Effect of inhibition of the mitochondrial ATPase on net myocardial ATP in total ischemia

J Mol Cell

Cardiol

23,

1383-1395

Efkt

Robert

(1991)

of Inhibition of the Mitochodria.l onNetMyocardid~inTotatIs&emia B. Jennings,

Keith

A. Reimer

AI&se

and Charles

Steenbergen

Department of Pathology, Duke University Medical Center, Box 3712, Durham, NC 22710, USA (Received II March 1990, accepted in revisedfonn 24July

1991)

R. B. JENNINGS, K. A. REIMER AND C. STEENBERGEN. Effect of Inhibition of the Mitochondrial ATPase on Net Myocardial ATP in Total Ischemia. Joumal ofMolccular and CclIukr Cardiology (1991) 23, 1383-1395. The effect of inhibition of the mitochondrial ATPase with oligomycin on the rate ofATP depletion and anaerobic glycolysis was studied in the totally ischemic dog heart. An oxygenated, buffered crystalloidal solution containing 10~~ oligomycin and 12 mM glucose was delivered at 100 mmHg pressure to the circumflex bed of the excised cooled heart. Buffered solution without oligomycin was delivered simultaneously to the anterior descending bed of the same heart. Little metabolic evidence of ischemia developed until the heart was made totally ischemic by incubating it in a sealed plastic bag at 37%. Successful inhibition of the mitochondrial ATPase was confirmed by the absence of both mitochondrial ATPase activity and the loss of respiratory control in mitochondria isolated from treated tissue. ATP, glycolytic intermediates and catabolites of the adenine nucleotide pool were measured in the control and treated beds at various intervals during 120 min of ischemia. Inhibition of the ATPase resulted in slowing of the rates of ATP depletion and anaerobic gfycolysis (estimated by lactate accumulation). Also, degradation of the adenine nucleotide pool occurred more slowly in the inhibited group. These data establish that about 35% of the ATP utilization observed during the first 90min of total ischemia in the canine heart is due to mitochondrial ATPase activity. KEY WORDS: Mitochondrial Demand for -P in ischemia.

ATPase;

Oligomycin:

Total

Introduction ATP utilization in acutely ischemic myocardium occurs through a number of reactions which continue while the tissue is ischemic. These include contractile activity, transport ATPases and other enzymatic reactions (Jennings and Reimer, 1981). The relative contribution of each to the total utilization of ATP by ischemic tissue has been difficult to estimate. It is clear that the limited contractile activity which continues during the first few minutes of ischemia is one important ATP consuming process (Lowe et al., 1979); eiimination of contractile activity by inducing K+ arrest prior to ischemia significantly slows the rate of ATP depletion (Jones et al., 1981). Other reactions which utilize ATP include ion transport ATPases. The Na/K ATPase of the sarcolemma, the Ca ATPases of the sarcolemma and sarcoplasmic reticulum and perhaps others all continue to function while the myocytes are ischemic. Additional enzymes which utilize ATP while the tissue is ischemic 0022-2828/91/121383

+ 13 $03.00/O

ischemia;

ATP;

Lactate;

Glycolytic

intermediates:

include fatty acid CoA synthetase, adenyl cyclase and the mitochondrial ATPase (Rouslin, 1983; Rouslin et al., 1986). The mitochondrial ATP synthetase is the macromolecular complex of proteins comprising the Fl-FO particles of mitochondria (Hinkle and McCarty, 1978, Kagawa, 1984). When the mitochondria are adequately energized, this enzyme synthesizes ATP from ADP and Pi. If the mitochondrial membrane potential falls, the reverse reaction can occur and ATP is hydrolyzed to ADP and Pi. This enzyme then is termed the mitochondrial ATPase (Kagawa, 1984). Both the synthetase and ATPase reactions of the Fl-FO particle are inhibited by oligomycin (Linnett and Beechey, 1979). The relative importance of the mitochondrial ATPase to tissue ATP consumption has been studied recently by Rouslin et al. (1986), who have concluded that most of the ATP hydrolysis observed in the totally ischemic heart is due to activity of this enzyme. @I 1991 Academic

Press

Limited

1384

R. B. Jennings

Using excised samples of myocardium pretreated in uivo with oligomycin, a specific inhibitor of the mitochondrial ATPase, and then exposed to total ischemia at 37%) he has shown that much less ATP depletion occurs in the treated than in the untreated myocardium over 20 min of total ischemia at 37°C. In this paper, we report the effect of oligomycin on net myocardial ATP, the rate of anaerobic glycolysis, the degradation of the adenine nucleotide pool @Ad) and glycogen metabolism in totally ischemic dog myocardium. In tissue treated with oligomycin, ATP depletion was slowed throughout the first 90min of ischemia, and at 90min there was much more ATP in treated than in untreated tissue. However, even in the oligomycintreated tissue, the ATP content of most hearts reached virtually zero after 120min of ischemia. Thus, these data confirm that the mitochondrial ATPase is an important, albeit not the only, cause of ATP hydrolysis during ischemia.

Materials

and

Methods

Experiment design The effect of total ischemia on the demand of the ischemic tissue for ATP with and without inhibition of the myocardial ATPase was tested in the totally ischemic excised dog heart maintained at 37%. Oligomycin-treated and untreated regions of the left ventricle were compared in the same hearts. The oligomycin was administered in oxygenated buffer into the left circumflex arterial bed while buffer alone was perfused into the left anterior descending arterial bed of a cooled heart. After completion of the infusion, the heart was placed in a sealed plastic bag and was immersed in a water bath. Glycogen, glycolytic metabolites and components of the adenine nucleotide pool were measured in samples excised from the treated and untreated tissue at 0, 10, 15,30,60,90 and 120 min. Confirmation that the oligomycin dose was adequate to inhibit aerobic respiration was established by showing that ADPstimulated mitochondrial oxygen consumption and mitochondrial ATPase function were depressed markedly in mitochondria isolated from the tissue treated with oligomycin.

et al. Animals Hearts from six healthy mongrel dogs which had been fasted overnight and allowed water au! libitum were used. Under intravenous pentobarbital anesthesia, an endotracheal tube was placed in position and respiration was maintained with a Harvard Model 607 respirator pump. Blood pH, kO,, and PO, were measured at intervals and were maintained in the physiologic range with 0, added to the room air entering the respirator (Murry et al., 1990). The thorax was opened through the left fourth intercostal space. The heart was suspended in a pericardial cradle and the circumflex artery was isolated under the left atria1 appendage. Two black silk ligatures were placed around the circumflex artery to facilitate rapid cannulation of the vessel for perfusion. After lo-15 min equilibration, the heart was excised quickly by cutting well above the atrioventricular groove. It was cooled for 2 to 3min in abundant ice-cold isotonic KC1 containing ice cubes composed of frozen isotonic KCl. After cooling to 13” to 18°C (measured with a thermister in the septum), the left main coronary and circumflex arteries were cannulated with polyethylene tubing and the vessels were perfused simultaneously at 100mmHg (Jennings et al., 1989). Buffer at room temperature (20” to 22°C) containing oligomycin and diIute Evans blue dye was infused into the circumflex bed while control buffer was infused into the remainder of the heart. The volume and duration of infusion were measured and the total dose of oligomycin was calculated. Buffer and oligomycin solution

A HEPES buffer of the composition used by Rouslin et al. (1986) was prepared. It contained (in mmoles): -118Na+, 4.7K+, 2.5Ca2+, 1.2Mg2+, 1.2KH,PO,, 200 HEPES pH 7.5, 0.5 EDTA, 25 NaHCOs and 12 glucose. Reagent grade chemicals were obtained from Fisher. HEPES, ethylene diamine tetraacetic acid (EDTA) and glucose all were obtained from Sigma. The buffer was aerated for 15 min with 95 % O2 and 5 % CO, at a rate of 5 l/min. The buffer then was split into two 1000 ml containers both of which were covered with parafilm after placing 95 % 0, and 5 % COT in the airspace over the solution.

Mitochondria

and

Oligomycin (Sigma) from streptomyces diastolochromogenes was dissolved (4.28 mg) in 10ml of 95% reagent grade ethyl alcohol immediately before adding it to the gassed buffer. The oligomycin used contained the A, B and C types of oligomycin with type A comprising 65 % of the mixture. The alcoholic oligomycin solution was added to 1OOOml of buffer 10 to 20min before it was infused into the heart. An identical bolus of ethyl alcohol alone was added to the buffer used to perfuse the control bed. In order to facilitate identification of the bed which had received the oligomycin, 50 mg of Evans blue dye also was added to the infusate. The oligomycin-treated myocardium turned uniformly light blue.

Total ischemia At the completion of the infusion, the serosal surface of the left ventricle was painted quickly with a dilute methylene blue solution so as to be able to identify the epicardial surface of the left ventricle in the samples taken for analysis. The left ventricle was opened quickly and the light blue oligomycin bed was examined. The bed stained evenly with Evans blue dye indicating that the perfusate was distributed uniformly. After freezing a sample of the treated and control beds for metabolites, the blue tissue was excised and weighed. The remainder of the left ventricle served as control tissue. A 1.5 to 2.0 g sample of subendocardial oligomycin-treated tissue was excised and was placed in ice-cold isotonic KC1 for isolation of mitochondria. A similar sized sample of septum was taken for isolation of mitochondria of untreated tissue. A thermister was placed in the anterior papillary muscle in order to record the temperature. The treated and untreated tissue was subjected to total ischemia using a previously described technique (Jennings et al., 1981). Briefly, blocks of myocardium were placed in two ziplock bags, one inside the other, which were closed in order to eliminate most of the air surrounding the tissue. The bags were incubated in a 38% waterbath. At each sampling time, the bag was removed, and a slice of tissue was frozen and treated as described in the next section.

ATP in Ischemia

1385 Metabolite

analysis

After selected intervals of ischemia, tissue samples weighing 250 to 500mg each were frozen in freon at liquid N, temperature and were dried in a Vertis Model 6 Freezemobile freeze-dryer. After the tissue was dried, the endocardium of the inner half was trimmed off and the tissue was ground into a fine powder in a mortar and pestle. Unpowderable tissue, which consists chiefly of fibrovascular septae, was removed before weighing the samples on a Mettler microbalance. They were transferred into perchloric acid for extraction of metabolites according to techniques described elsewhere (Jennings et al., 1985; Murry et al., 1990). Glucose-6-phosphate (Lamprecht and Trautschold, 1974), glucose- 1 -phosphate (Bergmeyer and Michal, 1974), 01 glycerol phosphate ((YGP) (Michal and Lang, 1974), lactate (Lowry and Passonneau, 1972) and glucose (Bergmeyer et al., 1974) were measured in neutralized perchloric acid by enzymatic techniques. Adenosine triphosphate (ATP) (Lamprecht and Trautschold, 1974) and creatine phosphate (CP) (Lamprecht et al., 1974) were measured enzymatically. ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (HX) and xanthine (X) were measured by high performance liquid chromatography using the technique described by Jennings et al. (1985). Glycogen was measured using amyloglucosidase (Keppler and Decker, 1974); in this technique, the powder was added directly to perchloric acid where it was further broken up with a 20s burst from a Type PT 10 polytron. After extraction of the glycogen, samples were digested with amyloglucosidase for 2 h at 37°C. Endogenous free-glucose was measured in the undigested homogenates. Tissue glycogen is reported as micromoles of glucose equivalents per gram dry weight after correcting for endogenous glucose. All results are reported as micromoles per gram dry tissue f the standard error of the mean. Since the powder picks up water from the air during weighing, three additional samples were dried to constant weight and the percent water calculated. The sample weights were corrected to the true dry weight before calculating the final results.

1386

R. B. Jennings

Mitochondrial isolation Pure mitochondria were isolated by a technique modified slightly ,from that described by Jennings et al. (1969). Subendocardial tissue was trimmed free of the endocardium. After weighing it quickly, the tissue was minced on the lip of a cold 50ml Tri-R homogenizer tube. It then was mixed in a freshly prepared solution consisting of 0.15 M KCl, W3M EGTA, and 0.01 M Tris HCl pH 7.6 containing 0.5 mg/ml of Nagarse (Sigma, Protease Type XXVII). One milliliter of homogenization fluid was added for each 100 mg muscle. After incubation for 10 min at O”C, the mince was homogenized at slow speed in a TRI-R homogenizer using a loosely fitting Teflon impregnated glass pestle. Complete homogenization was achieved in three to four strokes at a setting of 6 on the TRI-R homogenizer motor. The remainder of the isolation was as described previously (Jennings et al., 1969). The final pellet was rinsed and was resuspended by hand homogenization in 0.18~ KC1 using 10% of the volume of the initial homogenization. The final preparation was examined by phase contrast microscopy; if small myofibrillar fragments were present, the preparation was recentrifuged at low speed in order to clear mitochondria of the myofibrillar debris.

Mitochondrial

et al.

nique (Lowry et al., 1951) using a Sigma Protein Assay Kit (Procedure No. P5656).

Mitochondrial ATPase assay Mitochondrial ATPase activity in whole and sonicated mitochondria from the control and oligomycin-treated beds was assayed by a modification of the procedure by Kielley (1955). The reaction was carried out in 1 ml for 5 min at 28% and pH 7.5. The final concentrations were 5 mM ATP and 0.5 mM MgCl, in 0.2 M Tris buffer. The protein concentrations used were adjusted to hydrolyze less than 1 pmol ATP/5 min. Results are reported in micromoles of ATP hydrolyzed per 5 min per milligram of mg protein. Inorganic phosphate was measured by a modification of the procedure of Baginski et al. (1967). For the ATPase assay, mitochondria were totally disrupted by sonication using a Model LS75 Branson Instruments Sonifier (New Jersey) at a setting of 7.0. Phase microscopy was used to establish that no intact mitochondria were present. Total disruption required three 60 s bursts using a - 4% ice bath (KC1 and water ice) to keep the mitochondria cool during sonication. Also, the probe was cooled to 0°C between runs. The sonicated mitochondria exhibited no significant RCR.

function

Oxygen consumption was measured at 30% in a Model K-IC oxygraph (Gilson Medical Electronics, Middleburg, Wisconsin) in a 2 ml open type cell with a final volume of 1.56 ml. The final reaction mixture contained 112mM KCl, 10mM potassium phosphate buffer (pH 7.4) 1 .O to 2.0mg mitochondrial protein, 12OpM pyruvate and 64,~~ malate, and 3.2 mM ADP. The pyruvate was obtained from Cal Biochem; the malate was obtained from Nutritional Biochemicals Corporation and the ADP was obtained from Sigma. State 4 and state 3 rates of respiration were measured and the respiratory (acceptor) control ratio (RCR) was calculated. Results are nanoatoms atomic oxygen per milligram of mitochondrial protein per minute. RCR was calculated as the ratio of the rates of state 3 to state 4 respiration. Mitochondrial protein was measured by a modification of the Lowry tech-

Statistical methods The rates of fall of ATP and rise of lactate, respectively, were calculated from 15 to 90 min in the control and from 15 to 120 min in the oligomycin group. The slopes of the lines were compared by two-tailed paired t-tests. The same tests were used to compare means after various intervals of ischemia.

Results Eject of oligomycin treatment on mitochondrial function Oligomycin was infused successfully in all hearts. An average of 821 f 13 ml of buffer was infused into the control bed and 785 f 50 ml of buffer with oligomycin was perfused into the circumflex bed at a rate of 70 to lOOml/min over an average interval of 9.2 + 0.2 min. The

Mitochondria

TABLE

1.

Functional

characteristics

State 3 (mfiO/min/mg Control Oligomycin

390.8k

56.2

36.5f6.3b

and

ATE’

of cardiac mitochondria State 4 protein)

43.1

f8.3

40.2

f6.4

1387

in Ischemia

of control and oligomycin-treated

tissue”

Mitochondrial ATPase’ (pmol P,lmg protein/5’

at 28’C)

RCR 10.4

k2.0

0.68k

1.48+0.13

O.Ob

2.5b

a The substrate was pyruvate-malate. The means are the average of six hearts in all columns except for the ATPase where only five hearts are reported because of a technical error in the assay of phosphorus in one dog. b The probability that the means are the same using a two-tailed paired I-test is P
respectively. TABLE

Dog No.

2. Initial myocardial

metabolites in control and oligomycin-treated

Corrected glycogenb C

hearta

Glucose C

0

G6P 0

C

0

1

366.4

313.1

11.6

18.5

1.68

0.62

2 3 4

232.3 337.7 167.3

225.6 338.4 159.7

16.7 9.5 10.25

15.7 19.8 11.95

0.95 2.14 3.25

0.56 0.55 1.24

5

183.7

6

170.7

179.6 158.4

13.45 7.05

11.00 10.45

1.80 0.61

0.74 0.40

229.1

Mean S E.&f.

243.0 k35.9

k32.3

Mean S.E.M

312.1 k40.8

292.4 *34.2

Mean

173.9

169.9

S.E.M

*5.0

i5.9

11.43 k1.37

10.45 k1.64

1.74

0.69

0.39

0.12

1.59

0.58 0.03

High glycogen group (1, 2, 3) 12.60 k2.14

18.00 k1.21

0.35

11.13 kO.44

0.77

Low gbcogen group (4, 5, 6) 10.25 k1.85

1.89

0.80 0.243

a C, control; 0, oligomycin; G6P, glucose-6-phosphate h The corrected glycogen column contains an estimate of the glycogen of both perfused beds of the heart prior to excision. The measured glycogen is less than the initial glycogen because some glycogenolysis occurs during perfusion. This quantity can be estimated from the glycolytic intermediates remaining in the tissue. The quantity of glucose from glycogen required to produce the measured level of glycolytic intermediates is calculated by the following equation which includes a correction for the initial in uivo level of each intermediate in pmollg dry wt in aerobic myocardium. In this calculation, the contribution of the glucose in the perfusion media is counted as glycogen. Since the tissue concentration of intermediates observed in this study is similar to that observed in total ischemia when no perfusion had been used, it is clear that very little washout of intermediates occurred during the perfusion phase of this experiment (Jennings et al.. 1992). Corrected glycogen = Initial Glyc + [0.5(L-8)) + [0.5 (c&P-0.3)] + (G-l. 74) + (G6P-0.25) + (GIP-0.05) where Glyc = glycogen, L = lactate, GlP = glucose-l-phosphate, AGP = (Yglycerol-phosphate, G6P = glucose-6-phosphate.

mean dose of oligomycin was 0.25pmoYg of wet circumflex bed. This quantity of oligomytin was adequate to reduce state 3 mitochondrial respiration, to the state 4 rate (Table 1). Also, no mitochondrial ATPase activity was detectable (Table 1). These data taken together establish that the dose of oligomycin perfused into the heart was adequate to inhibit the mitochondrial ATPase.

Eject of perfusion on initial mycocardial metabolite levels The mean baseline glycogen of the control and treated beds of the six hearts used in this experiment was 243 f 35.9 and 229.1 f32.3pmol glucosyl units/g dry wt, respectively (Table 2). However, there was a wide range (158.4 to 366pmollg) among the six hearts. Essentially, the hearts fell into two

R. B. Jennings

1388

TABLE Dog No.

3.

et al.

tissue glycogen (pmol/g dry wt) after varying periods of total ischemia

Corrected

Minutes of total ischemia 0

10

313.1 225.6 338.4 160.0 179.6 158.3

275.1 205.9 245.2 67.7 116.5 78.8

229.2 k32.3

164.8 +33.3

366.4 232.3 337.7 167.3 183.7 170.7

297.4

233.3 *35.9

15

30

60

90

1‘Lo--

281.0 187.1 273.6 80.6 134.3 91.2

262.8 177.8 266.2 62.9 82.6 58.9

172.2 121.3 177.9 19.4 18.6 16.7

149.7 77.0 135.3 2.5 6.2 4.5

95.8

174.6 k35.9

151.9 k39.8

87.7 zt32.1

62.5 +27.8

34.4 k 17.2

166.1 260.9 86.5 137.2 89.5

Control 294.6 174.4 237.7 45.6 121.1 112.1

244.9 150.4 198.0 22.0 102.3 104.3

159.0 87.9 157.6 3.5 22.3 6.4

134.7 18.7 126.1 10.0 10.1 2.0

97.7 9.3

147.6 2.4 1.5 1.2

172.9 lt36.1

164.2 k37.0

137.0 k32.2

72.8 k29.8

50.2 k25.5

43.3 +25.9

Oligomycin-treated 5099

5124 5154 5110 5128 5143 Mean S.E.M.

5099

5124 5154 5110 5128 5143 Mean S.E.M.

classes, those in which the initial glycogen was high and those in which it was low. This difference in baseline glycogen content is important in terms of the effect of total ischemia on phosphate (“P) metabolism, because most of the -P utilized in totally ischemic myocardium is derived from GlP originating from glycogen (Jennings et al., 1981). Furthermore, exhaustion of the glycogen supply would be expected to result in an abrupt decrease in net ATP (Jennings et al., 1989). As shown in Table 3, cardiac glycogen decreased as the period of ischemia was extended. At 60min, cardiac glycogen was approaching zero in two of the three control hearts in the low initial glycogen group, while at 90min, tissue glycogen was close to zero in the oligomycin bed of all low glycogen hearts. Because of the relationship between low glycogen and rapid utilization of reserve “P, all metabolite levels reported in the remainder of this paper are from myocardial samples that had 7 pmol or more of glycogen in the tissue at the time of sampling. Note also that tissue glucose was increased over the 1.74 Pmol/g found in samples of blood perfused canine myocardium in vivo (Table 2).

27.7 77.2 0.7 4.1 0.8

Some of this increase can be explained by the 12 mM glucose in the perfusate. Regardless of its source, most of this glucose is utilized during total ischemia; it is reported as glycogen in Table 2 and 3. E$ect of oligomycin pre-treatment on net myocardial ATP and the CAd Totally ischemic myocardium pre-treated with oligomycin lost ATP at a significantly slower rate than control untreated tissue (Fig. 1) throughout the first 90 min of ischemia. Thus, the greatest difference in tissue ATP content was at 90 min when there was virtually no ATP in control tissue and more than 11 pmol of ATP in the treated tissue (P
Mitochondria

35-

b

< +x 30m $ 250 & m0 t

E E 2O c? 2 a

aad

ATP

in Ischemia

1389

Oligo.

Control

15IO5-

ohgo. Control

O-

L‘Bfi~~~~~~ 0 15 30 45 60 75 Total

ischemla

90

105120

135

I

60

30 Total

ischemlo

90

(nun)

(mm)

1. The effect of oligomycin (&go.) on the rate of ATP depletion (A ,a) and lactate accumulation ( A .o) is shown. Oligomycin slowed both the rise in lactate (PcO.05) and the fall in ATP (P
FIGURE

tide pool of the treated and control group are shown at sequential times in Figure 2. The retention of ATP in the oligomycin treated group was accompanied by a delay in the rate and magnitude of the increase in AMP, nucleosides and bases. Thus, these data in Figures 1 and 2 show that ATP consumption in total ischemia was much less in tissue in which the mitochondrial ATPase is inhibited from the onset of ischemia than in untreated control tissue. Since less ADP was produced, less AMP was produced via the action of adenylate kinase (myokinase) on ADP (‘Jennings and Steenbergen, 1985); the result was slower degradation of AMP via the action of 5’ nucleotidase and slower formation of adenosine and inosine.

Efect of oligomycin pre-treatment on anaerobic glycolysis Although inhibited

0

anaerobic glycolysis is partially in total ischemia, it continues to

FIGURE 2. The effect of oligomycin on adenine nucleotide pool constituents is shown. In non-rschemic tissue, this pool is composed primarily of ATP. 3 to 4pmol of ADP and very little AMP. With the onset of total ischemia, as net ATP decreased, nucleosides and bases accumulated. By 90 min of ischemia in control tissue, the adenine nucleotide pool was very small and was composed chiefly of AMP. The catabolites inosine (INO) and hypoxanthine (HX) were the dominant constituents. After 120 min of ischemia the constituents of adenine nucleotide catabolism were virtually identical in treated and untreated tissue. However, during the first 90min, oligomycin treatment markedly delayed adenine nucleotide catabolism. C, control; 0, oligomycin-treatment: XAN, xanthine; ADO, adenosine.

function at a significant rate. In fact, this metabolic pathway is the source of 80% or more of the -P formed during total ischemia (Jennings et al., 1981). The chief glycolytic enzyme inhibited by ischemia is glyceraldehyde phosphate dehydrogenase (Neely and Morgan, 1974; Rovetto et al., 1975). As a result, intermediates proximal to this reaction including G6P, GlP, and (rGP, accumulate in the tissue (Fig. 3). The concentration of each of these intermediates was higher and the peak concentration occurred earlier in the control tissue. This observation is consistent with the hypothesis that the demand of the ischemic tissue for -P was lower in the oligomycin-treated myocardium As expected based on the data in Figure 3 between 15 and 90 min of total ischemia, less glycogen was utilized in the oligomycin group than in control (Fig. 4). The greatest difference was noted at 60 min when 52.5 pmol more of

R. B. Jennings

et

al.

25

0

20

40

60

00

loo

I20

0 lschemia

20

40

60

80

loo

120

(min)

FIGURE 3. The changes in four substrates (G6P, GlP, uGP and glucose) involved in anaerobic glycolysis are plotted. These intermediates accumulated because they cannot be metabolized to CO, and H,O in the absence of oxygen, and because no washout is possible in the absence of perfusion. In each case, the intermediate reached its maximum level earlier in the control tissue than in the tissue treated with oligomycin. Thus, oligomycin treatment slowed the rate of intermediate accumulation resulting from anaerobic glycolysis.

glycogen was broken down to glucose in the control group (P= 0.006). After 30 min of ischemia the difference was only 30amol glucosyl equivalents/g dry wt, but this was still statistically significant (P= 0.018). Several aspects of the changes in glucose content (Fig. 3) are noteworthy. First, the’ initial glucose of the tissue was 10.7 pmollg dry wt, which is much higher than the mean value of 1.74 Fmol/g dry wt seen in blood-perfused control tissue (unreported data). This higher baseline value is because the oxygenated perfusate contained 12mM glucose (vs. the 5 to 6mM glucose found in blood), and there was

an increased extracellular space in hearts perfused with crystalloidal solutions. Much of the tissue glucose was utilized during the first 10min of total ischemia; this represents the equivalent of an 8 to 9 additional pmol of glucosyl units of glycogen (see Table 2). The second interesting feature is the progressive rise of glucose content of the ischemic tissue as the period of ischemia is extended. After decreasing from 11.4 to 7.5pmol/g dry wt, myocardial glucose rose to as high as 16.2pmollg dry wt at 60min in the control group. However, although tissue glucose increased with prolonged ischemia in the oligo-

Mitochondrir

and

180

1” E a

160-

i

120-

f

IOO-

140-

5

80-

5 ”

6040 20

Y 0

in Ischemia

1391

reduction in consumption, glycogenolysis and anaerobic glycolysis, as estimated by lactate accumulation, also proceeded at a slower rate. Reduced depletion of ATP also produced a delay in the degradation of the adenine nucleotide pool (Jennings and Reimer, 1981; Jennings et al., 1981) and because of the lower AMP, less adenosine and inosine were formed. Finally, the general slowing of energy metabolism observed in oligomycin-treated myocardium is qualitatively similar to that seen in hypothermia (Jones et al., 1981).

240

<

ATP

I

’ 20

40 Total

60 ischemio

80 (mln)

100

12(

FIGURE 4. Cumulative glycogen utilization estimated by the intermediates of anaerobic glycolysis found in the tissue in the oligomycin-treated (0) and control ( A ) tissue. The equation: Glyc. util. = 0.5 (L-8) + 0.5 (aGP - 0.5) (G - 1.74) + (G6P-0.25) + (GlP-0.05) yields total glycogen utilized (Murry et al., 1990). Note that less glycogen was utilized in the treated group during the first 90 min of total ischemia, but that the total amount utilized was the same in the treated and control groups after 120min ischemia. Each metabolite is corrected for the quantity found in aerobic tissue. (See Table 2 footnote for abbreviations used in above equation.)

mycin group, it did not reach the levels found in the control tissue. The source of the glucose is believed to be dephosphorylation of G6P, GlP or glycolipids (Murry et al., 1990), or release of glucose from glycogen by debranching enzyme with insufficient cytoplasmic ATP in the sarcoplasm late in total ischemia for phosphorylation to occur (Jennings et al., 1991).

Discussion

In this study, we confirmed that the mitochondrial ATPase plays a role in ATP depletion in the totally ischemic dog heart (Rouslin, 1983; Rouslin et al., 1986, 1990) by demonstrating that tissue treated with oligomycin, an inhibitor of ATPase, maintained a higher net ATP throughout the first 90min of ischemia than did control tissue. The inhibition of ATPase resulted in decreased consumption of -P. As a consequence of the

Relationship of oligomycin to the mitochondrial ATPase In myocardium in which mitochondrial ATPase has been inhibited by oligomycin, tissue ATP and presumably mitochondrial ATP was preserved. At least 20% of the ATP of the myocyte is in the mitochondrial compartment (Hohl et al., 1982; Geisbuhler et al., 1984); we assume that less mitochondrial ATP was lost during the anoxia of ischemia, so that net tissue ATP decreased more slowly in hearts treated with oligomycin than in control tissue. Since rapid transport of adenine nucleotides out of and perhaps into mitochondria is known to occur in ischemic tissue (Jennings et al., 1989), the compartment or compartments containing the increased ATP, as well as the source of the ATP being broken down in the mitochondria, was not established by our experiments. It is of interest that there is an endogenous inhibitor of the ATPase (Pullman and Monroy, 1963), with a molecular weight of 9500 Da (Frangione et al., 1981; Matsubara et al., 1981) present in the matrix space of the mitochondria. This inhibitor, when bound to the FO-Fl particles of mitochondrial cristae (Hinkle and McCarty, 1978; Kagawa, 1984), i.e. to the mitochondrial ATPase, acts like oligomycin, i.e. it slows or prevents ATP hydrolysis to ADP and Pi. Rouslin (1987) has shown that mitochondria of hearts of large animals, including rabbits, dogs and man, contain this inhibitor protein and that it becomes bound to ATP synthetase when the tissue becomes acidotic during ischemia. This partially explains why the rate of ATP depletion in control ischemic dog hearts is slower than that observed in rat or mouse hearts, i.e. less ATP

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R. B. Jennings

is consumed by the inhibited ATPase of the dog heart compared with the uninhibited ATPase of smaller hearts. The endogenous inhibitor has no effect on oxidative phosphorylation; synthesis resumes when the tissue is reoxygenated and the proton gradient is reestablished (Rouslin, 1983; Kagawa, 1984). Using isolated segments of canine heart pretreated with doses of oligomycin similar to those used in this experiment, or solely with doses of buffer adequate to prevent the development of acidosis when the myocardium was made ischemic, Rouslin et al. (1986) have estimated that nearly 90% of the ATP utilized in total ischemia could be accounted for by the action of the fully active mitochondrial ATPase. This conclusion was based on an analysis of the changes seen during the first 20 min of total ischemia, during which time the binding of the natural mitochondrial ATPase inhibitor was limited by maintaining tissue pH at around 7.0 with a combination of HEPES and bicarbonate buffer, In our experiments, on the other hand, we are comparing the effect of total inhibition of the mitochondrial ATPase with oligomycin to the effect of a partially inhibited ATPase on net myocardial ATP. The degree of partial inhibition by the natural inhibitor in severe ischemia in vivo in canine heart is about 50 % (Vander Heide et al., 1991); thus, the difference between the ATP depletion rate in control and oligomycin-treated tissue is significant, but much less marked, than it would be if the ATPase was fully active. Rouslin et al. (1990) have recently reported the results of an experiment in which they studied the effects of total inhibition of the mitochondrial ATPase with oligomycin delivered in a less strongly pH buffered solution, i.e. a solution that allowed the ischemic myocardium to become acidotic quickly. In this study, they found ATP depletion rates similar to those described in this paper, i.e. an overall reduction in ATP consumption of about 30%. We have estimated the degree of inhibition of the mitochondrial ATPase due to oligomytin using the rate of net myocardial ATP depletion between 15 and 90 min in the oligomycin group and 15 and 60min in the control group. These intervals were chosen because the supply of ATP and glycogen were not limiting during these times and because the

et al. 600

(a) T

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L

I

I

I

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FIGURE 5. (a)The cumulative changes in -I’ utilization in control (a) and treated ( A ) tissue. The differences are significant at P values varying from 0.001 to 0.02 at intervals between 15 and 90 min. (b)The rate of -P utilization during various intervals of ischemia. Inhibition by oligomycin significantly reduced (P- 0.001) the rate of-P utilization at all times. Note the high initial rate of -P utilization in both the treated and untreated tissue. The differences between 15 and 30, and 30 and 60min of ischemia are significantly different by a two-tailed paired ftest (P= 0.03). After the high initial rate, the tissue treated with oligomycin exhibited a constant slower rate between 15 and 90 min of total ischemia whereas the utilization rate progressively decreased in control tissue. The 4 utilization were 19,52 and 33% less than control between 0 and 15, 15 and 30, and 30 and 60min of ischemia. Thus, the mitochondrial ATPase accounts for a wasting of a significant proportion of the ATP utilized during total ischemia.

Mitochondria

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temperature of all hearts had reached 37’C at 15min. ATP was depleted at a rate of 0.316pmol ATP/g dry wt/min in control vs. 0.206 pmol ATPIg dry wt/min in the oligomytin group (P= 0.02). This represents a 35% reduction in the rate of ATP depletion brought about by pre-treatment with oligomycin. A more precise estimate of the effect of oligomycin on ATP utilization is obtained by estimating myocardial -P utilization (Jennings et al., 1981). This estimate is made by adding the amount of -P produced by anaerobic glycolysis to that contributed by reserves of -P during each interval of ischemia. This calculation assumes that anaerobic glycolysis, using GlP from glycogen, generates net 3pmol -P/pm01 glucosyl units which is converted into 2 kmol of lactate. This calculation is possible both because lactate is not further metabolized in the absence of 0, and because lactate is trapped in the tissue due to the lack of myocardial perfusion in this experimental model, The equation is: Wp utilization

= ACP + 2AATP 1.5 Alactate

+ AADP

+

This equation slightly overestimates the contribution of exogenous glucose to *P release since exogenous glucose converted to lactate yields 2 .O rather than 3 .O pmol ATP/ kmol lactate formed (Jennings et al., 1981). However, this quantity is negligible because only a small part of the glucosyl pool is exogenous glucose. The cumulative -P utilized is plotted in Figure 5(a). Note that cumulative utilization was lower even after 15 min of ischemia and the difference persisted throughout the study. The net rate of -P utilization per time interval is shown in Figure 5(b). At each of the intervals shown, the rate of -P utilization was significantly lower in the oligomycin-treated tissue. The degree of reduction in the rate of -P utilization brought about by oligomycin was 19 % , 52 % and 33 % between 0 to 15, 15 to 30 and 30 to 60 min of ischemia respectively. The curve shown in Figure 5(b) shows a high rate of -P utilization during the first 15 min in treated and untreated tissue even though oligomycin had been present in the treated tissue for 10 to 12min under aerobic conditions in a highly buffered, oxygenated perfusate containing glucose as substrate. This high initial rate of ATP utilization in the tissue

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treated with oligomycin suggests that there are one or several reactions consuming ATP at a high initial rate. Included in these reactions could be contractile activity. However, a similar high rate is seen in K’ -arrested, i.e. non-contractile hypothermic myocardium, when it is subjected to total ischemia (Jones et al., 1982). On the other hand, the oligomycin may not have been totally bound to the mitochondrial ATPase at the onset of total ischemia. This seems less likely because our measurements of ATPase activity were made from mitochondria isolated from tissue which had been placed in a large volume of ice-cold homogenization fluid almost immediately after completion of the ohgomycin infusion. Mitochondria isolated in this way exhibited total inhibition of the ATPase. Figure 5(b) also shows the decreasing rate of ATP utilization in the totally ischemic control myocardium. Binding of the endogenous inhibitor to the ATPase undoubtedly contributes to the decreased rate observed in the control tissue. Note that total inhibition of the ATPase by oligomycin further reduces the rate of ATP utilization over that brought about by the endogenous inhibitor, but does not prevent the loss of ATP. The perfusion technique used in these experiments was developed to allow the administration of a drug or toxin to a region of a heart in order to compare, in the same heart, the effect of the agent on ischemic metabolism (Jennings et al., 1989). The hearts were cooled to 13 to 17”C, and oxygenated buffer containing glucose as a substrate was used to slow ATP depletion during the infusion of the agent into the cool heart. Using these methods, there was little or no net loss of ATP and, when the perfusion was begun speedily, 50 % or more of the CP remained phosphorylated (data not shown). In the latter group of hearts, significant ischemic metabolism did not begin until the perfusion was stopped and the hearts were rewarmed (onset of normothermic total ischemia). However, the results were similar in hearts in which perfusion was begun more slowly and in which CP was depleted to 10 to 30 % of control.

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relationship between ATP depletion and the development of irreversible injury in the canine heart (Jennings et al., 1978; Jennings and Reimer, 1981). These have demonstrated clearly that ATP depletion, when present for an extended period of time, is associated closely with cell death. Additional evidence supporting a causal relationship is that maneuvers such as hypothermia (Jones et al., 1981) which preserve ATP, also prolong the duration of viability. Our results show that inhibition of the mitochondrial ATPase has a similar, but less marked, effect on ATP. It is theoretically possible that an agent could be developed which would transiently inhibit the mitochondrial ATPase and thereby slow ATP depletion and prolong tissue viability during regional ischemia or during the cardioplegia of open-heart surgery. The binding of inhibitor protein to the ATPase, which occurs early in acute regional ischemia, may play a role in preconditioning (Murry et al., 1990), the phenomenon in which myocardium made ischemic for 5 to 15min and then reperfused, is protected against depletion of ATP during subsequent episodes

et al.

of ischemia. If the inhibitor binds more quickly when the tissue is made ischemic a second time, it would have no effect on ATP synthesis 1984), but would slow ATP (Kagawa, depletion during subsequent episodes of ischemia. It also is possible that some inhibitor binding persists during reperfusion so that the onset of ischemia leads to less mitochondrial ATPase activity. These hypothesis are potential explanations for the data recently reported by Murry et al. (1990) in which it was shown that the metabolic changes of preconditioning are associated with a reduction in the demand of the tissue for -P.

Acknowledgements The authors are grateful to Mary L. Hill and Diane Magnuson for the excellent technical assistance they provided in performing the metabolite assays, and to Charles E. Murry, and to Spring Brooks who aided in the operative procedures. This work was supported in part by grant numbers HL23138, HL 27416 and 5R29 HL 39752 of the National Institutes of Health.

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JENNINGS RB, STEENBERGEN C JR (1985) Nucleotide metabolism and cellular damage in myocardial ischemia. Ann Rev Physiol47: 727-749. JONES RN, HILL ML, REIMER KA, WECHSLER AS, JENNINGS RB (1981) Effect of hypothermia on the relationship between adenosine triphosphate depletion and membrane damage. Surg Forum XXXII: 250-253. JONES RN, REIMER KA, HILL ML, JENNINGS RB (1982) Effect ofhypothermia on changes in high energy phosphate production and utilization in total ischemia. J Mol Cell Cardiol 14 [Suppl]: 123-130. KACAWA Y (1984) Proton motive ATP synthesis. In: Enn&cs, edited by L Emster. Amsterdam, Elsevier, pp 149-186. KEPPLER D, DECKER K (1974) Glycogen determination with amyloglucosidase, In: Methods of&eym& Ana(yr2s. edited by HU Bergmeyer. New York, Academic Press, pp 1127-1131. KIELLEY WW (1955) Mitochondrial ATPase. In: Mcthodr in Enrymolqy, edited by SP Colowick, NO Kaplan. New York, Academic Press, pp 593-595. LAMPRECHT W, STEIN P, HEINZ F, WEISSER H (1974) Creatine phosphate: determination with creatine kinase, hexokinase, and glucose-6- p h osphate dehydrogenase. In: Methods qf!fEnrymtic Analysis, edited by HU Bergmeyer. New York, Academic Press, pp 1777-1785. LAMPRECHT W, TRAUTSCHOLD I (1974) ATP: determination with hexokinase and glucose-6-phosphate dehydrogenase. In: Mcthodr ofEmymatic Analysis, edited by HU Bergmeyer. New York, Academic Press, pp2101-2110. LINNE~ PE, BEECHEY RB (1979) Inhibitors of the ATP synthetase system. In: Methods ofE’nrymolqy, L C’, edited by S Fleischer, L Packer. New York, Academic Press, ~~472-518. LOWE JE, JENNINGS RB, REIMER KA (1979) Cardiac rigor mortis in dogs. J Mol Cell Cardiol 11: 1017-1031. LOWRY OH, PASSONNEAU JR (1972) Measurement of enzyme activities with pyridine nucleotides. In: A FlexibleSystem of Enzymafic Analysis, edited by OH Lowry, JR Passonneau. New York, Academic Press, pp 94-201. LOWRY OH, ROSEBROUGH NJ, FARR HL, RANDALL RJ (1951) Protein measurement with the folin phenol reagent .I Biol Chem 193: 265-275. MATSURARA H, HASE T, HASHIMOTO T, TAGAWA K (1981) Amino acid sequence of an intrinsic inhibitor of mitochondrial ATPase from yeast. J Mol Cell Cardiol 90: 1159-1165. MICHAL G, LANG G (1974) L-(-)-Glycerol-3-phosphate. In: Methods ofE~ym&Analysis, edited by HU Bergmeyer. New York, Academic Press, pp 1415-1418. MURRY CE, RICHARD VJ, REIMER KA, JENNINGS RB (1990) Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during sustained ischemia. Circ Res 66: 913-931. NEELY JR, MORGAN HE (1974) Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Ann Rev Physio136: 413-459. PULLMAN ME, MONROY GC (1963) A naturally occurring inhibitor of mitochondrial adenosine triphosphatase. ,J Biol Chem 238: 3762-3769. ROUSLIN W (1983) Protonic inhibition of the mitochondrial oligomycin-sensitive adenosine 5’-triphosphatase in ischemic and autolyzing cardiac muscle, Possible mechanism for the mitigation of ATP hydrolysis under non-energizing conditions. J Biol Chem 258: 9657-9661. ROUSLIN W (1987) The mitochondrial adenosine 5’-triphosphatase in slow and fast heart rate hearts. AmJ Physio1252: H622-H627. ROUSLIN W, BROGE CW, GRUPP IL (1990) ATP depletion and mitochondrial functional loss during ischemia in slow and fast heart-rate hearts. Am J Physiol 259: H1759-H1766. ROUSLIN W, ERICSSON JLE, SOLARO RJ (1986) Effects ofoligomycin and acidosis on rates of ATP depletion in ischemir heart muscle. Am J Physiol250: H503-H508. ROVE?TO MJ, LAMBERTON WF, NEELY JR (1975) Mechanisms of glycolytic inhibition in ischemic rat hearts. Circ Res 37: 742-751. VANUER HEIDE RS, HILL ML, STEENBERGEN C JR, REIMER KA, JENNINGS RB (1991) Effect of reversible ischemia on mitochondrial ATPase activity in canine myocardium (abstr.). Circulation 84: (Suppl 4) H-306.