conversion of the rods to round-shaped cells. In the experimental preparations the number of metabolically inhibited rod cells (Exp rods) declined rapidly and cell squaring occurred. The higher initial number of rod cells in the initial calcium-free cell preparations resulted in a slightly higher number of square cells (triangles and circles) in the calcium-free inhibited group (open symbols). There were no differences in the rates of squaring or viable square to blue square conversions.
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the buffered calcium concentration in the T
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FIGURE 3. Effect of acute hypotonic swelling on the viability of metabolically inhibited square cells. The preparations were metabolically inhibited at time 0 with iodoacetic acid and amytal. By 15 min, 87.5% of rod cells in sample (a) and 89% of sample (b) had contracted into viable square-shaped cells that excluded trypan blue dye. At 30 min (arrow) the cells were resuspended into either isotonic (b) or hypotonic (a) media. The percentage of square cells that stained blue was significantly greater in hypotonic media than in isotonic, demonstrating that the cells had become osmotically fragile. Control cells (not shown) resuspended into hypotonic media demonstrated no significan; osmotic fragility. The differences at 35 and 60 min were statistically significant at P < 0.04 and P < 0.004, respectively.
of the cells during isotonic transfer of the cells, an increase in blue cells of 29% can be attributed to osmotic swelling alone. This is similar to the 3 1.2% blue cells obtained after 30 min of continuous hypotonic incubation. To rule out the possibility that hypotonic media caused cell damage through dilution of the ionic constituents rather than through osmotic effects the experiment was repeated, but the isotonic media was diluted with a 340 mM mannitol solution, instead of distilled water, to maintain the osmolarity of the buffer. Resuspension with isotonic media with reduced ionic concentration caused no increase in the relative percentage of blue cells over that of controls transferred to normal isotonic media (data not shown). Calcium-free incubation with EG TA and injury To ensure significant reduction of media calcium levels during calcium-free incubations, the experiments were repeated but with the addition of 1 mM EGTA to the calcium-free media. Assuming an initial calcium concentration in calcium-free media of 50 ,u~ and A-EGTA of 5 x lop7 (Grynkiewicz et al., 1985),
media can be calculated at below 2.5 nM. Control cells were also incubated in the absence of added calcium with EGTA in order to rule out deleterious effects of EGTA in the absence of metabolic inhibition. The results in Figure 4 show that EGTA did not affect the rate of cell death of inhibited cells or the viability of control cells. A.23187 and calcium contracture A23 187 is a calcium ionophore and increases the permeability of cell membranes to calcium. Although an increased influx of calcium into control cells would be expected to cause a calcium-induced contracture and cell rounding, the results (Fig. 5(a), (b)] demonstrate that control cells were not significantly affected by A23187. The lack of apparent effect of A23187 was postulated to be due to mitochondrial accumulation of the excess calcium influx with resultant maintenance of a low cytosolic free calcium level. This hypothesis was tested by incubating A23 187-treated cells in the presence of a mitochondrial inhibitor. The rationale was that anaerobic glycolysis would provide sufficient ATP to support active calcium-induced contracture and prevent rigor, while mitochondrial uptake of calcium would be inhibited. These conditions would allow the increased influx of calcium caused by A23187 to raise the cytosolic free calcium level while ATP levels were still sufficiently high to support active contraction. The result would be a calcium-induced rounding of cells. The results of the experiment were that mitochondrial inhibition alone caused a delayed contracture of rod-shaped mytocytes into a mixture ofround and square forms [Fig. 5(c)]. After 20 min of mitochondrial inhibition without A23187, there was an increase of 14.8% round cells and 26% square cells. In contrast, after 20 min of incubation of cells with mitochondrial inhibition and A23187 the number of round cells increased by 37% (P < 0.001 as compared to mitochondrial inhibition alo.ne) with only a small (6.8%) increase in square cells [Fig. 5(d)]. The markedly increased rate of A23 187-induced rounding of cells with inhibited mitochondria is indirect evidence that A23187 was capable of elevating cellular calcium levels under the experimental conditions used in this study.
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Time
(mln)
FIGURE 4. Effect of addition of 1 mM EGTA to the calcium-free incubation media during isotonic incubations of control non-inhibited cells (circles) and metabolically inhibited cells, in calcium-containing (closed symbols) and calcium-free (open symbols) incubations. The rates of squaring and conversion of viable square cells (triangles) to nonviable (blue) square cells (boxes) were nearly identical in calcium-free or calcium-containing media during metabolic inhibition.
on the rates of cell death was compared to a control sample of metabolically inhibited cells in the presence of calcium alone. The results shown in Figure 6 indicate that there was no difference in the rates at which cells squared before the addition of A23187 or the rates at which cells converted to blue-staining forms after A23187 was added.
Time (mm1
FIGURE 5. Effect of the calcium ionophore A23187 and mitochondrial inhibition on contracture of rodshaped cells into round cells in calcium-containing media. Control preparations with no inhibitors present showed little rounding in the presence (b) or absence (a) of the ionophore. Cells with mitochondrial inhibition, but allowed to continue anaerobic glycolysis in the absence of A23187 (c) developed a slowly developing conversion of rod cells to a mixture of square and round cells. In contrast, similarly inhibited cells in the presence of A23187 (d) rapidly contracted into round forms, confirming a calcium-induced contracture in the presence of the ionophore.
A23187 and cell death The influence of ionophore-induced intracellular loading and depletion of calcium in calcium-containing and calcium-free media
Morphologic
changes
Light microscopy of plastic embedded sections showed that control myocytes in calciumcontaining and calcium-free media were elongated. Membrane protrusions were rarely seen on control cells [Fig. 7 (a)]. Round cells in both control and metabolically inhibited preparations had a single dense contraction band and often fragmented membranes or large membrane blebs. Myocytes from metabolically inhibited preparations were contracted into square shapes. Square cells frequently had polyploid or dome-like blebs of the lateral or the intercalated disc membranes [Fig. 7(b)]. There were no apparent differences between inhibited cells in calcium-containing and calcium-free media (Fig. 7(c)].
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60 v) 5 50 0 s 40 E i! 30 l? 20
Time FIGURE metabolically containing
(min)
6. Effect of the calcium ionophore A23187 on the rates of squaring inhibited (IAA plus amytal) cells in calcium-free ( q ), calcium-free (A) media. There were no differences among the three experimental
Discussion It was important to the design of these experiments that the rates of ATP depletion in metabolically inhibited myocytes in calciumfree and calcium-containing media coincided. The similar rates of ATP depletion with or without extracellular calcium allowed a direct comparison of the effects of calcium on injury independent of its effects on cell energy demand. Energy consumption of quiescent calcium tolerant myocytes in the presence of calcium is low unless the cells are stimulated to beat (Haworth et al., 1983), and the major energy utilization is to maintain the calcium and ionic equilibrium of the cell. In the absence of extracellular calcium, energy is not utilized to extrude excess calcium influxes, but ATP utilization by the sodium pump is increased (Cheung et al., 1982) as the cell extrudes sodium entering via the “sodium conducting calcium channels” (Haworth et al., 1982; Hohl et al., 1983). The end result is that the energy demands of isolated myocytes in the presence or the absence of calcium are similar. This is made manifest in metabolically inhibited myocytes in calcium-containing and calcium-free media having a similar time interval to the onset of severe ATP depletion (Altschuld et al., 1981; Ganote et al., 1984;
and cell death during incubations of plus 1 rnM EGTA (A) and in calciumgroups.
Haworth et al., 1981, 1988; Haworth and Berkoff, 1985; Piper, 1988) and hence rigor contracture (squaring). In analogy to intact hearts and tissue slices (Jennings et al., 1986; Steenbergen et al., 1985, 1987a; Ganote and Vander Heide, 1987), isolated myocytes develop membrane blebbing (Buja et al., 1985) and osmotic fragil.ity (Ganote and Vander Heide, 1988) as manifestations of irreversible injury. Although loss of membrane integrity occurs during isotonic incubations, osmotic swelling of irreversibly injured cells (but not control cells) increases the rate of cell death by exposing the progressively increasing fragility of cells at an earlier time (Vander Heide and Ganote, 1987; Jennings et al., 1986). The point of cell death is considered to be the time of rupture of the plasma membrane (Steenbergen et al., 1985, 1987a), and can be determined either by measurement of enzyme release from cells (Rajs et al., 1980; Murphy et al., 1982) or (as in this study) the onset of trypan blue permeability (Altschuld et al., 1980, 1981; Rajs et al., 1980). In these experiments both the rates of cell squaring of metabolically inhibited cells (as a marker of rigor contracture due to severe ATP depletion) and onset of trypan blue permea-
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FIGURE 7. Light microscopy of toluidine blue-stained, plastic embedded sections of myocytes from experimental protocol 2 after 60 min of inhibition. All cells were incubated in hypotonic media but fixed in isotonic glutaraldehyde. Non-inhibited control cells (a) were elongated and had intact cell membranes. Occasional round cells with either blebbed or ruptured membranes were also present. Inhibited cells (b, c) were contracted into rectangular or square shapes and often had polypoid of dome-like blebs (arrows) on the lateral or intercalated disc surfaces. Cells in calciumcontaining media (b) were morphologically indistinguishable from those in calcium-free media (c). x 480.
bility (as a marker of cell death) were similar in calcium-containing and calcium-free media. In hypotonic incubations the rates of cell squaring remained similar, but the rate of cell death increased. Similarly, myocytes in-
cubated in isotonic inhibiting solution and then acutely swollen with hypotonic buffer demonstrated osmotic fragility. These results demonstrate that increased fragility of myocytes occurs during metabolic inhibition and
Calcium
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that extracellular calcium depletion does not affect the process. Nominally calcium-free media has been estimated to be contaminated by up to 5 x 1o-5 M calcium. The addition of 1 mM EGTA would reduce this to a value less than about2.5 x lo-* M (Grynkiewicz et al., 1985)) an amount below the range of normal cytosolit calcium ( 10 - 5 to 10 - ’ M). Since addition of EGTA to calcium-free media did not alter the rate of cell death it can be concluded that calcium influxes from the extracellular space are not necessary for development of irreversible injury in isolated myocytes under conditions of metabolic inhibition. Calcium movements and their significance in severely denergy depleted cells are not fully understood. It has been reported that ATP depletion inhibits calcium influx in isolated myocytes, thereby questioning whether or not cytosolic free calcium actually rises during this critical phase of injury (Haworth et al., 1987). There is evidence that cytosolic free calcium can be predictably controlled in the presence of a calcium ionophore (Haworth et al., 1988; duBel1 et al., 1988; Li et al., 1987). The ionophore A23187, which selectively permeabilizes cells to calcium, was therefore tested for effects on cell injury. As a control, the drug was added to respiring cells where elevation of cytosolic calcium in the presence of ATP would be expected to produce a calciuminduced contracture of the cells into round forms (Altschuld et al., 1980, 1985; Lambert et al., 1986). However, rounding did not occur when control cells were incubated with A23187. It was reasoned that the absence of rounding was due to mitochondrial accumulation of the excess calcium influx at a rate sufficient to maintain a low cytosolic calcium level. To show that A23187 actually permeabilized cells to calcium it was necessary to inhibit mitochondrial respiration Ernster et al., 1963). Myocytes can produce ATP by either anaerobic or aerobic metabolism. When only one source is inhibited development of rigor contracture is delayed. In such preparations the relative rates of ATP production and consumption in individual cells results in a mixture of square cells and round cells (Haworth et al., 1981; Altschuld et al., 1980), rather than the predominantly square cell population seen with total metabolic inhi-
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bition (Ganote and Vander Heide, 1988). It was reasoned that if A23187 were rendering the plasma membrane permeable to calcium, then during the early period of mitochondrial inhibition (when there was still sufficient ATP being generated by anaerobic glycolysis to support calcium-induced contracture) the rapid rounding of cells would provide indirect evidence of an increased calcium influx. Such an effect would not be expected if myocytes were totally depleted of ATP (squared) prior to addition of A23 187, since no ATP would be available to support a calcium-induced contracture (rounding) even in the presence of elevated cytosolic calcium. In these experiments rapid conversion of rod cells to roun.d cells in the presence of mitochondrial inhibition suggests that the ionophore did increase under our experimental calcium flux conditions. Without continuous direct monitoring of cytosolic free calcium, incubation of myocytes in calcium-free media alone could not rule out the possibility that a rise of cytosolic calcium, due to redistribution of intracellular calcium secondary to metabolic inhibition could contribute to cell injury (Cobbold and Allshire, 1988; Haworth et al., 1981; Steenbergen et al., 1987b; Walsh and Tormey, 1988a, b). However, this seems unlikely since similar preparations of myocytes loaded with the fluorescent calcium probe Furashowed no increase in intracellular free calcium following ATP depletion with amytal and CCCP in a calcium-free medium (fig. 2 of Li et al., 1988). We have conducted Furaexperiments under conditions similar to those used in this study. In the presence of the non-fluorescence iontophore BrA23187 (Q Li and R. Altschul(d, unpubl. res.) cells developed no measurable rise of calcium from control levels in calciumfree media, while 50 min after squaring of cells in 1.12 mM calcium the cytosolic calcium had risen to 10 PM. That in the present experiments neither calcium loading nor depletion of metabolically inhibited cells in the presence of A23187 affected the rate of cell injury suggests that cytosolic free calcium levels may not directly control the intracellular processes leading to irreversible injury. These results do not rule out an influence of calcium on anoxic or ischemic injury. Even in isolated myocytes the processes causing
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irreversible injury could require only micromolar levels of cytosolic calcium in sequestered amounts that could still be present under our experimental conditions. In addition, calcium influxes into calcium depleted, sodium-loaded cells mediated through Na-Ca exchange at the time of reoxygenation could potentiate reoxygenation contracture (Shine and Douglas, 1978; Kohl et al., 1982; Piper et al., 1985; Stern et al., 1985; Lambert et al., 1986). Although results of these experiments will require confirmation using direct methods of calcium measurement in single cells and intracellular calcium chelation, these data indicate that neither increasing nor depleting
calcium during the period of severe energy depletion significantly modifies the rate of development of cell death of metabolically inhibited isolated myocytes.
Acknowledgements This study was supported by a grant AHA 86 674 from the American Heart Association. The excellent technical assistance of Walter Glogowski is appreciated. Most of the work for this study was conducted at the School of Medicine, Northwestern University, Chicago, IL, USA.
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