J Mol

Cell

Cardio122,

1655181

An in vitro

Richard

(1990)

Model Isolated

S. Vander

Department

of Myocardial Adult Rat

Ischemia Myocytes

Heidet, Dianne Rim, and Charles E. Ganotel

Charlene

Utilizing

M. Hohl*

of Patkology,

*Department

Northwestern University Medical Sckool, Chicago, Illinois, USA of Physiological Chemistry, Ohio State University, Columbus, Ohio, USA

(Received 12 May 1989, accepted in revisedform

10 October 1989)

R. S. VANDER HEIDE, D. RIM, C. M. HOHL AND C. E. GANOTE. An In Vitro Model of Myocardial Ischemia Utilizing Isolated Adult Rat Myocytes. Journal of Molecular and Cellular Cardiolo~ (1990) 22, 165-181. Isolated adult rat myocytes were used to develop an in vitro model of myocardial ischemia. Freshly isolated myocytes were spun into a cell pellet to limit extracellular volume. Excess supernatant was removed and the pellet was covered with mineral oil and incubated in a temperature controlled water bath. After various periods ofincubation, cells were analyzed for adenine nucleotide levels, lactate accumulation, rate of cell death, and cell morphology. Adenine nucleotide profiles after 60 min incubation at 37°C showed marked depletion ofadenosine triphosphate (ATP) and large increases in adenosine monophosphate (AMP), a d enosine, inosine, and lactate and no significant difference in levels of inosine monophosphate. These results are consistent with ischemic conditions. Reduction of the incubation temperature to 34 and 30°C slowed the rate of cell squaring and the onset of cell death. Resuspension of ischemic cells after 30,45,60 and 90 min incubation in hypotonic buffer (170 mosmol) to induce acute cell swelling caused an increase in the number of non-viable cells at each time point. Control cells and ischemic cells incubated less than 30 min did not show increases in non-viable cells when subjected to hypotonic swelling. Morphological analysis revealed that isolated myocytes respond to ischemia in a heterogeneous fashion and exhibit changes at both light and electron microscopic levels similar to those seen in other ischemic models. These results indicate that pelleted isolated adult rat myocytes may be a useful in vitro model to study myocardial ischemic cells injury. KEY WORDS:

Ischemia;

Ischemic

injury;

Isolated

myocyte;

Introduction Ischemia can be defined as the profound reduction of energy production in the presence of a restricted extracellular space. As a consequence of the limited extracelhdar space ceils are unable to rid themselves of various catabolic products of cell metabolism, some of which may be detrimental to continued cell survival (Neely and Grotyohann, 1984). In contrast, anoxia (substrate free) can be defined as the cessation of of energy production in the presence of an unrestricted extracellular space. With the ability to freely exchange metabolites with the extracellular fluid, anoxic cells, although energy depleted, could rid

Osmotic

fragility;

Membrane

injury.

themselves of potentially harmful waste products. Therefore to study the harmful effects of ischemia, above and beyond the effects of severe energy deprivation, it is necessary to limit the extracellular space in which the ischemic cells reside. Previous studies of myocardial ischemia have, until recently, focused primarily on events which occur after the onset of irreversible injury. Indeed with the advent of clinical methods to acutely reperfuse ischemic myocardium (e.g. streptokinase) many recent studies have reexamined the issue of reperfusion injury and ways to preserve and even to salvage ischemic tissue at risk (Engler et al.,

tPlease address all correspondence to: Richard S. Vander Heide, Department of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710, USA. $Present address: Department of Pathology, East Tennessee State Univeristy, Johnson City, TN 37614, USA. 0022-2828/90/020

165 + 17 $03.00/O

0 1990 Academic

Press Limited

166

R. S. Vander

1986; Simpson et al., 1988b). However the precise series of events which precede the point at which an individual cell can be considered to be irreversibly injured is not known. A cell can be considered to be irreversibly injured when its plasma membrane is disrupted Uennings and Reimer, 1981). At this point the cell is no longer capable of maintaining normal ionic gradients necessary for its survival. It is likely that disruption of the plasma membrane is a final step in the transition from reversible to irreversible injury since disruption has been shown to be closely related to lethal injury. However, it is possible that a series of events occurs prior to loss of membrane integrity that could render a myocardial cell incapable of maintaining homeostasis in its normal environment. Such a cell would also be defined as irreversibly injured. Studies from Jennings and his colleagues using incubated slices of ischemic dog myocardium and from our laboratory using perfused anoxic rat hearts and anoxic isolated adult rat myocytes have suggested that irreversible injury may result from two interrelated steps (Jennings et al., 1986; Vander Heide and Ganote, 1987). In the first step a yet undetermined lesion occurs in the cytoskeletal support apparatus of the cell that renders the cell fragile and thereby susceptible to physical stress. In the second step abrupt cell swelling (or other physical stresses to the cell) causes the plasma membrane to rupture thereby rendering the cell irreversibly injured. One major advantage of this hypothesis is that it provides a unifying explanation for not only ischemic myocardial injury but also many in vitro models of injury that have been used to study various aspects of ischemic injury (Hearse and Humphrey, 1973; Vander Heide et al., 1986a). To study specifically the cytoskeleton and its interaction with the plasma membrane is difficult in the in vivo dog &hernia model because of the complicating influences of connective tissue, vascular elements and other contaminating proteins present in intact tissue. In addition it is difficult to acutely stress intact myocardium to assess cell fragility without introducing the potentially complicating influences of no reflow and oxygen-derived free radicals. In vitro perfused anoxic rat hearts circumvent some of these problems but are

Heide

et al.

obviously not a model of ischemia since the cells by definition have access to an unlimited extracellular space. Cultured adult isolated myocytes offer the advantage of looking directly at myocyte changes without the influence of other contaminating tissues. In addition it is easy to acutely stress isolated myocytes by varying the incubation conditions. However limiting extracellular space would be difficult under culture conditions. Furthermore, significant changes occur in isolated myocyte structure and protein composition after they are placed in long-term culture (Haddad et al., 1988; Simpson et al., 1988a). Therefore if isolated myocytes were to be used as a model of ischemia they would have to be used within hours of isolation to insure that the cells reflect the normal state of cellular protein composition during the experiment. Isolated adult rat,myocytes would seem to offer significant advantages in studying myocardial ischemia but there have been to date no descriptions of an ischemic adult myocyte model. For isolated myocytes to be a useful model it must first be established that the cells in vitro respond to ischemia similarly to ischemic cells in vivo. This report describes the major features of a new model of myocardial ischemia utilizing freshly isolated adult rat myocytes. Methods

and Materials

Isolated myocyte preparation Male Sprague-Dawley rats weighing 250 to 300 g were anesthesized by intraperitoneal injection of 55 mg/kg sodium pentobarbital (Diamond Laboratories, Des Moines, IA). Following intravenous administration of 2000 IU sodium heparin (Elkin-Sinns, Cherry Hill, NJ), hearts were removed and immersed in ice-cold calcium-free perfusion buffer which contained NaCl (90 mM), KC1 (30 mM), NaHCOs (25 mM), KHsP04 (1.2 mM), bovine serum albumin (BSA) (1 mg/ml) (Pentex Fraction V), MgClz (1.2 mM), glucose (11 mM), taurine (60 mM), creatine (20 mM), and a complete amino acid solution. Final osmolarity of the buffer was 340 m osmol as measured by freezing-point depression. The heart was flushed with 5 ml of ice-cold buffer and then cannulated on a pump-driven Langendorff apparatus and perfused in a non-recirculating

Ischemia

and Isolated

mode with calcium-free buffer for 5 min at 37°C. After 5 min, collagenase (Class II, Worthington Biochemical, Freehold, NJ) was added to a final concentration of 1.25 mg/ml and the hearts were perfused in a recirculating mode until they became soft (45 to 60 min). The hearts were then removed from the apparatus and minced in 5 ml of perfusion buffer containing 4% w/v BSA. After gentle dispersion and filtering through a nylon mesh, calcium was added to the cells in a step-wise fashion to a final concentration of 1.12 mM. The cells were then collected and subjected to a series of centrifugation steps designed to increase the proportion of viable rod-shaped cells and discard non-viable cells as previously described (Vander Heide et al., 198613). After the final wash the remaining cells were suspended in 10 ml of the final wash buffer. Approximately 12 million cells were obtained from each isolation. All experiments were conducted on the day of cell isolation. Cell pellet formation To insure uniform conditions for metabolic studies, a standard procedure was used to form packed cell pellets. Briefly, cells were washed free of BSA and diluted to approximately 2 mg/ml. Precisely 1 ml of cells was placed into microcentrifuge tube so that each tube would contain 2 mg protein. The tubes were then spun lightly to allow the cells to sediment. Precisely 900 ~1 of the supernatant was removed to obtain a final concentration of 20 mg/ml in each microcentrifuge tube. Tubes were flushed with nitrogen gas, capped and incubated for the appropriate time at 37°C. Following incubation, cells were sampled for metabolic analysis as described below. In morphologic experiments cells were pelleted as above but varying amounts of supernatant were removed relative to the cell size and prior to incubation a 1 ml layer of mineral oil was placed over the cells to exclude room air from the pellet.

Measurement Intracellular adenosine,

of cellular adenine nucleotides levels of ATP, ADP, AMP, IMP, and inosine were measured at Ohio

Myocytes

167

State University using HPLC techniques previously described (Altschuld et al., 1987). All results are expressed as nmol/mg cell protein. Lactate was measured using the Bergmeyer method (Bergmeyer, 1963). Results are expressed as nmol/mg protein.

Morphologic studies Cells were counted by light microscopy and were determined to be rod, round, or square using criteria previously described (Ganote and Vander Heide, 1988). Individual cells were further classified as being viable or nonviable based upon their ability to exclude the vital dye trypan blue. For electron microscopic analysis cells were fixed by transferring 0.25 ml of the cell suspension into isotonic (isotonic fixation) or a 1: 1 dilution (hypotonic fixation) of a fixative solution containing 2% glutaraldehyde, 0.05 M lysine, and 0.005 M EGTA in 0.05 M cacodylate buffer pH 7.4. Cells were fixed at room temperature for 1 to 2 min and then spun at approximately 4000 g for 30 s to pellet the cells. This step was repeated once with a change of fresh fixative. After the second spin, the cells were transferred into the same fixative solution without lysine. The cells were allowed to stand at room temperature in this fixative for the remainder of a 15-min total fixation period. After 15 min the cells were spun at 4000 g, the fixative was removed and the cells were washed twice with 0.05 M cacodylate buffer. Folllowing washing, the cells were post-fixed in pH 7.4 2% osmium tetraoxide prepared in 0.05 M cacodylate buffer for 15 min at 4°C. After post-fixation the cells were washed three times with cold distilled water and stained en bloc with 2% uranyl acetate overnight at 4°C. Cells were then dehydrated and processed by routine techniques. Blocks were trimmed and cut at 1 pm. for light microscopy. Sections showing properly oriented cells were identified and sectioned for routine electron microscopy. Prior to viewing the sections were mounted on copper grids and counterstained with saturated uranyl acetate (20 min) and lead citrate (5 min). Sections were examined and photographed at 60 keV with a JEOL 100 CX electron microscope.

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R. S. Vander

Statistics Differences between sample means were tested for using a two-tailed paired Student’s t-test. A P value of less than 0.05 was considered to be statistically significant. A separate cell isolate was used for each experiment. Values in the figures are group means plus or minus standard error of the mean.

Experimental design The experiments in this study were designed to test the hypothesis that the isolated adult rat heart my+ocyte can be used as a model to study certain aspects of myocardial ischemia. As a result, we chose to evaluate three responses to myocardial ischemia that have been well documented in in viva models and other in vitro models. The first set of experiments determined the effect of ischemia (cell pellet formation) on cellular adenine nucleotide levels and lactate production after 15, 30, and 60 min incubation at 37°C. Cells were harvested and spun into pellets as described above. At time zero and after 15, 30, and 60 min of ischemic incubation cells were sampled and analyzed utilizing HPLC. The second experiment determined the effect of ischemic incubation temperature on cell morphology and viability. After cell isolation the total yield was split into two equal groups. One group of freshly isolated cells was spun into an ischemic pellet, the supernatant removed, and placed under a layer of mineral oil prior to incubation at 30, 34, or 37°C in a still water bath. The second group (control cells) was placed in an open 50 ml Nalgene beaker and incubated in a shaking water bath at the same temperature. Cells were sampled at zero time, at 15 min, and then every 30 min for a total of 120 to 180 min. For analysis of morphology and viability cells were removed from the appropriate incubation container, diluted into isotonic incubation media containing 6 mM amytal to prevent reoxygenation induced cell rounding, and then mixed on a glass microscopic slide with an equal volume of 1% glutaraldehyde in modified Tyrodes solution to which 0.5% trypan blue dye had been added (Ganote and Vander Heide, 1988). At least 300 cells were

Heide

et al.

examined on each slide from multiple areas of the slide within 15 min of sampling. A total of 15 cell isolations were used to achieve an n value of 5 at each of the three temperatures. The final group of experiments examined the effect of an acute hypotonic stress on cells subjected to a period of ischemic incubation. Freshly isolated cells in isotonic buffer were split into two groups. One group was spun into an ischemic pellet and incubated at 37°C for a total of 120 min. At 0, 15, 30,60, 90, and 120 min cells were removed from the pellet and resuspended in hypotonic (final wash buffer diluted 1: 1 with distilled water) buffer containing amytal and examined for cell morphology and viability. The other group served as a control. This group was spun into a pellet, incubated at 37°C and sampled at the same time intervals as the experimental group. However, after sampling the control cells were suspended in isotonic buffer prior to analysis for cell morphology and viability. Electron microscopic studies A separate group of experiments was conducted to investigate specifically the morphological effects of ischemia and the imposition of a physical stress on previously ischemic myocytes. In these experiments cells were spun into a pellet and incubated for 60 min at 37°C. At 0, 30 and 60 min the cells were sampled and resuspended in isotonic (340 mosmol) or hypotonic (170 mosmol) buffer for 5 min before cell counts were performed. The sampling media contained 6 mM amytal to reduce cell rounding due to reoxygenation during dilution. A separate group of non-schemic cells was exposed to hypotonic swelling as a control experiment. At each time interval cells from isotonic and hypotonic incubations were fixed and processed for electron microscopic analysis. Results

Nucleotide data At the initiation of the metabolic studies, cell isolates contained an average of 76.5 + 4.5% rod cells, no square cells and an overall viability of83.0 + 2.5%. As a consequence of the experimental protocol, the initial sample (t = 0) was not analyzed until other samples

Ischemia

TABLE

1. Levels of various metabolites

Incubation time (min) 0

ATP 12.1

ADP 3.21

and

Isolated

(nmol/mg)

AMP 1.76

169

Myocytes

protein; mean +

IMP 0.293

at various incubation

s.E.M.)

ADO

IN0

2.16

1.82

kO.41

f0.65

+0.09

+0.91

+0.29

+1.8

3.48 f 0.09

0.56 f0.06

0.20 +0.03

2.90 kO.48

3.38 +0.45

30

7.28 +0.95

3.94 kO.45

2.67 +0.27

0.20 +0.04

4.58 +0.36

4.77 +0.36

60

2.4" +0.27

kO.11

5.79" 50.32

0.37 +0.03

f0.20

k2.1

15

13.5

2.31

5.6"

6.43"

+0.11

times

ALAC

283.7 f 7.92 512.7 t-39.9 810.3

118.27

Myccytes suspended at a concentration of 2 mg/ml in HEPES-buffered KHB, pH 7.4 containing 11 rn~ glucose were spun into a pellet to achieve a final concentration of 20 mg/ml, flushed with nitrogen gas, and incubated at 37°C for times indicated. At indicated times cells were sampled and sedimented through bromododecane into perchloric acid, extracted, neutralized, and analyzed by HPLC as previously described. aP < 0.05 vs. control. Abbreviations: ATP, adenosine triphosphate; ADP, adenosine dephosphate; AMP, adenosine monophosphate; IMP, inosine monophosphate; ADO, adenosine; INO, inosine; LAC, lactate. R = 3 for e&h tjme period.

had been gassed with nitrogen and the incubation started (approximately 5 min). Consequently initial ATP values are lower than would be expected from our usual preparations (average ATP immediately after isolation = 28.4 nmol/mg protein; Altschuld et al., 1987). During the ischemic incubation levels of ATP declined progressively from an initial level of 12.1 & 2.0 to 2.4 nmol/mg protein after 60 min. During the incubation period there was a concomitant increase in the of AMP from 1.76 + 0.65 to amount 5.79 + 0.32 nmoles/mg protein (P < 0.02). Levels of adenosine and inosine also significantly increased during the 60-min ischemic incubation while the level of inosine monophosphate (IMP) remained constant (Table 1). As expected the level of lactate greatly increased from an initial value of approximately 50 to 810 + 18.3 nmol/mg protein after 60 min of ischemia. Cell count data At the initiation of the experiments the isolates contained an average of 74.2 + 1.7% rod cells with 89.7 f 2.0% of the cells viable by trypan blue exclusion. Preliminary studies investigated the role of cell pellet size on cell morphology. Varying the thickness of the cell pellet

over a six-fold range from 0.3 to 2 cm did not influence the rate or extent of cell squaring and/or cell death. Therefore in subsequent morphologic studies a 2-cm pellet was used to assure enough cells for repeated morphological analysis. In control, oxygenated cells in suspension at 37°C the proportion of rodshaped cells gradually decreased to 62.8 f 0.58% after 120 min of incubation. After 120 min there were virtually no square cells present (1.4 + 0.58%) and no trypan blue staining (non-viable) square cells. Nonviable cells were all round cells and represented 22% of the total incubate after 120 min. In contrast, the pelleted ischemic cells exhibited changes soon after incubation (Fig. 1). After 30 min at 37°C there were 67% rod cells, 13% round cells and nearly 8% square cells. All non-viable cells were blue round cells. By 60 min there was a dramatic increase in the proportion of square cells to 61 y. of the total isolate which was mirrored by a decline in the number of rod cells to 3% of the isolate. Further incubation of the ischemic cells resulted in slowly increasing numbers of non-viable round and square cells with a corresponding decrease in the number of viable round and square cells. After 120 min the final cell isolate contained 0% rod cells, 19.2 + 2.5% round cells, 24.6 + 3.7% blue rounds, 43.4 ) 4.8%

170

R. S. Vander

viable square cells, and 12.8 f 2.8% blue squares. The effect of hypothermia on ischemic myocytes was determined by incubating pelleted myocytes at 34°C and 30°C (Fig. 1). At 34°C control cell suspensions contained (at time zero) 74.8 + 1.8% rods and were 91.4 &- 0.8% viable. After 120 min of incubation the isolate contained 63.2 &- 3.7% rod, 15.4 + 2.1% round, 20.6 f 2.3% blue rounds, and 0.7 + 0.36% viable square cells; values not significantly different from control cells incubated at 37°C. After 60, 90, and 120 min of ischemic incubation there were no significant differences in the proportion of various cell types between 34 and 37°C. Although not statistically significant, the time to peak squaring was delayed from 60 min at 37°C to 90 min at 34°C. After 90 min 37” incubates contained significantly more blue squares than 34°C cells (P < 0.05) indicating that cell death was occurring earlier at higher incubation temperatures. At 30°C cells incubated under ischemic conditions showed significant differences from ischemic cells incubated at 37°C. Although the largest proportion of viable square cells (62.8 + 2.2%) was

Heide

et al.

not significantly different from the 37°C incubate (61.2 f 3.5%), the time to peak squaring (60 min at 37°C; 90 min at 34°C) was delayed further to 120 min. After 60 min, in contrast to 37°C incubates, most of the cells were still viable rods (77.6 + 3.8) while no square cells were observed. After 120 min of ischemia 30°C incubated contained significantly fewer numbers of non-viable cells than 37°C incubates (P < 0.005) indicating that hypothermia delayed the onset of cell death.

Cellfragility In intact tissue both anoxia and ischemia cause an increase in myocyte fragility that can be demonstrated by inducing a physical stress. To determine if isolated myocytes also exhibit an increased fragility following an ischemic insult, myocytes were stressed by acutely swelling them after various periods of ischemic incubation. Cells swollen immediately after isolation showed no significant increase in non-viable cells. However after 60 min of ischemia the imposition of a physical stress (ceil swelling) caused a significant increase in the number of blue squares (Fig. 2). The difference in the number of non-viable squares between acutely swollen aand control

50 % 405 :: 30s 0 E zo? a” 0

30

60

90 Time (min)

FIGURE 1. Effect of temperature on rate of cell squaring and rate of cell death in pelleted isolated adult myocytes. Although the maximum proportion of square cells at each temperature was constant, lowering the ischemic incubation temperature from 37 to 30°C slowed both the rate of squaring and the rate of cell death (as indicated by proportion of blue squares). Non-pelleted control cells (oxygenated) incubated for 120 min at 37°C are shown at the top of the graph. Ischemic cells were suspended in anoxic (6 rnrd amytal) isotonic buffer for 3 min prior to cell counting. Blue squares are permeable to the vital dye trypan blue indicating rupture of the cell membrane. n = 5 for each temperature.

IO-

0

30

60 Time

90 (min)

120

-! I#50

FIGURE 2. Effect of acute hypotonic cell swelling on pelleted ischemic isolated myocytes incubated at 37°C. At each indicated time point, ischemic cells were resuspended in either isotonic (Iso; 340 mosmol) or hypotonic (Hypo; 170 mosmol) buffer for 3 min prior to cell counting. After 30 min ischemic incubation, cells stressed by acute hypotonic swelling contained significantly more blue square cells indicating that ischemic isolated myocytes develop osmotic fragility as the duration of ischemic incubation increases. n = 5 for each time point.

Ischemia

and Isolated

ischemic myocytes increased with increasing duration of ischemic incubation (Fig. 2). After 120 min of ischemia at 37°C the incubates swollen with hypotonic buffer contained a total of approximately 55% square cells, 81 y0 of which (44.3 + 3.0%) were non-viable. In contrast, control ischemic cells contained the same proportion of total square cells (56.2%) but only 21 y0 (12.8 + 2.8%) of the squares were non-viable (P < 0.001). In the experiments designed to specifically investigate the morphological changes associated with ischemia and cell swelling, at each time period (30 and 60 min) the swollen ischemic cells exhibited a significantly higher number of blue squares than the corresponding isotonic cells (P < 0.001; data not shown).

FIGURE 3. Light viability and appear

Myocytes

171

Morphologic studies Figure 3 shows the appearance of freshly fixed trypan blue stained myocytes under various experimental conditions. The majority of cells in control isolates were rod-shaped cells and excluded trypan blue (Fig. 3). Ischemic incubates contained a mixture of cell types (Fig. 4). After 60 min ischemia at 37°C most of the cells were viable square cells but a small proportion of square cells stained with trypan blue indicating that these cells were no longer viable. Ischemic incubates acutely stressed by hypotonic swelling contained increased numbers of non-viable square cells. Nearly all of these cells contained a variable number of large, usually domed shaped blebs (Fig. 5).

micrograph of freshly fixed control rod-shaped myocytes. Rod cells exclude trypan blue indicating relaxed with well defined striations. Two non-viable round cells are also shown. x 875.

172

R. S. Wander Heide

et al.

FIGURE 4. Low power light micrograph of freshly fixed isolated myocytes after 60 min of ischemic There is a heterogeneous population of cells. The majority of cells present are viable square cells (non-stained) some non-viable squares and rounds are seen. Note the absence of rod-shaped cells. x 350.

FIGURE 5. Higher power light micrograph ischemic conditions and resuspended in hypotonic, is non-viable. Note the prominent blebs located surface of the cell. x 1400.

of square cell. anoxic buffer. over intercalated

Cells

were

incubated

for

30 minutes

incubation. although

at 37°C

under

This cell is permeable to trypan blue indicating that it disk regions and occasional blebs seen on the lateral

Ischemia

and

Isolated

The blebs almost invariably occurred on the ends of cells overlying the intercalated disk. Occasional cells contained what appeared to be lateral membrane blebs but it could not be excluded that these were also intercalated disk-associated membrane blebs. Blebs were also commonly seen on non-viable blue cells but were rarely identified on viable rod cells. The morphologic changes observed in ischemic cells at the light microscopic level were confirmed and extended by electron microscopic analysis. Swollen control rod cells exhibited intact sarcolemmal membranes and appeared relaxed with prominent I-bands and well-aligned sarcomeres (Figs. 6 and 7). There appeared to be an increased space between intracellular organelles and prominent scalloping of the sarcolemmal membrane indicative of cell swelling but mitochrondria appeared condensed and did not exhibit matrix densities. Overall these cells appeared very similar to swollen control myocytes from intact hearts. In contrast, ischemic myocytes exhibited changes early after ischemic incubation. After 30 min isotonic ischemia there was

Myocytes

173

a mixed population of cells present. Figure 8 shows a square cell with an intact sarcolemmal membrane. The sarcomeres are contracted with thickening of Z-bands and loss of I-bands. The nuclear chromatin is clumped and the mitochondria appear swollen and contain occasional amorphous matrix densities. A small intercalated disk bleb is present at the end of the cell which appeared to result from the lifting of the membrane from the underlying terminal ends of the sarcomeres. The space enclosed by the bleb is filled with flocculant material. The intact membrane surrounding the bleb suggests that this was a viable square cell. Acute swelling of ischemic cells resulted in a significantly larger number of non-viable square cells than present in isotonic ischemic preparations. Figure 9 shows a square cell that was exposed to hypotonic buffer after 30 min ischemia. The cell appears contracted and mitochondria exhibit amorphous and linear matrix densities indicative of ischemia. A large break in the sarcolemmal membrane is evident on the lateral edge of the cell suggesting

FIGURE 6. Etectron micrograph of freshly isolated adult myocyte subjected scarcolemmal membrane is intact and the sarcomeres appear relaxed with prominent between cellular organelles indicating that the cell has been swollen. Mitochondria exhibit amorphous matrix densities. x 4375.

to hypotonic cell swelling. The I-bands. There is increased space appear condensed and do not

R. S. Vander

Heide

et al.

FIGURE 7. Higher power electron micrograph of acutely swollen control myocyte. The sarcolemmal membrane intact and exhibits scalloping between Z-line attachment sites indicative of cell swelling. The sarcomeres are aligned with prominent I-bands. Glycogen is seen evenly distributed throughout the sarcoplasm. x 13 125.

is w ell

is FIGURE 8. Electron micrograph of isolated myocyte after 30 min ischemia at 37°C. The sarcolemmal membrane intact. The sarcomeres are contracted with loss of I-bands and thickening of Z-lines indicating that this cell is in rig or us contracture. The nuclear chromatin is clumped and the mitochondria appear slightly swollen with rare amorpho matrix densities. Note the small intercalated disk bleb on the end of the cell. The membrane overlying the bleb appea iTS intact which indicates that this was probably a viable square cell. x 2625.

Ischemia

FIGURE sarcomeres amorphous membrane

and Isolated

Myocytes

9. High power view of square cell subjected to hypotonic swelling after 30 min ischemia at 37 ‘“C. The are contracted with prominent thickening of Z-lines. The mitochondria are swollen and exhibit p* .ominent (small arrows) and linear matrix densities (thin arrows). There is a large break in the sarc olemmal (thick arrows) overlying the mitochondria indicating that this was a non-viable square. x 18 375.

this is a non-viable cell. The outer membrane of the mitoochondria also appears ruptured underneath the membrane break. After 60 min &hernia the changes were more severe than after 30 min and a larger proportion of square cells contained blebs. In addition, of cells that contained blebs, a larger proportion showed breaks in the bleb membrane indicating that the cell was non-viable. Figure 10 shows an ischemic cell incubated for 60 min and resuspended in isotonic buffer. The cell appears contracted and the mitochondria show variable amounts of swelling and occasional flocculant matrix densities. Although the sarcolemmal membrane appears intact, there is a large intercalated disk bleb present on the end of the cell that appears ruptured. The bleb space shows occasional mitochondria which appear adherent to the underlying cell lattice. Figure 11 shows a cell incubated for 60 min prior to resuspension in hypotonic buffer (acute cell swelling). The myofibrils are

hypercontracted and the mitochondria are massively swollen with disruption of cristae and outer membranes. Intercalated disk blebs are present on both ends of the cell with associated flocculant material and free mitochondria. The bleb membrane on the larger end of the cell exhibits a large break. The sarcolemmal membrane appears disrupted in numerous places on the lateral edge of the cell.

Discussion The purpose of this study was to determine if isolated adult myocytes, a relatively new model used extensively to characterize biochemical and electrophysiological properties of normal myocardium, can be a useful model to study myocardial ischemia. Four welldocumented responses of mycoardium to ischemia were used to compare the response of isolated adult rat myocytes to other established models of ischemia.

176

R. S. Vander

FIGURE 10. Electron micrograph ofan isolated The cell appears contracted with loss of glycogen amorphous densities can be found. The sarcolemmal end of the cell appears ruptured (arrows) indicating

Heide et al.

myocyte resuspended in isotonic buffer after 60 min ischemia at 37°C. stores. The mitochondria appear massively swollen but only rare membrane appears intact but a large intercalated disk bleb on the that this cell was probably a non-viable square cell. x 2625.

FIGURE 11. Electron micrograph of ischemic myocyte subjected to hypotonic 37OC. The mitochondria are massively swollen with disruption of both inner and blebs are present on both ends of the cell with the larger bleb showing a prominent membrane appears to be disrupted in numerous places along the lateral membrane.

swelling after outer membranes. break (arrows). x 2625.

60 min ischemia at Intercalated disk The sarcolemmal

Ischemia

and

Isolated

Cellular metabolism Perhaps the most important response of myocardium to ischemia is the profound loss of energy stores that occurs during the period of ischemia. Following the onset of ischemia, a reproducible series of changes occurs in the adenine nucleotide pool of myocytes: there is a rapid loss of creatine phosphate which is followed by a more gradual loss of ATP (Braasch et al., 1968; Jennings and Reimer, 1981). As the ATP levels decline there is a concomitant redistribution of other nucleotides: levels of AMP, adenosine, inosine, and to a much lesser degree IMP rise. Levels of lactate also rapidly accumulate following the onset of ischemia. Prolonged periods of ischemia cause a depletion of the total adenine nucleotide pool which reflects the degradation of adenine nucleotides to adenosine, inosine, hypoxanthine and xanthine through the action of 5’-nucleotidase, adenosine deaminase, and adenylate or AMP deaminase (Berne and Rubio, 1974; Jones et al., 1976; Altschuld et al., 1987; Hohl et al., 1989). In this study pelleting of isolated myocytes followed by incubation at 37°C resulted in marked changes in the levels and proportions of adenine nucleotides as shown in Table 1. There was progressive decline in ATP levels associated with a rise in AMP, adenosine, inosine, and a large rise in lactate. The levels of IMP showed a small but insignificant rise after 60 min ischemia. The level of lactate measured in this model is much higher than that usually observed in models of severe canine in vivo ischemia. In the canine heart levels of lactate after severe ischemia rarely exceed 300 pmol/g dry w. Although the reason for this difference is not readily apparent, there are at least two plausible mechanisms. The incubation medium that the isolated myocytes are suspended in prior to pelleting contains 11 mM glucose. This provides the cells with a readily available source of substrate for anaerobic glycolysis which may allow the accumulation of large amounts of lactate. However, for lactate to accumulate requires that anaerobic glycolysis continue uninhibited throughout the ischemic interval. In canine in viuo ischemia anaerobic glycolysis is eventually inhibited thereby limiting the total potential lactate accumulation. It is possible that in the

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isolated mvocyte model of ischemia anaerobic glycolysis is not inhibited or inhibited to a lesser extent thereby allowing a large accumulation of lactate. Regardless, these changes are consistent with those seen in other models of myocardial ischemia (Jennings and Reimer, 1981). Although the changes in the adenine nucleotide pool seen in ischemic isolated myocytes are similar to those of other ischemic models, it may be argued that the changes seen are not specific for ischemia. Many models of substrate-deprived anoxia both in intact hearts and in isolated myocytes have been shown to produce a rapid decline in cellular energy levels and a redistribution of adenine nucleotides similar to that seen in However, some ischemic models. recent evidence has suggested that anoxia can be distinguished from ischemia based on subtle qualitative differences in the adenine nucleotide pool. Recent studies using isolated rat myocytes have documented that under conditions of rapid metabolically-induced anoxia there is a large accumulation of AMP -hat under anoxic conditions is converted to IMP through the action of AMP deaminase (Altschuld et al., 1987; Hohl et al., 1989). In contrast, cells that are allowed to incubate with rotenone present to stimulate anaerobic glycolysis prior to rapid de-energization do not accumulate large amounts of IMP. It was hypothesized that anaerobic glycolysis inhibits AMP deaminase thereby allowing accumulated AMP to be dephosphorylated by 5’-nucleotidase to adenosine. The conclusion that anoxia and ischemia in rat myocardium may be differentiated based on the relative concentration of IMP has been supported in studies of perfused rat hearts (Humphrey et al., 1984). In this study the authors showed that after 60 min of high flow anoxia, ATP had declined from 22.08 to 0.80 ,umol/g dry wt while the level of IMP increased from 0.18 to 8: 15 pmol/g dry wt. In contrast, in hearts subjected to 60 min of &hernia the level of ATP declined from 22.08 to 1.76 ,umol/g dry wt while the level of IMP increased from 0.18 to only 2.02 pmol/g dry wt. The mechanism by which AMP deaminase and ii’-nucleotidase are regulated and/or altered under conditions of anoxia or ischemia is unknown at present. However the results indicate that in rat

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myocardium the adenine nucleotide pool profile after 60 min incubation in a pellet are more consistent with &hernia than with anoxia.

Hypothermia Hypothermia is known to protect myocardium from ischemic injury (Hearse et al., 1975, 1980; Jones et al., 1982). The most likely mechanism responsible for hypothermic protection involves slowing the depletion of high energy phosphates from the cell. Indeed, the rate of degradation of the adenine nucleotide pool in ischemia has been shown to be temperature dependent; as the temperature is lowered the rate of ATP depletion is slowed accordingly (Jones et al., 1982). Since the onset of ischemic contracture and the onset of ischemic injury are both dependent on a critical level of cellular ATP, the decreased rate of energy loss from hypothermia results in a delay in the onset of both ischemic contracture and ischemic injury. In isolated adult myocytes it has been shown that the transition from rod shape to square shape is associated with a critical reduction of cellular ATP levels (Haworth et al., 1981; 1988; Haworth and Berkoff, 1985; Altschuld et al., 1985). Therefore, in isolated myocytes the onset of cell squaring can be considered analagous to the onset of ischemic contracture in intact hearts. Furthermore, as square cells are subjected to further periods of ischemia or energy deprivation, the proportion of square cells that stain with the vital dye trypan blue increases (Ganote and Vander Heide, 1988). Since trypan blue staining correlates with enzyme release and is an indicator of loss of membrane integrity, it seems rational to assume that blue squares resulting from ischemia are indicative of ischemic cell death. In this study decreasing the temperature of the ischemic incubation from 37 to 30°C delayed both the onset of “ischemic contracture”, in this case the onset of cell squaring, and the onset of ischemic injury (blue squares) suggesting that depletion of cellular energy levels in isolated myocytes is delayed by lowering the incubation temperature; results are consistent with other documented models of myocardial ischemia.

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Morphology The ultrastructure of normal myocardium and the structural changes which occur as a result of ischemic injury are well known and are the subject of many excellent review articles (Jennings and Ganote, 1974; Kloner et al., 1974a, b; Sommer and Johnson, 1979). In the present study morphological changes in isolated myocytes after a period of ischemia were similar to those observed in other models of in vivo and in vitro ischemia. After a period of ischemia the cellular glycogen was depleted, the mitochondria were massively swollen and exhibited occasional prominent amorphous and linear matrix grandules, the nuclear chromatin was clumped, many cells contained prominent blebs, and in some cells obvious defects in the sarcolemmal membrane were apparent. However the sarcomeres are contracted in in vitro models in contrast to the prominent I-bands and N-bands which are seen in vivo. This difference is presumably due to the systolic stretching of the noncontractile ischemic tissue in vivo which is not present in vitro. In addition, in vitro models usually show fewer amorphous mitochondrial densities after similar periods of ischemia Wennings and Hawkins, 1980). Interestingly, subsarcolemmal blebs are common to both in uivo and in vitro models of ischemia; however intercalated disk-associated blebs are more prominent in in vitro models while lateral blebs predominante in in vivo models. The significance of this is unknown. The time course of ischemic injury in isolated myocytes was also consistent with other models of ischemic injury. In this study, 30 min ofischemic incubation produced a heterogeneous population of cells. Most cells were still in the rod-shaped configuration and only 10 to 15% of cells had undergone the transition into the square shape indicating a critical loss of cellular ATP levels. After 60 min ischemia a more homogeneous population of viable square cells existed with the appearance of small numbers of non-viable blue squares. These observations suggest that ischemic injury in isolated myocytes is a heterogenous process; the entire incubate exhibits a gradual increase in the number and proportion of square and non-viable square

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cells. Furthermore ischemic injury in isolated myocytes is a graded process;the extent and severity of ultrastructural changes and the proportion of cells containing blebs increased as the duration of ischemia was prolonged (Fig. 8 to 11). Myocytefragilio Increased myocyte fragility following a critical period of anoxia or ischemia has been demonstrated in in vitro dog heart slices, perfused rat hearts, and most recently in isolated adult rat myocytes following chemical anoxia (Steenbergen et al., 1985; Vander Heide and Ganote, 1987; Ganote and Vander Heide, 1988). In thesestudiesthe increasein fragility has been shown to be dependent on both the duration of the ischemic or anoxic insult and the strength of the induced physical stress.At the time increase myocyte fragility can be demonstrated, changesin the cytoskeletal support system, specifically loss of vinculin and alpha-actinin immunofluorescence staining, can be demonstrated (Steenbergenet al., 1987; Ganote and Vander Heide, 1987). However the precise causefor the lossof staining and the specific mechanism responsible for the increasedfragility is not currently known. In the present study isolated myocytes subjected to a period of in vitro ischemiadeveloped increased fragility similar to that demonstrated in other model systems.At each time period studied, cells acutely stressedby suspension in hypotonic buffer (cell swelling) showed an increasedproportion of non-viable squares when compared to cells sampled at the same time and suspendedin isotonic buffer. In addition, the increase in cell fragility was graded with time; as the duration of the ischemic interval increased the difference in the responseto stressbetween the two groups increased.Control cellsincubated for the same amount of time and subjected to a similar stressdid not show increased proportions of blue cells.

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limited extracellular space subjected to a period of incubation undergo biochemical and morphological changesthat are similar to the changes observed in several other welldocumented models of myocardial ischemia. Furthermore, ischemic myocytes respond to two different manipulations, one protective (hypothermia) and one detrimental (acute cell swelling), in a similar manner to other documented modelsof ischemia. Isolated myocytes possessseveral advantageswhich will make them uniquely suited to study certain aspectsof myocardial ischemic injury. First, isolated myocytes possessthe inherent advantages of an in uitro model system. In vitro modelsallow more precisecontrol of experimental conditions and manipulations than in vivo systems.In addition, the myocyte preparations are nearly pure populations of myocytes thereby allowing for exact measurement of nucleotide levels and other metabolic parameters free from any confounding influences of diffusion problems or of other cell types which are present in intact preparations. Finally isolated myocytes provide a model of ischemia in which it is uniquely possibleto acutely stressischemicmyocytes (cell swelling) without the potentially complicating influences of no-reflow, oxygen-induced injury, and oxygen free radical production present in other in vivo modelsof ischemia (Hearse et al., 1973; Kloner et al., 1974a; McCord, 1985). To determine precisely when irreversible injury occurs and which, if any, interventions may delay or prevent ischemic injury it is necessary to be able to test for cell viability without introducing new forms of cell injury or accelerating already preexisting cell injury.

Limitations There are limitations to the isolated myocyte model which may limit its applicability in studying certain aspectsof ischemia. Because the cells are isolated from each other it is impossible to study function and functional recovery during and after ischemia or the Usefulnessand limitations of ischemicisolated modulating effects that mechanical motion myocytes may have on ischemic injury. Furthermore, Usefulness because it is an in vitro system it lacks the The results of this study have shown that possible modulating effects that vascular tisisolated adult rat myocytes with a severely sue, the glycocalyx, neurohumoral substances,

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and leukocytes and platelets may contribute to the overall mechanism of ischemic injury (Reimer et al., 1985; Kammermeier and Rose, 1988). Although these factors undoubtedly play a role in ischemic injury, isolated myocytes, when properly utilized, provide a valuable model to study specific aspects of ischemia.

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thermic protection, (3) demonstration of osmotic fragility, and (4) characteristic electron microscopic changes. In contrast to in vivo ischemic models where lateral subsarcolemmal blebs predominate, isolated adult myocytes develop blebs preferentially over intercalated disk regions. Isolated myocytes may provide a new, useful, in vitro model of ischemic myocardial cell injury.

Summary Pelleted isolated adult rat myocytes respond to ischemia similarly to other in viva and in vitro ischemic models in four important respects: ( 1) changes in adenine nucleotide profile and lactate accumulation, (2) response to hypo-

Acknowledgements Supported in part by AHA 86-647 and HL36240. The authors are grateful to MS Nancy Hall for her invaluable help in the preparation of the manuscript.

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