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Effects of reperfusion on myocardial wall thickness, oxidative phosphorylation, and Ca2+ metabolism following total and partial myocardial ischemia
Effects of reperfusion on myocardial wall thickness, oxidative phosphorylation, and Ca*+ metabolism following total and partial myocardial ischemia Coronary artery reperfusion following acute myocardial ischemia may salvage ischemic jeopardized cells. We studied the effects of early brief reperfusion on totally ischemic and on partially ischemic myocardium of open-chest pigs. In 10 animals, coronary flow was reduced to 0% for 30 minutes and was followed by 10 minutes reperfusion (group A). In another 10 animals, coronary flow was reduced to 25% of the baseline value for 30 minutes followed by 10 minutes of reperfusion (group 6). In another eight animals coronary flow was reduced to 25% of the baseline value for 60 minutes and followed by 10 minutes of reperfusion (group C). Results showed that a brief lO-minute period of reperfusion of ischemic myocardium after total occlusion caused abnormal diastolic wall thickening with only partial return of systolic wall thickening. However, reperfusion of ischemic myocardium after partial occlusion, whether 30 or 60 minutes, caused little diastolic wall thickening and a partial return of systolic thickening. A marked elevatton of myocardial Ca*+, a decrease in mitochondrial adenosine triphosphate (ATP) production and cellular ATP concentration, and a reduction in the rate of Ca” uptake by sarcoplasmic reticulum vesicles occurred in the totally ischemic myocardium but not in the partially ischemic myocardlum. These results demonstrate that reperfusion of ischemic myocardium after 1 hour of coronary flow reduction to 25% of baseline is less damaging than reperfusion after a 30-minute total coronary occlusion, and suggest that preexisting states affecting coronary flow need to be evaluated in assessing the outcome of reperfusion. (AM HEART J 1986;112:1238.)
Chun Fu Peng, Ph.D., J. Lynn Davis, M.D., Marvin and Karl D. Straub, M.D., Ph.D. Little Rock, Ark.
Current interest in the use of thrombolytic agents in acute myocardial infarction’ and in reflow after coronary spasm and angioplasty have focused attention on the functional and metabolic aspects of reperfusion of ischemic myocardium. Although effects of reperfusion on regional myocardial performance,2*3 biochemical function? 5 and ultrastructural integritF7 in the totally ischemic model have been extensively studied, little is known of the effects of reperfusion on partially ischemic myocardium. A few reports have studied segmental
From Medical Research Service, John L. McClelan Hospital and the Departments of Biochemistry and of Arkansas for Medical Sciences. This work was supported by Veterans Administration and by a grant from the American Heart Association, Received accepted
for publication May 5, 1986.
Reprint requests: John L. McClelan Rock, AR 72205.
1238
Dec.
2, 1985;
revision
Chun Fu Peng, Ph.D., Medical Memorial Veterans Hospital,
Memorial Medicine,
Veterans University
Merit Review funds Arkansas affiliate. received
Apr.
1, 1986;
Research Service (151), 4300 W. 7th St., Little
L. Murphy,
M.D.,
mechanical functions,’ and regional lactate balancelO in ischemic myocardium after subtotal reduction of coronary arterial flow. The effects of reperfusion on biochemical aspects including oxidative metabolism, cellular Ca2+ content, and high energy compound levels under these conditions have not been well evaluated. This study was designed to investigate the effects of reperfusion on wall motion, oxidative phosphorylation, and Ca2+ metabolism in ischemic myocardium that was produced by partial coronary artery occlusion and compare these results to that of a totally ischemic reperfused myocardium. METHODS Experimental protocol. The pig was chosen as the experimental model for this study because its major coronary distribution, collateral circulation, and blood supply of the conduction system are similar to those of man and because of our experience with this model.“,” Pigs weighing 45 to 50 kg were fasted overnight prior to experiments and were anesthetized intravenously with 6% phenobarbital. Respiration was maintained with a
Volume
112
Number
6
Harvard pump with room air supplemented with 100% 0, to maintain an arterial oxygen tension above 100 mm Hg. A midline sternal splitting thoracotomy was performed and the heart was suspended in a pericardial cradle, exposing the distribution of the left anterior descending coronary artery. Myocardium was made ischemic by reversibly occluding the left anterior descending coronary artery at the site of the first or second diagonal branch with a vascular occluder (In Vivo Metric Systems, Healdsburg, Calif.). Coronary flow of the left anterior descending artery was assessed by an electromagnetic flow probe (Carolina Medical Electronics, Inc., King, N.C.) before, during, and after total and partial occlusion. The flow probe was calibrated before use. The flow was constantly recorded every 10 seconds during the occlusion and reperfusion period. The average of these recordings was used as indicative of tlow during the occlusion and perfusion period. In 10 animals, the coronary artery was completely occluded by reducing coronary flow 100% (group A) for 30 minutes followed by 10 minutes of reperfusion. In the other 18 pigs, the coronary artery was partially occluded by reducing coronary flow to 25% of baseline value. Ten were occluded for 30 minutes (group B) and eight were occluded for 60 minutes (group C), followed by 10 minutes of reperfusion in all animals. If ventricular fibrillation developed during the occlusion or reperfusion period, electrical countershock was used with the defibrillator set at 20 to 50 Wsec. The ECG (lead II) and arterial pressure through a femoral artery cannula were continuously monitored on a physiologic recorder and continuously tape recorded. Immediately prior to killing of each animal, the coronary artery was again occluded at the original site momentarily (less than 30 seconds) as a rapid intravenous bolus of 2.5 ml of 10% alphazurine 2-G blue dye (patent blue) was given. This dye was used to separate perfused from nonperfused myocardium’3 and has been shown to have no adverse effect on mitochondrial energy production or sarcoplasmic reticulum Ca*+ transport function4 Immediately after dye injection, the heart was removed and taken to a cold room where samples were excised from nonischemic, ischemic, and/or ischemic-reperfused areas of the left ventricle, with the surface anatomy and dye stain as guidelines. Echocardiographic methods. Left ventricular wall thickness was measured serially before and at 5-minute intervals after occlusion and reperfusion by placing a 5.0 MHz ultrasound transducer directly over the involved left ventricle. The transducer was coupled to an Echoline 20A ultrasonoscope (Smith, Kline & French, Philadelphia, Pa.) and data were recorded on a strip-chart recorder (Honeywell Test Instruments, Denver, Coio.). To avoid pressure artifact and distortion of the heart wall, the transducer washeld lightly by a finger cot in contact with the epicardial surface over the center of the ischemicand nonischemicareaof left ventricle, with the coronary artery surface anatomy as a landmark. This finger-held method resulted in reproducible echocardiographic data by assuring perpendicular orientation of the transducer with
of reperfusion
Effects
on ischemic
1239
Tr,-\‘vc:dium
Table I. Coronary flow determination beftjre and during occlusion and after reperfusion* ~..._... -.. cI ._ __---\ileragP /loll: Baseline flow before occlusion (mUmin
Groups
GroupA
during wperfuswn imllmin! --____.
19 t_ 4
I)
5’7.I iz
22 r 6
(O”, b+ 5.6 f (1.7
(;wo”~ iI 5:i + x f 240 I’, it
h = 10)
GroupB (n = 10) GroupC (n = 8)
Average ]fou during occ~lusio/: (ml/m/w _----_.._ .-
20 It 7
(25.3’. it 5.: ’ I.(i {2.5.:,‘!I! -.-
50 ‘. 1:i I:!:‘, )t
*vaiuesaremean-+SD. ‘rNumhers
shown in parentheses
are percent 01
hri:ne
v&w
respect to the epicardial surface.’ Wall thickness was measuredin millimeters during maximal systolic thickening and at end diastole. Each measurementrepresented the mean of five consecutive cardiac cycles Biochemical procedures. Tissue samples from nonischemic and ischemic-reperfused left ventricle were homogenized with a Polytron PTlO-135 (Brinkmann Instruments Inc., Westbury, N.Y.) at a setting of 7 for 15 secondsat &second intervals in a medium containing 250 mmol/L sucrose and 20 mmol/L Tris buffer (pH 7.5). Mitochondria were isolated by a method previously described.* After final washing, mitoch
1240
Peng et al.
American
II. Left ventricular wall thickness recorded by echocardiography during ischemic and ischemic-reperfused periods* Table
30 min Baseline Group
A
Systolic thickness
15.0 + 3.0
Diastolic thickness
5.7 + 1.6
S/D ratio
2.6 + 0.5
Group B Systolic thickness
15.0 + 2.5
Diastolic thickness
5.4 1 2.5
S/D ratio
2.7 2 0.4
Baseline Group
occlusion
10 min. reperfusion
3.9 + 1.0 (26%)t 3.8 $- 0.6 (67”G)t 1.0 * 0.1 (40%H
11.0 + 3.2 ( 72%)t 10.4 + 2.4 (182% )t 1.1 k 0.3 ( 46%)t
8.0 t 3.8 (539o)t 5.2 t 2.2 (96So)t 1.5 k 0.6 (56% )‘I’
11.0 + 3.6 ( 739GI.t 6.6 k 2.5 (122% )t 1.7 -+ 0.4 ( 63%)t
60 min occlusion
10 min reperfusion
C
Systolic thickness
15.0 + I.1
Diastolic thickness
5.8 _t 1.2
S/D ratio
2.6 +- 0.4
8.7 r 2.6 (60%)t 5.6 k 1.8 (96%)t 1.5 + 0.3 (65% )t
10.6 t 1.9 ( 72%)t 6.6 f 1.8 (113%)t 1.6 + 0.3 ( 70%)t
*Values are mean 2 SD. TNumbers
shown
in parentheses
are percent
of baseline
values.
were suspendedin buffered 600 mmol/L KC1 and recentrifuged at 35,000 x g for 30 minutes. The pellets were then washed and suspendedin 20 mmol/L Tris-maleate (pH 7.0) and 160mmol/L KC1 or 160mmol/L NaCl with a final protein concentration of 4 mg per milliliter. Protein concentration of sarcoplasmic reticulum vesicles was determined by Lowry’s method.lgThe rates of Ca2+uptake by isolated sarcoplasmicreticulum vesicleswas measured in a spectrophotometer (Aminco DW2, American Instrument Company, Silver Spring, Md.) with murexide as a CaZ+indicator.** The incubation medium contained 160 mmol/L KCl, 20 mmol/L Tris-maleate buffer (pH 7.0), 10 mmol/L MgSO,, 0.133 mmol/L CaCl,, 0.33 mmol/L oxalate, and 0.3 mmol/L murexide. Sarcoplasmic reticulum vesicles,Ca2+,and adenosinetriphosphate were added to the reaction mixture (final volume of 3 ml) to initite Ca*+ accumulation at 23” C. Dual wavelength measurements were performed at a wavelength pair of 542 and 507 nm. Tissue adenosine triphosphate (ATP) content was determined on duplicate tissue samples obtained by plunge biopsy with a renal biopsy needle (Travenol Labs, Inc., Deerfield, Ill.) just before killing the animal. The needle was cooled in liquid nitrogen, plunged into the heart to obtain tissue,and immediately returned to a vial containing liquid nitrogen. Tissue for ATP determination wasthus removed and frozen within 3 to 5 seconds.These
December 1986 Heart Journal
sampleswere extracted in boiling water and ATP content was determined by the firefly luciferase reaction.2” RESULTS Coronary
flow and left ventricular
wail motion.
Table
I shows the average coronary flow in each group (mean + SD). The average flow was I9 + 4 mI/min for group A, 22 +- 6 ml/min for group B, and 20 +- 7 ml/min for group C prior to coronary occlusion. After coronary occlusion, blood flow was recorded as 0 ml/min for group A, 5.6 + 0.7 ml/min for group B, and 5.1 4 1.6 ml/min for group C during the occlusion period. Hence blood flow was 100% reduced in group A and approximately 75 % reduced in group B and C. After 10 minutes’ reperfusion, coronary blood flow was 300% of the baseline (57 + 14 ml/min) for group A, 240% of the baseline (53 + 8 ml/min) for group B and 250% of the baseline (50 4 13 ml/min) for group C; hence a hyperemic coronary flow response was observed in all three groups. Table II demonstrates left ventricular wall thickness measurements during ischemic and reperfused periods. Systolic thickness decreased from 15 mm before occlusion to approximately 4 mm after 30 minutes of occlusion in the totally ischemic myocardium (group A), to 8 mm after 30 minutes of partial coronary occlusion (group B), and to 8.7 mm after 60 minutes of partial coronary occlusion (Group C). A small decrease (group A) or little change (groups B and C) in diastolic wall thickness during the ischemic period compared to the baseline value was observed. Reperfusion after 30 minutes of a total coronary occlusion (group A) resulted in a systolic wall thickness of 11 mm (72 % of baseline) and a marked increase in diastolic wall thickness from 5.7 to 10.4 mm (182% of baseline). Reperfusion after 30 minutes of partial coronary occlusion (group B) resulted in a partial return of systolic thickness of 11 mm (73% of baseline) and a slight increase in diastolic thickness from 5.4 to 6.6 mm (122% of baseline). Reperfusion after 60 minutes of partial coronary occlusion (group C) resulted in a systolic thickness of 10.6 mm (72% of baseline) and a minimal increase in diastolic thickness from 5.8 to 6.6 mm (113% of baseline). An abnormality of ventricular wall motion can also be observed in the alteration of the systolic thickness/diastolic thickness ratio (S/D ratio). The S/D ratio for group A was 2.6 before occlusion, 1.0 after 30 minutes of occlusion, and 1.1 after 10 minutes of reperfusion. The S/D ratio for group B was 2.7 before occlusion, 1.5 after 30 minutes of occlusion, and 1.7 after 10 minutes of reperfusion. The S/D ratio for group C was 2.6 before occlusion,
Volume Number
Table
112 6
Effects of reperfusion
on ischemic
m.vl~cnrdium
1241
III. Mitochondrial oxidative phosphorylation in nonischemicand ischemic-reperfusedmyocardium” .~_..--.-~ ...___._-~--___-._~-. _ -.-Grwps
NO.
Group A Nonischemic
(10)
lschemir-reperfused
State 3 respiration Olmglmin)
(nanoatoms
(10)
Group H Nonischemic
(10)
I<<-.pimtlc-?
AUPIO
196 zk 13 p < 0.001 107 -+ 38 (55T)t
3.0 t 0.3
?~I
2.7 2 0.4 (9OCc )t
,qr
188 -+ 19
3.0 r 0.2
p < 0.005 Ischemic-reperfused Group c Nonischemic
(10)
159 i 19 (8SPC~ )t
2.9 k 0.3 197’f tt
( 8)
207 k 20
2.9 -+ 0.2
-_____ ADP = adenoside ‘Values are mean
diphosphate. + SD. +Numbers
2.8 iY 0.1 (96°C )t
161 zk 17 (78@r’)t
( 8)
$
‘$1
., 1 < ti.001 ; ,-,’ :. i < 0.01
p < 0.005 Ischemic-reperfused
t.,ri iriii ratio _ ._._._ - - -...----.--
_. ~ (,;;; -/ I-’ r:
:
,a < 0.01 _ / t ! (8X’, It
--__I_ shown
in parentheses
are percent
of control
1.5 after 60 minutes of occlusion, and 1.6 after 10 minutes of reperfusion. The S/D ratio observed in groups B and C indicates a less severe abnormality of ventricular wall motion than in group A. Biochemical results. Table III shows the results of oxidative phosphorylation by mitochondria in the three groups of animals. Mitochondria from group A showed an ATP production rate of 55% of the control (107 vs 196 nanoatoms O/mg/min) as determined from the state 3 respiration. Mitochondria from group B showed an ATP production rate of 85% of the control (159 vs 188 nanoatoms O/mg/ min), and mitochondria from group C showed an ATP production rate of 78% of the control (161 vs 207 nanoatoms O/mg/min). Mitochondria from the partially ischemic myocardium thus have higher rates of ATP production than the totally ischemic myocardium. There was no significant difference in ADP/O ratio among the three groups. The respiratory control ratio in group A showed marked differences between the nonischemic and ischemic myocardium, suggesting that those mitochondria from the totally ischemic myocardium suffered severe damage. Fig. 1 shows the results of determination of tissue Ca2+ content in the three groups of animals. In group A, the ischemic myocardial Ca*+ increased to 340% of the control (4.4 for the ischemic vs 1.3 rcmol/gm wet tissue weight for the nonischemic myocardium). A significant elevation of myocardial Ca2+ (p < 0.001) in the totally ischemic myocardium was observed. In groups B and C, however, only a nonsignificant elevation of tissue Ca2’ was observed
value.
in the partially ischemic myocardium when compared to the control. Fig. 2 demonstrates the ATP level in the ischemic myocardium compared to the nonischemic myocardium. The ATP level of ischemic myocardium was 42 +- 16% of baseline in group A, 72 + 12% of baseline in group B, and 68 + 14% of baseline in group C (p < 0.001 when group A is compared with group B or C), respectively. The return of ATP content after 10 minutes of reperfusion was greater in the partially ischemic myocardium than in the totally ischemic myocardium. Fig. 3 shows the rate of Ca2+ uptake by sarcoplasmic reticulum vesicles in the ischemic myocardium compared to the nonischemic myocardium. The rate of Ca2+ uptake by sarcoplasmic reticulum vesicles was 57 r 15% of baseline in group A, 85 r 14% of baseline in group B, and 86 + 19%> of baseline in group C @ < 0.001 when group A was compared to group B or C), respectively. DISCUSSION
In humans, a total coronary occlusion may predict a less favorable result from reperfusion than a partial occlusion.22~2” It has been postulated that total coronary occlusion, in some instances, may be intermittentz4 and therefore more favorable for reperfusion. The presence of coronary collaterals when associated with complete coronary occlusion may set the stage for better results of reperfusion.2” Therefore, it is pertinent to investigate the effect of reperfusion after partial and total coronary occlusion in the experimental animal.
December
1242
Peng et al
American
Heart
1986 Journal
P
--
70
..
60
--
50
--
T
40.. 30
.-
20
.-
I o--
Group
Group
A
8
Group
C
Group
content in the nonischemic and ischemicreperfused myocardium. Group A, p < 0.001 between the nonischemic and the ischemic-reperfused myocardium; groups B and C, p < 0.05 between the nonischemic and ischemic-reperfused myocardium. Fig.
1. Ca*+
T
P (0.001
f
roup A
--
Group
i-
: B
roup
C
2. ATP level in ischemic-reperfused myocardium. The ATP level is expressed as a percent of the control value (from the nonischemic myocardium).
Fig.
Banka et al9 reported that reduction in coronary flow to 50% of the control level resulted in regional contraction abnormalities that worsened as flow was further reduced. Waters et al.‘O demonstrated that flow reduction to 40% of the control level produced no significant changes in regional lactate balance or segmental mechanical function. Further reductions in flow, however, caused progressive decreases in both lactate balance and segmental wall shortening
A
Fig. 3. Rate of Ca*+ vesicles isolated from The rate is expressed (from the nonischemic
Group
0
Group
C
uptake by sarcoplasmic reticulum ischemic-reperfused myocardium. as a percent of the control value myocardium).
during ejection.‘O Wyatt et al? observed that segmental wall function was preserved when flow was reduced to 30 % to 70 % of control levels but deteriorated rapidly with further small decreases in coronary flow. Our study also demonstrated that changes in wall thickening and motion occur when coronary flow is significantly reduced to 25% of baseline. Our results showed striking differences between total and partial occlusion models in the amount of Ca2+ accumulated by the ischemic myocardium. The extent of intracellular Ca2+ deposition may relate to the magnitude of thickening in the ischemic-reperfused myocardium. 4 Reperfusion after 1 hour of partial coronary flow results in less Ca2+ accumulation and less diastolic wall thickening than reperfusion after 30 minutes of total coronary occlusion. Maintaining a reduced flow may affect the outcome of reperfusion by preventing the influx of Ca2+ into the ischemic zone. A significant improvement of mechanical recovery and increased content of highenergy phosphate compound has been reported when ischemic rabbit interventricular septa was reperfused with blood containing reduced amounts of Ca2+.2’ It is known that mitochondria accumulate Ca2+ when cytoplasmic Ca2+ is elevated, resulting in the inhibition of ADP phosphorylation.26 Thus, the magnitude of Ca2+ accumulation at an early stage of reperfusion may be an important factor in determining the ultimate functional and metabolic status of the ischemic-reperfused myocardium.
Volume Number
112 6
Effects of brief reperfusion in the ischemic myocardium may predict the results of longer periods of reperfusion. A significantly increased regional diastolic wall thickness in the canine left ventricle was observed after 60 minutes of reperfusion following 60 minutes of total coronary occlusionz7 Increases in regional wall thickness and Ca2+ accumulation were also observed in a 30-minute totally ischemic pig ventricle after 2 hours of reperfusion4 Several reports have demonstrated that many days or even weeks may be required for the return of ultrastructural, functional, and biochemical characteristics to normal after 15 minutes of total coronary occlusion.28,“g This phenomenon has been described as stunned myocardium.30 Stunned myocardium, which implies potential recovery, may be irreversibly injured if there is marked diastolic wall thickness and high concentration of calcium during early reperfusion. Haendchen et a1.27recently showed that increased end-diastolic wall thickness early after reperfusion correlated directly with the transmural extent of necrosis. The ischemic segments that exhibited more than 25% increase in end-diastolic wall thickness early after reflow did not show recovery of function after 7 days of reperfusion.27 In contrast, segments with less than a 25% increase in end-diastolic wall thickness during early reperfusion exhibited at least partial functional recovery after 7 days. Our results suggest that irreversible injury occurred in ischemic myocardium that was produced by 30 minutes of total coronary artery occlusion but not by 30 minutes or 1 hour of partial occlusion, since diastolic wall thickness increased 82% in the former and 13% to 22 % in the latter. This is further illustrated by the S/D ratio, which showed ineffective wall motion in the totally occluded reperfused group compared to the partially occluded groups. Clinical relevance of our results must be interpreted with caution. Our measurements were taken from the central ischemic area, presumably the site of the worst injury. The time of reperfusion was short. Despite these limitations, our data emphasize that in the evaluation of thrombolysis and angioplasty in the acutely ischemic period, partial flow states are likely to result in a better outcome than those associated with total coronary artery occlusion. In conclusion, experimental data in pigs with acute coronary artery occlusion followed by a brief reperfusion indicate some noteworthy differences between the totally ischemic and partially ischemic myocardium. A significantly increased regional enddiastolic wall thickness and myocardial calcium accumulation occurred in the former but not in the
Effects
of reperfusion
on ischmic
rn\*ltr
w-dirts
1243
latter after a brief period of early reperfusion. These wall thickness changes and calcium deposition may be associated with irreversibly damaged myocardiurn, and therefore may be useful as an index to predict myocardial salvage. Partial coronary occlusion prolongs the time during which reperfusion may be beneficial. While it may seem obvious that partial occlusion causes less damage than total occlusion, the evaluation of this damage as a function of time and blood flow should be determined. It is not yet clear that the damage in partial occlusion is by the same biochemical and functional sequence as in the total occlusion model. REFERENCES I.
2.
3.
4.
5.
6. 7. 8.
9.
10.
11,
12.
13.
14.
15.
Markis JE, Malagold M, Parker A, Silvertmm KJ, Barry WI-I, Als AV, Paulin S, Grossman W, Braunwald E: Myocardial salvage after intracoronary thrombolysis with streptokinasr in acute myocardial infarction. N Engl ,I Med 1981;305:777. Hevndrickx GR. Millard RW. McRitchie Rd. Maroko PR. Vainer SF: Regional myocardial functional and electrophyciological alterations after brief coronary arterv occlusion in conscious dogs. J Clin Invest 1975;56:978. Apstein CS, Mueller M, Hood WB
December
1244
16. 17. 18. 19. 20.
21. 22.
23.
Peng et al.
phosphorylation. I. Kinetic8 of oxygen utilization. J Biol Chem 1955;217:383. Sparrow MP, Johnstone BM: A rapid-method for extraction of Ca and Mg from tissue. Biochim Biophys Acta 1964; 90:425. Willis JB: Analysis of biological materials by atomic absorption spectroscopy. In Blick D, editor. Methods of biochemical analysis. New York: Interscience, 1963:l. Harigaya S, Schwartz A: Rate of calcium binding and uptake in normal animal and failing human cardiac muscle. Circ Res 1969;25:781. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265. Strehler BL: Adenosine&‘-triphosphate and creatine phosphate: Determinations with luciferase. In Bergmeyer HU, editor. Methods of enzymatic analysis. New York: Academic Press, 1963:559. Shine KI, Doublas AM Low calcium reperfusion of ischemic mvocardium. J Mol Cell Cardiol 1983;15:251. R&rtrop KP, Feit F, Blanke H, Stecy P, Schneider R, Rey M, Horowitz S. Goldman M. Karsch K, Leibnan H. Cohen M, Siegel S, Sanger J, Slate; J, Gorlin R, Fox A, Fagerstrom R, Calhoun F: Effects of intracoronsry streptokinase and intracoronary nitroglycerin infusion on coronary angiographic patterns and mortality in patients with acute myocardial infarction. N Enel J Med 1984:311:1457. Rogers WJ, Hood WP Jr, Mantle JA, Baxley WA, Kirklin JK, Zorn GL, Nath HP: Return of left ventricular function after reperfusion in patients with myocardial infarction: Importance of subtotal stenoses or intact collaterals. Circulation 1984;69:338.
American
Heart
1986 Journal
24. Davies GJ, Chievchia S, Maseri A: Prevention of myocardial infarction by very early treatment with intracoronary streptokinase. N Engl J Med 1984;311:1488. 25. Smalling RW, Fuenter F, Matthews MW, Freund GC, Hicks CH, Reduto LA, Walker WE, Sterling RR, Gould KL: Sustained improvement in left ventricular function and mortality by intracoronary streptokinase administration during evolving myocardial infarction. Circulation 1983;68:131. 26. Peng CJ, Murphy ML, Kane JJ, Straub KD: Alteration in calcium metabolism in mitochondria isolated from ischemic and reperfused myocardium. In: Recent advances in studies on cardiac structure and metabolism. vol 11. Baltimore: University Park Press, 1977:533. 27. Haendchen RV, Corday E, Torres M, Maurer G, Fishbein MC, Meerbaum S: Increased regional end-diastolic wall thickness early after reperfusion: A sign of irreversible damaged myocardium. J Am Co11 Cardiol 1984;3:1444. 28. Deboer LWV, Ingwall JS, Kloner RA, Braunwald E: Prolonged derangements of canine myocardial purine metabolism after a brief coronary artery occlusion not associated with anatomic evidence of necrosis. Proc Nat1 Acad Sci USA 1980;77:5471. 29. Kloner RA, Ellis SG, Lange R, Braunwald E: Studies of experimental coronary artery reperfusion: Effects on infarct size, myocardial function, biochemistry, ultrastructure and microvascular damage. Circulation 1983;68:1-8. 30. Braunwald E, Kloner RA: Editorial: The stunned myocardiurn-prolonged, postischemic ventricular dysfunction. Circulation 1982;66:1146.
Chun Fu Peng, Ph.D., J. Lynn Davis, M.D., Marvin and Karl D. Straub, M.D., Ph.D. Little Rock, Ark.
Current interest in the use of thrombolytic agents in acute myocardial infarction’ and in reflow after coronary spasm and angioplasty have focused attention on the functional and metabolic aspects of reperfusion of ischemic myocardium. Although effects of reperfusion on regional myocardial performance,2*3 biochemical function? 5 and ultrastructural integritF7 in the totally ischemic model have been extensively studied, little is known of the effects of reperfusion on partially ischemic myocardium. A few reports have studied segmental
From Medical Research Service, John L. McClelan Hospital and the Departments of Biochemistry and of Arkansas for Medical Sciences. This work was supported by Veterans Administration and by a grant from the American Heart Association, Received accepted
for publication May 5, 1986.
Reprint requests: John L. McClelan Rock, AR 72205.
1238
Dec.
2, 1985;
revision
Chun Fu Peng, Ph.D., Medical Memorial Veterans Hospital,
Memorial Medicine,
Veterans University
Merit Review funds Arkansas affiliate. received
Apr.
1, 1986;
Research Service (151), 4300 W. 7th St., Little
L. Murphy,
M.D.,
mechanical functions,’ and regional lactate balancelO in ischemic myocardium after subtotal reduction of coronary arterial flow. The effects of reperfusion on biochemical aspects including oxidative metabolism, cellular Ca2+ content, and high energy compound levels under these conditions have not been well evaluated. This study was designed to investigate the effects of reperfusion on wall motion, oxidative phosphorylation, and Ca2+ metabolism in ischemic myocardium that was produced by partial coronary artery occlusion and compare these results to that of a totally ischemic reperfused myocardium. METHODS Experimental protocol. The pig was chosen as the experimental model for this study because its major coronary distribution, collateral circulation, and blood supply of the conduction system are similar to those of man and because of our experience with this model.“,” Pigs weighing 45 to 50 kg were fasted overnight prior to experiments and were anesthetized intravenously with 6% phenobarbital. Respiration was maintained with a
Volume
112
Number
6
Harvard pump with room air supplemented with 100% 0, to maintain an arterial oxygen tension above 100 mm Hg. A midline sternal splitting thoracotomy was performed and the heart was suspended in a pericardial cradle, exposing the distribution of the left anterior descending coronary artery. Myocardium was made ischemic by reversibly occluding the left anterior descending coronary artery at the site of the first or second diagonal branch with a vascular occluder (In Vivo Metric Systems, Healdsburg, Calif.). Coronary flow of the left anterior descending artery was assessed by an electromagnetic flow probe (Carolina Medical Electronics, Inc., King, N.C.) before, during, and after total and partial occlusion. The flow probe was calibrated before use. The flow was constantly recorded every 10 seconds during the occlusion and reperfusion period. The average of these recordings was used as indicative of tlow during the occlusion and perfusion period. In 10 animals, the coronary artery was completely occluded by reducing coronary flow 100% (group A) for 30 minutes followed by 10 minutes of reperfusion. In the other 18 pigs, the coronary artery was partially occluded by reducing coronary flow to 25% of baseline value. Ten were occluded for 30 minutes (group B) and eight were occluded for 60 minutes (group C), followed by 10 minutes of reperfusion in all animals. If ventricular fibrillation developed during the occlusion or reperfusion period, electrical countershock was used with the defibrillator set at 20 to 50 Wsec. The ECG (lead II) and arterial pressure through a femoral artery cannula were continuously monitored on a physiologic recorder and continuously tape recorded. Immediately prior to killing of each animal, the coronary artery was again occluded at the original site momentarily (less than 30 seconds) as a rapid intravenous bolus of 2.5 ml of 10% alphazurine 2-G blue dye (patent blue) was given. This dye was used to separate perfused from nonperfused myocardium’3 and has been shown to have no adverse effect on mitochondrial energy production or sarcoplasmic reticulum Ca*+ transport function4 Immediately after dye injection, the heart was removed and taken to a cold room where samples were excised from nonischemic, ischemic, and/or ischemic-reperfused areas of the left ventricle, with the surface anatomy and dye stain as guidelines. Echocardiographic methods. Left ventricular wall thickness was measured serially before and at 5-minute intervals after occlusion and reperfusion by placing a 5.0 MHz ultrasound transducer directly over the involved left ventricle. The transducer was coupled to an Echoline 20A ultrasonoscope (Smith, Kline & French, Philadelphia, Pa.) and data were recorded on a strip-chart recorder (Honeywell Test Instruments, Denver, Coio.). To avoid pressure artifact and distortion of the heart wall, the transducer washeld lightly by a finger cot in contact with the epicardial surface over the center of the ischemicand nonischemicareaof left ventricle, with the coronary artery surface anatomy as a landmark. This finger-held method resulted in reproducible echocardiographic data by assuring perpendicular orientation of the transducer with
of reperfusion
Effects
on ischemic
1239
Tr,-\‘vc:dium
Table I. Coronary flow determination beftjre and during occlusion and after reperfusion* ~..._... -.. cI ._ __---\ileragP /loll: Baseline flow before occlusion (mUmin
Groups
GroupA
during wperfuswn imllmin! --____.
19 t_ 4
I)
5’7.I iz
22 r 6
(O”, b+ 5.6 f (1.7
(;wo”~ iI 5:i + x f 240 I’, it
h = 10)
GroupB (n = 10) GroupC (n = 8)
Average ]fou during occ~lusio/: (ml/m/w _----_.._ .-
20 It 7
(25.3’. it 5.: ’ I.(i {2.5.:,‘!I! -.-
50 ‘. 1:i I:!:‘, )t
*vaiuesaremean-+SD. ‘rNumhers
shown in parentheses
are percent 01
hri:ne
v&w
respect to the epicardial surface.’ Wall thickness was measuredin millimeters during maximal systolic thickening and at end diastole. Each measurementrepresented the mean of five consecutive cardiac cycles Biochemical procedures. Tissue samples from nonischemic and ischemic-reperfused left ventricle were homogenized with a Polytron PTlO-135 (Brinkmann Instruments Inc., Westbury, N.Y.) at a setting of 7 for 15 secondsat &second intervals in a medium containing 250 mmol/L sucrose and 20 mmol/L Tris buffer (pH 7.5). Mitochondria were isolated by a method previously described.* After final washing, mitoch
1240
Peng et al.
American
II. Left ventricular wall thickness recorded by echocardiography during ischemic and ischemic-reperfused periods* Table
30 min Baseline Group
A
Systolic thickness
15.0 + 3.0
Diastolic thickness
5.7 + 1.6
S/D ratio
2.6 + 0.5
Group B Systolic thickness
15.0 + 2.5
Diastolic thickness
5.4 1 2.5
S/D ratio
2.7 2 0.4
Baseline Group
occlusion
10 min. reperfusion
3.9 + 1.0 (26%)t 3.8 $- 0.6 (67”G)t 1.0 * 0.1 (40%H
11.0 + 3.2 ( 72%)t 10.4 + 2.4 (182% )t 1.1 k 0.3 ( 46%)t
8.0 t 3.8 (539o)t 5.2 t 2.2 (96So)t 1.5 k 0.6 (56% )‘I’
11.0 + 3.6 ( 739GI.t 6.6 k 2.5 (122% )t 1.7 -+ 0.4 ( 63%)t
60 min occlusion
10 min reperfusion
C
Systolic thickness
15.0 + I.1
Diastolic thickness
5.8 _t 1.2
S/D ratio
2.6 +- 0.4
8.7 r 2.6 (60%)t 5.6 k 1.8 (96%)t 1.5 + 0.3 (65% )t
10.6 t 1.9 ( 72%)t 6.6 f 1.8 (113%)t 1.6 + 0.3 ( 70%)t
*Values are mean 2 SD. TNumbers
shown
in parentheses
are percent
of baseline
values.
were suspendedin buffered 600 mmol/L KC1 and recentrifuged at 35,000 x g for 30 minutes. The pellets were then washed and suspendedin 20 mmol/L Tris-maleate (pH 7.0) and 160mmol/L KC1 or 160mmol/L NaCl with a final protein concentration of 4 mg per milliliter. Protein concentration of sarcoplasmic reticulum vesicles was determined by Lowry’s method.lgThe rates of Ca2+uptake by isolated sarcoplasmicreticulum vesicleswas measured in a spectrophotometer (Aminco DW2, American Instrument Company, Silver Spring, Md.) with murexide as a CaZ+indicator.** The incubation medium contained 160 mmol/L KCl, 20 mmol/L Tris-maleate buffer (pH 7.0), 10 mmol/L MgSO,, 0.133 mmol/L CaCl,, 0.33 mmol/L oxalate, and 0.3 mmol/L murexide. Sarcoplasmic reticulum vesicles,Ca2+,and adenosinetriphosphate were added to the reaction mixture (final volume of 3 ml) to initite Ca*+ accumulation at 23” C. Dual wavelength measurements were performed at a wavelength pair of 542 and 507 nm. Tissue adenosine triphosphate (ATP) content was determined on duplicate tissue samples obtained by plunge biopsy with a renal biopsy needle (Travenol Labs, Inc., Deerfield, Ill.) just before killing the animal. The needle was cooled in liquid nitrogen, plunged into the heart to obtain tissue,and immediately returned to a vial containing liquid nitrogen. Tissue for ATP determination wasthus removed and frozen within 3 to 5 seconds.These
December 1986 Heart Journal
sampleswere extracted in boiling water and ATP content was determined by the firefly luciferase reaction.2” RESULTS Coronary
flow and left ventricular
wail motion.
Table
I shows the average coronary flow in each group (mean + SD). The average flow was I9 + 4 mI/min for group A, 22 +- 6 ml/min for group B, and 20 +- 7 ml/min for group C prior to coronary occlusion. After coronary occlusion, blood flow was recorded as 0 ml/min for group A, 5.6 + 0.7 ml/min for group B, and 5.1 4 1.6 ml/min for group C during the occlusion period. Hence blood flow was 100% reduced in group A and approximately 75 % reduced in group B and C. After 10 minutes’ reperfusion, coronary blood flow was 300% of the baseline (57 + 14 ml/min) for group A, 240% of the baseline (53 + 8 ml/min) for group B and 250% of the baseline (50 4 13 ml/min) for group C; hence a hyperemic coronary flow response was observed in all three groups. Table II demonstrates left ventricular wall thickness measurements during ischemic and reperfused periods. Systolic thickness decreased from 15 mm before occlusion to approximately 4 mm after 30 minutes of occlusion in the totally ischemic myocardium (group A), to 8 mm after 30 minutes of partial coronary occlusion (group B), and to 8.7 mm after 60 minutes of partial coronary occlusion (Group C). A small decrease (group A) or little change (groups B and C) in diastolic wall thickness during the ischemic period compared to the baseline value was observed. Reperfusion after 30 minutes of a total coronary occlusion (group A) resulted in a systolic wall thickness of 11 mm (72 % of baseline) and a marked increase in diastolic wall thickness from 5.7 to 10.4 mm (182% of baseline). Reperfusion after 30 minutes of partial coronary occlusion (group B) resulted in a partial return of systolic thickness of 11 mm (73% of baseline) and a slight increase in diastolic thickness from 5.4 to 6.6 mm (122% of baseline). Reperfusion after 60 minutes of partial coronary occlusion (group C) resulted in a systolic thickness of 10.6 mm (72% of baseline) and a minimal increase in diastolic thickness from 5.8 to 6.6 mm (113% of baseline). An abnormality of ventricular wall motion can also be observed in the alteration of the systolic thickness/diastolic thickness ratio (S/D ratio). The S/D ratio for group A was 2.6 before occlusion, 1.0 after 30 minutes of occlusion, and 1.1 after 10 minutes of reperfusion. The S/D ratio for group B was 2.7 before occlusion, 1.5 after 30 minutes of occlusion, and 1.7 after 10 minutes of reperfusion. The S/D ratio for group C was 2.6 before occlusion,
Volume Number
Table
112 6
Effects of reperfusion
on ischemic
m.vl~cnrdium
1241
III. Mitochondrial oxidative phosphorylation in nonischemicand ischemic-reperfusedmyocardium” .~_..--.-~ ...___._-~--___-._~-. _ -.-Grwps
NO.
Group A Nonischemic
(10)
lschemir-reperfused
State 3 respiration Olmglmin)
(nanoatoms
(10)
Group H Nonischemic
(10)
I<<-.pimtlc-?
AUPIO
196 zk 13 p < 0.001 107 -+ 38 (55T)t
3.0 t 0.3
?~I
2.7 2 0.4 (9OCc )t
,qr
188 -+ 19
3.0 r 0.2
p < 0.005 Ischemic-reperfused Group c Nonischemic
(10)
159 i 19 (8SPC~ )t
2.9 k 0.3 197’f tt
( 8)
207 k 20
2.9 -+ 0.2
-_____ ADP = adenoside ‘Values are mean
diphosphate. + SD. +Numbers
2.8 iY 0.1 (96°C )t
161 zk 17 (78@r’)t
( 8)
$
‘$1
., 1 < ti.001 ; ,-,’ :. i < 0.01
p < 0.005 Ischemic-reperfused
t.,ri iriii ratio _ ._._._ - - -...----.--
_. ~ (,;;; -/ I-’ r:
:
,a < 0.01 _ / t ! (8X’, It
--__I_ shown
in parentheses
are percent
of control
1.5 after 60 minutes of occlusion, and 1.6 after 10 minutes of reperfusion. The S/D ratio observed in groups B and C indicates a less severe abnormality of ventricular wall motion than in group A. Biochemical results. Table III shows the results of oxidative phosphorylation by mitochondria in the three groups of animals. Mitochondria from group A showed an ATP production rate of 55% of the control (107 vs 196 nanoatoms O/mg/min) as determined from the state 3 respiration. Mitochondria from group B showed an ATP production rate of 85% of the control (159 vs 188 nanoatoms O/mg/ min), and mitochondria from group C showed an ATP production rate of 78% of the control (161 vs 207 nanoatoms O/mg/min). Mitochondria from the partially ischemic myocardium thus have higher rates of ATP production than the totally ischemic myocardium. There was no significant difference in ADP/O ratio among the three groups. The respiratory control ratio in group A showed marked differences between the nonischemic and ischemic myocardium, suggesting that those mitochondria from the totally ischemic myocardium suffered severe damage. Fig. 1 shows the results of determination of tissue Ca2+ content in the three groups of animals. In group A, the ischemic myocardial Ca*+ increased to 340% of the control (4.4 for the ischemic vs 1.3 rcmol/gm wet tissue weight for the nonischemic myocardium). A significant elevation of myocardial Ca2+ (p < 0.001) in the totally ischemic myocardium was observed. In groups B and C, however, only a nonsignificant elevation of tissue Ca2’ was observed
value.
in the partially ischemic myocardium when compared to the control. Fig. 2 demonstrates the ATP level in the ischemic myocardium compared to the nonischemic myocardium. The ATP level of ischemic myocardium was 42 +- 16% of baseline in group A, 72 + 12% of baseline in group B, and 68 + 14% of baseline in group C (p < 0.001 when group A is compared with group B or C), respectively. The return of ATP content after 10 minutes of reperfusion was greater in the partially ischemic myocardium than in the totally ischemic myocardium. Fig. 3 shows the rate of Ca2+ uptake by sarcoplasmic reticulum vesicles in the ischemic myocardium compared to the nonischemic myocardium. The rate of Ca2+ uptake by sarcoplasmic reticulum vesicles was 57 r 15% of baseline in group A, 85 r 14% of baseline in group B, and 86 + 19%> of baseline in group C @ < 0.001 when group A was compared to group B or C), respectively. DISCUSSION
In humans, a total coronary occlusion may predict a less favorable result from reperfusion than a partial occlusion.22~2” It has been postulated that total coronary occlusion, in some instances, may be intermittentz4 and therefore more favorable for reperfusion. The presence of coronary collaterals when associated with complete coronary occlusion may set the stage for better results of reperfusion.2” Therefore, it is pertinent to investigate the effect of reperfusion after partial and total coronary occlusion in the experimental animal.
December
1242
Peng et al
American
Heart
1986 Journal
P
--
70
..
60
--
50
--
T
40.. 30
.-
20
.-
I o--
Group
Group
A
8
Group
C
Group
content in the nonischemic and ischemicreperfused myocardium. Group A, p < 0.001 between the nonischemic and the ischemic-reperfused myocardium; groups B and C, p < 0.05 between the nonischemic and ischemic-reperfused myocardium. Fig.
1. Ca*+
T
P (0.001
f
roup A
--
Group
i-
: B
roup
C
2. ATP level in ischemic-reperfused myocardium. The ATP level is expressed as a percent of the control value (from the nonischemic myocardium).
Fig.
Banka et al9 reported that reduction in coronary flow to 50% of the control level resulted in regional contraction abnormalities that worsened as flow was further reduced. Waters et al.‘O demonstrated that flow reduction to 40% of the control level produced no significant changes in regional lactate balance or segmental mechanical function. Further reductions in flow, however, caused progressive decreases in both lactate balance and segmental wall shortening
A
Fig. 3. Rate of Ca*+ vesicles isolated from The rate is expressed (from the nonischemic
Group
0
Group
C
uptake by sarcoplasmic reticulum ischemic-reperfused myocardium. as a percent of the control value myocardium).
during ejection.‘O Wyatt et al? observed that segmental wall function was preserved when flow was reduced to 30 % to 70 % of control levels but deteriorated rapidly with further small decreases in coronary flow. Our study also demonstrated that changes in wall thickening and motion occur when coronary flow is significantly reduced to 25% of baseline. Our results showed striking differences between total and partial occlusion models in the amount of Ca2+ accumulated by the ischemic myocardium. The extent of intracellular Ca2+ deposition may relate to the magnitude of thickening in the ischemic-reperfused myocardium. 4 Reperfusion after 1 hour of partial coronary flow results in less Ca2+ accumulation and less diastolic wall thickening than reperfusion after 30 minutes of total coronary occlusion. Maintaining a reduced flow may affect the outcome of reperfusion by preventing the influx of Ca2+ into the ischemic zone. A significant improvement of mechanical recovery and increased content of highenergy phosphate compound has been reported when ischemic rabbit interventricular septa was reperfused with blood containing reduced amounts of Ca2+.2’ It is known that mitochondria accumulate Ca2+ when cytoplasmic Ca2+ is elevated, resulting in the inhibition of ADP phosphorylation.26 Thus, the magnitude of Ca2+ accumulation at an early stage of reperfusion may be an important factor in determining the ultimate functional and metabolic status of the ischemic-reperfused myocardium.
Volume Number
112 6
Effects of brief reperfusion in the ischemic myocardium may predict the results of longer periods of reperfusion. A significantly increased regional diastolic wall thickness in the canine left ventricle was observed after 60 minutes of reperfusion following 60 minutes of total coronary occlusionz7 Increases in regional wall thickness and Ca2+ accumulation were also observed in a 30-minute totally ischemic pig ventricle after 2 hours of reperfusion4 Several reports have demonstrated that many days or even weeks may be required for the return of ultrastructural, functional, and biochemical characteristics to normal after 15 minutes of total coronary occlusion.28,“g This phenomenon has been described as stunned myocardium.30 Stunned myocardium, which implies potential recovery, may be irreversibly injured if there is marked diastolic wall thickness and high concentration of calcium during early reperfusion. Haendchen et a1.27recently showed that increased end-diastolic wall thickness early after reperfusion correlated directly with the transmural extent of necrosis. The ischemic segments that exhibited more than 25% increase in end-diastolic wall thickness early after reflow did not show recovery of function after 7 days of reperfusion.27 In contrast, segments with less than a 25% increase in end-diastolic wall thickness during early reperfusion exhibited at least partial functional recovery after 7 days. Our results suggest that irreversible injury occurred in ischemic myocardium that was produced by 30 minutes of total coronary artery occlusion but not by 30 minutes or 1 hour of partial occlusion, since diastolic wall thickness increased 82% in the former and 13% to 22 % in the latter. This is further illustrated by the S/D ratio, which showed ineffective wall motion in the totally occluded reperfused group compared to the partially occluded groups. Clinical relevance of our results must be interpreted with caution. Our measurements were taken from the central ischemic area, presumably the site of the worst injury. The time of reperfusion was short. Despite these limitations, our data emphasize that in the evaluation of thrombolysis and angioplasty in the acutely ischemic period, partial flow states are likely to result in a better outcome than those associated with total coronary artery occlusion. In conclusion, experimental data in pigs with acute coronary artery occlusion followed by a brief reperfusion indicate some noteworthy differences between the totally ischemic and partially ischemic myocardium. A significantly increased regional enddiastolic wall thickness and myocardial calcium accumulation occurred in the former but not in the
Effects
of reperfusion
on ischmic
rn\*ltr
w-dirts
1243
latter after a brief period of early reperfusion. These wall thickness changes and calcium deposition may be associated with irreversibly damaged myocardiurn, and therefore may be useful as an index to predict myocardial salvage. Partial coronary occlusion prolongs the time during which reperfusion may be beneficial. While it may seem obvious that partial occlusion causes less damage than total occlusion, the evaluation of this damage as a function of time and blood flow should be determined. It is not yet clear that the damage in partial occlusion is by the same biochemical and functional sequence as in the total occlusion model. REFERENCES I.
2.
3.
4.
5.
6. 7. 8.
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Markis JE, Malagold M, Parker A, Silvertmm KJ, Barry WI-I, Als AV, Paulin S, Grossman W, Braunwald E: Myocardial salvage after intracoronary thrombolysis with streptokinasr in acute myocardial infarction. N Engl ,I Med 1981;305:777. Hevndrickx GR. Millard RW. McRitchie Rd. Maroko PR. Vainer SF: Regional myocardial functional and electrophyciological alterations after brief coronary arterv occlusion in conscious dogs. J Clin Invest 1975;56:978. Apstein CS, Mueller M, Hood WB
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phosphorylation. I. Kinetic8 of oxygen utilization. J Biol Chem 1955;217:383. Sparrow MP, Johnstone BM: A rapid-method for extraction of Ca and Mg from tissue. Biochim Biophys Acta 1964; 90:425. Willis JB: Analysis of biological materials by atomic absorption spectroscopy. In Blick D, editor. Methods of biochemical analysis. New York: Interscience, 1963:l. Harigaya S, Schwartz A: Rate of calcium binding and uptake in normal animal and failing human cardiac muscle. Circ Res 1969;25:781. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265. Strehler BL: Adenosine&‘-triphosphate and creatine phosphate: Determinations with luciferase. In Bergmeyer HU, editor. Methods of enzymatic analysis. New York: Academic Press, 1963:559. Shine KI, Doublas AM Low calcium reperfusion of ischemic mvocardium. J Mol Cell Cardiol 1983;15:251. R&rtrop KP, Feit F, Blanke H, Stecy P, Schneider R, Rey M, Horowitz S. Goldman M. Karsch K, Leibnan H. Cohen M, Siegel S, Sanger J, Slate; J, Gorlin R, Fox A, Fagerstrom R, Calhoun F: Effects of intracoronsry streptokinase and intracoronary nitroglycerin infusion on coronary angiographic patterns and mortality in patients with acute myocardial infarction. N Enel J Med 1984:311:1457. Rogers WJ, Hood WP Jr, Mantle JA, Baxley WA, Kirklin JK, Zorn GL, Nath HP: Return of left ventricular function after reperfusion in patients with myocardial infarction: Importance of subtotal stenoses or intact collaterals. Circulation 1984;69:338.
American
Heart
1986 Journal
24. Davies GJ, Chievchia S, Maseri A: Prevention of myocardial infarction by very early treatment with intracoronary streptokinase. N Engl J Med 1984;311:1488. 25. Smalling RW, Fuenter F, Matthews MW, Freund GC, Hicks CH, Reduto LA, Walker WE, Sterling RR, Gould KL: Sustained improvement in left ventricular function and mortality by intracoronary streptokinase administration during evolving myocardial infarction. Circulation 1983;68:131. 26. Peng CJ, Murphy ML, Kane JJ, Straub KD: Alteration in calcium metabolism in mitochondria isolated from ischemic and reperfused myocardium. In: Recent advances in studies on cardiac structure and metabolism. vol 11. Baltimore: University Park Press, 1977:533. 27. Haendchen RV, Corday E, Torres M, Maurer G, Fishbein MC, Meerbaum S: Increased regional end-diastolic wall thickness early after reperfusion: A sign of irreversible damaged myocardium. J Am Co11 Cardiol 1984;3:1444. 28. Deboer LWV, Ingwall JS, Kloner RA, Braunwald E: Prolonged derangements of canine myocardial purine metabolism after a brief coronary artery occlusion not associated with anatomic evidence of necrosis. Proc Nat1 Acad Sci USA 1980;77:5471. 29. Kloner RA, Ellis SG, Lange R, Braunwald E: Studies of experimental coronary artery reperfusion: Effects on infarct size, myocardial function, biochemistry, ultrastructure and microvascular damage. Circulation 1983;68:1-8. 30. Braunwald E, Kloner RA: Editorial: The stunned myocardiurn-prolonged, postischemic ventricular dysfunction. Circulation 1982;66:1146.