Canine myocardial ischemia: Defect in mitochondrial electron transfer complex I

Journal

of Molecular

Canine

and Cellular

Cardiolou

Myocardial Electron

(1980)

12, 639-645

Ischemia: Transfer

Defect in Mitochondrial Complex I*

1. Introduction When blood flow to a region of the heart is interrupted by the sudden and complete occlusion of a coronary artery, the myocardial cells which are rendered ischemic cease to contract within seconds [4, 121. Such cells remain viable only if the blood flow is restored within approximately 15 min. If it is not, irreversible cell injury progresses and becomes largely complete by 60 min of coronary artery occlusion

[12, 131. One relatively early and prominent feature of the irreversible cell damage is the swelling and disruption of mitochondrial inner membrane structures [S-9] and the concomitant impairment of phosphorylating respiration, particularly with NAD-linked substrates [8, 91. For more than 20 years attempts have been made to identify specific biochemical defects in isolated mitochondria from ischemic myocardium which might account directly for the observed impairment of their state 3 respiratory activity with NAD-linked substrates [2, 8-12, 141. In the aggregate, these earlier studies have demonstrated a variety of biochemical alterations in mitochondria, but none of these defects appears to be directly relatable both causally and quantitatively to the observed impairment of energylinked functions in mitochondria isolated from ischemic myocardium. In the study reported here, we identify a lesion in the NADH-coenzyme Q reductase segment of the electron transport chain, i.e., in the activity of electron transfer complex I, which appears to account quantitatively for the observed loss of phosphorylating respiratory activity of mitochondria from ischemic canine myocardium.

2. Materials Mongrel dogs pentobarbital (model 607). administered

of either sex weighing (30 mg/kg). Ventilation During the operation under positive pressure.

* This work was supported by Public 0022-2828/80/060639+ 07 $02.00/O

Health

and Methods

20 to 35 kg were anesthetized with sodium was controlled with a Harvard respirator room air supplemented with oxygen was Electrocardiographic leads were attached to Service grants HL 22619 (IVB) 0 1980 Academic Press Inc.

and HL (London)

23558. Limited

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the limbs to identify ST segment changes indicative of &hernia. After a groin incision Tygon tubing was advanced retrogradely through the femoral artery to the thoracic aorta, secured in place and connected to a Statham P23Db transducer to measure systemic arterial pressure. A left thoracotomy was performed through the fourth intercostal space, the pericardium opened and the heart exposed. Ultrasonic segment length transducers were inserted with circumferential orientation in pairs into the mid myocardium (5 to 8 mm beneath the epicardial surface), one pair in the left circumflex coronary artery distribution area (ischemic) and the other in the left anterior descending coronary artery distribution area (non-ischemic) of the LV with a spacing of 1.5 to 2.0 cm between the transducers in each pair. Myocardial segment lengths between the transducers in each pair were measured continuously by the untrasonic transit time principle [1, 191 to verify the absence of contractile activity in the ischemic region. All physiological data were recorded continuously on a Brush recorder (model 200). The left circumflex artery was dissected free of surrounding tissue 2 cm distal to its origin and a ligature placed around it and later tightened to occlude the artery. In one group of dogs the circumflex artery was occluded for 60 min. In a second group it was occluded for 30 min. At the end of the occlusion period, the animal was sacrificed by rapid removal of the whole heart which was placed immediately in ice-cold 0.9:/, NaCl until contractile activity ceased. A transmural sample of the posterior LV wall coincident with the visible zone of epicardial cyanosis was dissected free, trimmed of fat and connective tissue and placed in ice-cold 180 mM KCl, 10 mM EGTA. A transmural sample of non-ischemic anterior LV wall separate from the ischemic area by at least 1 cm was also removed and treated identically. Mitochondria were prepared from both samples essentially according to the method of Sordahl et al. [18]. All steps were carried out at ice bucket temperatures. Briefly, ischemic and control myocardium were drained and then minced finely. Approximately ten volumes of ice-cold isolation medium containing 180 mM KCI, 10 mM EGTA (Tris), pH 7.4, 0.5:/, bovine serum albumin (SigmaFraction V) and 10 mM Hepes-KOH buffer, final medium pH of 7.4, was added to the tissue mince. This was homogenized as gently as possible by four 2-s passes with a Polytron type PT lo/35 tissue homogenizer (Brinkman Instruments) set at a low speed just sufficient to fully homogenize the tissue mince fragments in the 8 s total employed. The homogenates were filtered through two layers of cheese cloth and centrifuged at 450 g for 10 min. The low speed supernatants were decanted through two layers of cheese cloth and centrifuged at 10 000 g for 10 min. The tops of the 10 000 g pellets were rinsed carefully to remove any light or fluffy material (usually more apparent with ischemic material) and the mitochondrial pellets were then suspended in the isolation medium using glass homogenizers with loose fitting Teflon pestles at as high a final protein concentration as was practicable (c. 40 mg/ml) .

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IN

MITOCHONDRIAL

ELECTRON

TRANSFER

COMPLEX

I

641

State 3 and state 4 respiratory rates, respiratory control ratios and P/O ratios were measured polarographically using a Gilson model K-IC Oxygraph equipped with a Clark oxygen electrode. Approximately 1.5 mg of mitochondrial protein was used per assay and the assay medium contained 0.25 M sucrose, 10 mM Hepes-KOH, pH 7.4, 2.5 mM phosphate, 6.25 mM glutamate and 6.25 mM malate minus (state 4) and plus (state 3) 536 nmol of ADP. When used, the medium contained 6.25 mM Na succinate and 1 pg of rotenone per assay. NADH-coenzyme Q reductase was assayed spectrophotometrically by measuring the rotenone-sensitive initial rate of oxidation of NADH at 340 nm at a full scale sensitivity of 0.1 O.D. unit. The 1.5 ml reaction mixture contained 120 pmol of Tris-HCl buffer, pH 8.0, 5.0 pmol of KCN, 1.5 pmol of NADH and 40 pg of oxidized coenzyme Qz. The reaction was begun by the addition of 100 ~1 of mitochondria which had been diluted to 2.0 mg/ml and sonicated a total of 45 s. Reduced coenzyme Q cytochrome c reductase and cytochrome c oxidase assays were performed as described earlier [17] using mitochondria which had been swollen for 20 min at 30°C in 0.1 M phosphate buffer, pH 7.4. Protein was assayed by the method of Lowry et al. [15]. The coenzyme Qz was a generous gift supplied by F. Hoffmann LaRoche Br Company, Basal, Switzerland. The cytochrome c and NADH were Sigma Chemical Company Type III horse heart and yeast material, respectively. All other chemicals used were of reagent grade.

3. Results

and

Discussion

The extent of the decrease observed in NAD-linked substrate-supported respiratory activity of mitochondria after 60 min of coronary artery occlusion varied widely from zero to 66% in numerous experiments performed. This wide variation in mitochondrial functional impairment was due to the naturally occurring large variation in the riehness of collateral circulation to the circumflex distribution area of the left ventricle in this model [3, 161. This in turn dictated the severity of the ischemia obtained in any given experiment. The present study was concerned with the identification of the mitochondrial defect(s) underlying the observed loss of state 3 respiration and not with the relationship between degree of blood flow reduction and extent of biochemical functional impairment. Accordingly, the data reported here are from only those experiments in which relatively severe ischemia was achieved, i.e., from those which showed a reduction in mitochondrial state 3 respiratory activity with glutamate plus malate of 20”; or more by 30 min, and of 40% or more by 60 min of circumflex artery occlusion. The succinate-supported respiration data and the enzymatic activity data were compiled from these same experiments. These animals also consistently showed a significant elevation in ST segment voltage on electrocardiograms. In addition (see Table 1 for representative data), there was a complete loss of systolic shortening in the region supplied by the left circumflex coronary artery. It fell from an

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average of 19.6”: to 0.0~~ of end diastolic segment length upon coronary artery occlusion and this loss of regional cardiac contraction persisted throughout the ischemic period. In the non-ischemic region supplied by the left anterior descending coronary artery, systolic shortening remained nearly normal throughout the ischemic period. Heart rate (155 beats/min) and mean arterial pressure ( 126 mmHg) also remained unchanged.

TABLE

1. Regional

EDLt ESL; AJ4 %SS II

myocardial

mechanics

Non-ischemic

(LAD)

region

Control

60 min

occlusion

15.9 12.6 3.3 20.4

+ & * f

0.34 0.43 0.27 1.78

in anesthetized

16.7 5 13.6 f 3.2 & 19.0 f

0.76 0.89 0.36 2.46

open

Ischemic

chest

(circumflex)

Control 15.8 12.9 2.9 19.6

f * f i

* All data in this table are averages f S.E.M. of four experiments. conditions are described in the Materials and Methods section. 7 End diastolic myocardial segment length in mm. $ End systolic myocardial segment length in mm. 8 EDL-ESL. (( [AL/EDL] x 100.

dogs* region

60 min occlusion 1.65 2.22 0.61 5.11 The

16.0 16.1 -0.1 0.0

i f & 5

experimental

2.42 2.7 0.35 2.31 design

and

The oxygraph data reported in the upper half of Table 2 show that 60 min of circumflex coronary artery occlusion caused a 51% drop in the phosphorylating respiratory activity of mitochondria isolated from severly ischemic myocardium when the NAD-linked substrates, glutamate plus malate, were used. There was also a 53% decrease in the respiratory control of the ischemic organelles. The latter was due almost entirely to decreased state 3 rates, the state 4 rates remaining nearly constant at 19,to 20 nA O,/ min/mg. The P/O ratios decreased by only 10% indicating that the coupling efficiency of the lowered respiratory activity of the ischemic mitochondria remained high. The addition or uncouplers such as dinitrophenol, however, did not at all augment the glutamate plus malate supported oxygen uptake rate of ischemic mitochondria above the impaired state 3 rate reported in Table 2 (data not shown). This suggests that any decrease in the activity of either the mitochondrial ATPase or of the adenine nucleotide translocase which may have occurred concomitantly was not rate limiting, in vitro, to mitochondrial phosphorylating respiration in these experiments. In contrast to respiratory rates supported by glutamate plus malate, those supported by succinate (plus rotenone) decreased by only 14% after 60 min of coronary artery occlusion. The greater stability of succinate supported respiration in myocardial ischemia had been noted before [IO, 14, 201, but no specific

DEFECT

IN MITOCHONDRIAL

ELECTRON

TRANSFER

COMPLEX

643

I

mechanism for this differential had been demonstrated by earlier workers. Our observations on the greater stability of succinate supported respiration together with the observations of earlier workers suggested to us the possibility of a lesion in electron flow on the substrate side of ubiquinone. Accordingly, we measured TABLE

2. Mitochondrial functional and enzymatic changes caused by occlusion of the left circumflex coronary artery in mongrel dogs*

Control State 3 QO, (glutamate + malate)? State 3 QO, (succinate + rotenone)t Respiratory Control (glutamate + malate) P/O Ratio (glutamate + malate) Complex I (NADH-CoQ reductase): Complex I I I (CoQH,-c reductase) z Complex IV (cytochrome c oxidase):

60

min

Ischemic/ control

Ischemic

187 f 14 109 & 8

91 + 9 94 + 8

0.49 0.86

* *

0.03 0.02

9.8 + 0.4 2.96 j, 0.05 0.100 5 0.013 2.41 f 0.20 3.14 & 0.09

4.6 & 0.4 2.63 f 0.07 0.055 * 0.010 2.08 & 0.24 2.74 .+ 0.09

0.47 0.90 0.55 0.85 0.85

* + f * f

0.04 0.02 0.04 0.04 0.05

* All data in this table are averaaes + S.E.M. of eight exoeriments. conditions are described in the Ma&i& and Methods section. t State 3 QO, expressed as nAt0m.s 0, consumed/min/mg. z Enzyme activities expressed as pmol/min/mg. -

I

The

.

of

experimental

design

and

100-

35 $ 50 s 0 NADH-COP 0 State

o1

Reductose

3 QO,

I

I

I

0

30

60

Ischembo

(mln)

FIGURE 1. Time course of decreases in state 3 respiration with glutamate plus malate and of NADH-CoQ reductase activity. The 30 min points are the averages f S.E.M. of six experiments; the 60 min points are the averages & S.E.M. of the same eight experiments reported in Table 2. The experimental conditions are described in the Materials and Methods section.

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MILLARD

the enzyme activities of the individual complexes of the electron transport chain from NADH-coenzyme Q reductase to cytochrome c oxidase. Of these activities, only that of complex I was observed to decrease commensurately (lower half of Table 2) and concomitantly (Figure 1) with state 3 respiration supported by NAD-linked substrates. Moreover, consistent with the greater stability of respiratory activity with succinate (plus rotenone), the activities of both complexes III and IV decreased to the same small extent as did succinate-supported state 3 respiration. Succinic dehydrogenase activity is known to be stable during the first four to five hours of myocardial ischemia [IO] and was found to be unaffected in these studies (data not shown). The data reported here show clearly that the different mitochondrial electron transfer complexes exhibit different liabilities during the ischemic process. Electron transfer complex I is the most labile of these and, in that 75Ob of the reducing equivalents from the oxidation of fatty acids (the preferred substrates of cardiac mitochondria) are channeled through this enzyme complex, its decreased activity constitutes a central lesion in aerobic energy metabolism in ischemically damaged myocardial cells. Acknowledgements

The authors gratefully acknowledge the valuable critical comments Schwartz and Abdul Matlib. We also wish to thank Mr Billy-Joe Paul Harper for their competent technical assistance. W. ROUSLIN* Department of Pharmacology Universi~ of Cincinnati

AND

of Drs Arnold Rice and Mr R. W. MILLARD

and Cell Biophysics, College of Medicine, 231 Bethesda Avenue, Ohio 45267, U.S.A.

REFERENCES 1. BUGGE-ASPERHEIM, B., LERAAND, S. & KIIL, F. Local dimensional changes of the myocardium measured by ultrasonic technique. Scandinavian Journal of Clinical Laboratory Investigation 24, 361-371 (1969). 2. CALVA, E., MUJICA, A., NUNEZ, R., AOKI, K., BISTENI, A. & SODI-PALLARES, D. Mitochondrial biochemical changes and glucose-KCl-insulin solution in cardiac infarction. American Journal of Physiology 221, 7 l-76 (1966). 3. Cox, J. L., PASS, H. I., WECHSLER, A. S., OLDHAM, H. N. & SABISTON, D. C. Coronary collateral blood flow in acute myocardial infarction. Journal of Thoracic and Cardiovascular Surgery 69, 117-125 (1975). 4. FISHER, V. J., MARTINO, R. A., HARRIS, R. S. & KAVALER, F. Coronary flow as an independent determinant of myocardial contractile force. American Journal of Physioloa271, 1127-1133 (1969). * Request for reprints.

DEFECT

5.

HERDSON,

changes (1969). 6.

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10. 11. 12. 13.

14.

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17.

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20.

MITOCHONDRIAL

P. B., KALTENBACH, in dog myocardium

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TRANSFER

COMPLEX

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615

J. P. &JENNINGS, R. B. Fine structural and biochemical American Journal of Patholog 57, 539.-557 during autolysis.

R. B., BAUM, J. H. & HERDSON, P. B. Fine structural changes in myocardial injury. Arch&s of Pathology 79, 135-l-t3 (1965). JENNINGS, R. B. & GANOTE, C. E. Structural changes in myocardium during acutc ischemia. Circulation Research 34-35 (Supplement IIIj 156-l 72 (1974-j. JENNINGS, R. B. & GANOTE, C. E. Mitochondrial structure and function in acutr myocardial ischemic injury. Circulation Research 38, (Supplement I), 80-91 (1976). JENNINGS, R. B., HERDSON, P. B. & SOMMERS, H. M. Structural and functional abnormalities in mitochondria isolated from ischemic dog myocardium. Luborotory investigation 20, 548-557 ( 1969). JENNINGS, R. B., KALTENBACH, J. P. & SMETHERS, G. W. Enzymatic changes in acutt myocardial ischemic injury. Archives of Pathology 64, 1 O-l 6 ( 1957). JENNINGS, R. B., KALTENBACH, J. P. & SOMMERS, H. M. Mitochondrial metabolism in ischemic injury. Archives of Patholop 84, 15-19 (1967). JENNINGS, R. B., SOMMERS, H. M., HERDSON, P. B. & KALTENBA~H, J. P. Ischemic injury of myocardium. Annals of the New York Academy of Sciences 156, 61-78 ( 196!, 1. JENNINGS, R. B., SOMMERS, H. M., SMYTH, G. :\., FLASK, H. A. & LINN, H. Myocardial necrosis induced by temporary occlusion of a coronary artery in the doa. .4rchiw o1/’ Pathology 70, 68-78 (1960). KAHLES, A., GORING, G. G., NORDBECK, H., PREUSSE, C. J. & SPIEKERMANN. P. (;. Functional behavior of isolated heart mitochondria after in situ ischemia. Ba.\ic Research in Cardiology 72, 563-57-I (1977). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J, Protein mrasurement with the folin reagent. Journal ofBiological Chemittry 193, 265-275 (1951). PRINZMETAL, M., BERGMAN, H. C., KRUGER, H. E., SCHWARTZ, L. L., SIMKIN, B. & SOBIN. S. S. Studies on the coronary circulation. III. Collateral circulation of beating human and dog hearts with coronary occlusion. American Heart Journal 35, 689-717 (1918). ROUSLIN, W. Oxygen dependence of promitochondrial and cytoplasmic protein synthesis in the formation of electron transfer complexes III and IV in adapting bakers’ yeast. Archioes of Biochemistry and Biophysics 168, 685-692 ( 1975). SORDAHL, L. A., JOHNSON, C., BLAILOCK, Z. R. & S~HWAR,IZ, A. The mitochondrion. In Methods in Pharmacology, Schwartz, A., Ed. Vol. 1, Chap. 8, pp. 247.-286. New York : Appleton-Century-Crofts ( 197 1). THEROUX, P., FRANKLIN, D., Ross, J., JR. & KEMPER, W. S. Regional myocardial function during acute coronary artery occlusion and its modification by pharmacologic agents in the dog. Circulation Research 35, 896-908 ( 1974). WACHSTEIN, M. & MEISEL, E. Succinic dehydrogenase activity in myocardial infarction and in induced myocardial necrosis. American Journal of PatholoD 31, 353-365 (1955). JENNINGS,

ischemic

7.

IN