Oxygen-Induced Enzyme Release After Irreversible Myocardial Injury Effects of Cyanide itl Perfused Rat Heart.s Charles E. Ganote, MD, Jonathan Worstell, BS. and John P. Kaltenbach, PhD
The effects of 5 mMf potassium cyanide (KC-N) on creatine phosphokinase (CPK) release and cellular morphology were studied. Rat hearts were perfused with substratedeficient media gassed with 02 or N2 (02 medium. N2 medium) at 37 C, and effluent was collected for creatine phosphokinase analysis. Tissue fixation was with glutaraldehyde for light and electron microscopy. Experiments included the follow-ing: a) continuous perfusion with 02- or N2-medium in the presence of KCN: b) 45 or 60 minutes of perfusion with N2-medium followed by 02-medium for 15 or 180 minutes, respectively: c) 45 minutes of perfusion with N2-medium with KCN- added 15 minutes before reoxygenation with 02-medium plus KCN: (4) 60 minutes of N2-medium plus KCN- followed by 02-medium plus KCN- for 180 minutes: d) as a control for irreversible injury. 21 minutes of perfusion with calcium-free 02-medium followed by 2.5 mMI calcium-02-medium ("calcium paradox"). The following results were seen: a) Initial CPK release occurred about 30 minutes later from hearts perfused with 02-medium plus KCN- than from hearts perfused with N2-medium plus KCN. b) Upon reoxygenation after either 45 or 60 minutes of anoxia. hearts had a sudden peak of oxygen-induced CPK release. NMost irreversibly injured cells were massively swollen and had sarcolemmal defects and contraction bands. Reversibly injured cells in the same hearts resembled normal my-ocardium. A previously unrecognized third population of cells is described. These cells were characterized by contraction bands but were not swollen, had intact sarcolemma. and contained both normal and damaged mitochondria with intramatrical calcium accumulation granules. It could not be determined if these cells were rev ersibly injured or in an early stage of irreversible injury. c) KCN- added 15 minutes before reoxygenation of hearts after 45 minutes of anoxia inhibited the sudden peak of oxygen-induced CPK release but not a slow sustained release. Small to moderate numbers of cells in these hearts contained contraction bands. d) After 60 minutes. KC_N completely inhibited both oxygen-induced CPK release and contraction band formation. e) Addition of calcium to calcium-free hearts caused both massive CPK release and contraction band formation. It is concluded that: the beginning of CPK release from oxygenated KCNinhibited hearts requires about 30 minutes longer than from anoxic hearts: KCN- can inhibit both oxy gen-induced CPK release and contraction bands in irreversibly injured rat myocardial cells: sudden contracture of myocardial cells as occurs in the calcium paradox can result in massive CPK release: contraction bands occur in nonswollen cells, hence contraction bands can occur independently of massive cell swelling or membrane rupture. It is postulated that there may be two stages of irreversible myocardial injurn-: a) loss of control of contraction and b) progressive loss of mitochondrial and membrane integrity. (Am J Pathol 84:327-350, 1976) From the Department of Pathohlogz. N rth%ettern 1-nlit -rlitx \ledical sch-l (icO()I 111111i Supp,)rted in part h! (;rant HL-19000 from the National Ilnstitiutet of Health Accepted for publicatmn April 1 19:6 F anote. Dtepartmetrit 4 Pathowloiz. N()rth%%eterT Address reprint requests to Dr (Chalres E. t1ni-ersitx \ledical Scho(l. 303 East Chicaizo A'enue. Chicaco. IL 606i11 327
328
GANOTE ET AL
American Journal of Pathology
STUDIES OF THE PATHOGENESIS of cell injury have shown that brief ischemic or anoxic stress causes cells to pass into a state of reversible injury. The term anoxia is used here to mean a reduction in oxygen tension such that mitochondria can no longer function but the cells remain exposed to constantly renewed extracellular fluid. Ischemia is a reduction of blood flow to an area of tissue to a level below that necessary to meet normal oxygen demand. Total ischemia is absence of arterial flow. During anoxia, mitochondria of reversibly injured cells stop functioning and cells accelerate anaerobic metabolism, with stores of intracellular glycogen being the principal substrate.1'2 With this inefficient metabolic pathwav and depletion of glycogen, adenosine triphosphate (ATP) levels fall,3 and the cells swell,"5 lose potassium,6 and accumulate sodium, water,"5 and perhaps calcium. If blood flow or oxygen is restored, reversiblv injured cells recover.5 However, when anoxia or ischemia is prolonged, there is a transition from reversible to irreversible injury. This transition has been called the "point of no return,"8 and once it has been passed the cell is considered dead. Reoxygenation or reflow of blood to irreversibly injured cells does not affect survival. In studies of nonmyocardial cells 4.5s8-12 the onset of irreversibility has been at or about the time of nonspecific increases in cell membrane permeability. Prior events seem at least potentially reparable.'3 Increased membrane permeabilitv allows influx of calcium into the cells, causing uncoupling of mitochondria and escape of vital intracellular materials. 1'2 The subsequent events constitute necrosis, which is degeneration of cellular components usually aided by release of Iysosomal enzymes.5'1o Anoxic injury to isolated perfused hearts can result in irreversible injurv with changes strikingly similar to those occurring in vivo following ischemic injur of dog myocardium.'5-18 In both experimental models, reversiblv injured myocardial cells show minimal structural changes of cellular swelling, mitochondrial swelling, and loss of normal matrix granules. Reflow of blood or reoxygenation results in rapid functional recovery and survival of these cells.15'18 With continued injury, myocardial cells develop amorphous densities in mitochondrial matrix spaces, clumping of nuclear chromatin, and terminal increases in cell membrane permeability which indicate cell death.18 With either reflow of blood or reoxygenation, changes in irreversibly injured myocardial cells are rapid: heat production increases abruptly,'9 contraction bands form,16 2 cells swell,'9'2' and sodium and calcium accumulate.' Large blebs of edema fluid occur beneath the sarcolemma,15'17'2' and defects in cell membranes are demonstrable bv electron microscopy.8 15 Enzymes, including creatine phosphokinase (CPK),
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are released from the irreversiblv injured cells and mitochondria accumulate calcium in the form of granular dense bodies."156'20.22 In injured myocardium, defects have been shoxN-n in mitochondrial function, calcium metabolism, cell volume control, and membrane permeability. To define the primary cause of cell death, -e have sought the earliest event or events that dictate the onset of irreversibility. In the present study, the effects of cy anide on ex ents occurring after reoxy genation of irreversibly injured, in vitro perfused rat hearts are reported. Cyanide inhibits the acute rapid changes occurring upon reoxy genation. Assuming that irreversible myocardial injury and increased membrane permeability are related events and that cvanide acts as an inhibitor of mitochondrial function,23 27 the data suggest that there are two stages of irreversible myocardial injury. The first stage is perhaps unique to contractile cells and consists of conditions causing uncontrolled hypercontracture upon reoxygenation and rapid increases of cell membrane permeability. The second stage appears to be a more slox lv developing cellular degeneration e-entuating in cell death. Materials and Methods Hearts Xvere obtained from male, specific pathogen-free Sprague-Da.-ley rats wseighing 200 to 400 g that svere fed ad libitutn. Hearts mvere rapidly remox-ed from animals anesthetized by intraperitoneal injection of 40 mg kg sodium pentobarbital iDiabutal. Diamond Laboratories, Inc.. Des Moines. Io va) and immersed in ice-cold perfusion fluid. After dissection, hearts 'sere blotted and sseighed, and the aortas 'vere cannulated on the perfusion apparatus.
Experimental Design All hearts svere perfused for an initial 13-minute period mvith an oxygenated medium. The follosving experiments mvere performed a) Perfusion of hearts mvith substrate-free media containing 3 mMn KCN' gassed 'vith either 953% 02-3% C02 or 953c \-2-5c CO2 for 240 minutes b) Anoxic perfusion for 43 minutes follom -ed by reoxx-genation for either 13 minutes without KCN.- or 73 minutes in the presence of 3 m\M KC\ X hich "-as added 13 minutes before reoxy genation. c Anoxic perfusion for 60 minutes folloNved bx- reox -genation for 120 minutes ssithout KCN or I80 minutes in the presence of 3 m\I KC\ wshich "vas added at the beginning of the anoxic perfusion period. d Perfusion of hearts for 21 minutes Xwith oxygenated calcium-free medium containing glucose and 0. I mn EDTA follomved b\ a similar medium but X ith 2.3 mMn calcium for an additional 13 minutes. e Anoxic perfusion of hearts for 60 minutes. Experimental '-alues are expressed as mean ± SE. where N = 6. except ,-here so noted. At the end of each experiment hearts Nvere fixed bx- retrograde perfusion of glutaraldehyde for morphologic studies. Perfusion Apparatus The perfusion X"as accomplished on a modified double-resen-oir. double-heart. nionrecirculating Langendorf 2s apparatus. The apparatus and techniques used "vere the same
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as previously described 15 Both reservoirs vvere maintained at 37 C in a water bath. Fluid w as pumped through tx-gon tubing to glass condensing tubes. Temperature as controlled by a thermal probe placed in an aortic cannula. The probe, connected to a Yjellomv Springs Instruments (Mlodel 73) temperature controller. maintained the fluid passing through the aortic cannula at 37 ± 0.3 C. Pressure -as recorded on a Grass Model 3 polygraph Perfusion could be rapidly s -itched from either reservoir. In experiments requiring a third medium, the unused reserxoir was emptied, rinsed, and refilled mvith the new medium. Flov- rates vvere individuallb measured b- timed collection of effluent in a graduated cx-linder. After the 13-minute control perfusion period. flow rates averaged 19.6 ± 3.6 ml min ± SD. After 13 minutes of anoxic perfusion. the flov rates averaged 14.9 ± 1.3. Perfusion Fluid
Perfusion fluid for the control period of perfusion "vas a Krebs-Henseleit-bicarbonate medium 2' containing 2.3 m\I calcium and 10 m\1 D-glucose gassed with 93% 02-5% CO2. The medium for anoxic perfusion Xvas equilibrated Xvith 93% N3-3% CO2 and had the same ionic composition except that 10 m\M mannitol replaced glucose. The medium for reoxxgenation also contained 10 m\M mannitol in place of glucose. Five millimolar KCN was added to either the anoxic or reoxygenation medium. Osmolalit! "as 290 to 293 mOsmoles liter, and pH "as 7.3 to 7.4. All media "vere gas equilibrated for at least 43 minutes prior to beginning the experimentt Creatine Phosphokinase Analysis
One- to t%vo-milliliter samples of effluent "vere collected serially in glass or plastic vials and stored in crushed ice until the end of the experiment. Creatine phosphokinase activity -as measured on 0. I-ml aliquots bx- the method of Oliver 3" in a Beckman DU spectrometer using reagents obtained from Boehringer-NMannheim GMBH (\annheim. German-). The results of CPK activity expressed in international units per minute per gram initial heart vveight "ere obtained from the follom-ing formula: IU min g=
CPK acti'-it'-
IU liter) X flomv (ml
min) X
1000 ml
Initial heart %veight (g)
Total CPK release "as obtained bx- meighing enzx-me release curves cut from standard graph paper
Morphologic Studies At the end of each experiment, hearts "vere fixed bx- perfusion of I % glutaraldehx-de in m(odified Tvrode's solution.3' Folloving fixation, the apex and atria were discarded. Four equally spaced horizontal sections of the heart "vere taken and processed for light microscopy. In addition, 0 3-mm sections v"ere cut from tissue adjacent to the light microscope sections and "vere placed in glutaraldehx-de for an additional 1 hour. Following postfixation in I % osmium tetroxide. one group of blocks "vas soaked in aqueous uranyl acetate. a second group "as left unstained Follo' ing dehx-dration and treatment Xvith propylene oxide, blocks "vere flat-embedded in Epon S12 (Shell Chemical Co., Nemv York. N.Y.. Semi-thin sections were stained with toluidine blue for light microscopy. and areas were selected for thin sectioning using diamond knives. Thin sections of tissue stained en bloc Xvere mounted on plain copper grids and examined directly. Tissue not stained before
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embedding s-as stained wsith 7,.7` aqueous uran\ l acetate prior to exaamination in an Hitachi HU-12 electron microscope. Estimation of Cell Damage
U-sing light microscopy of hematoxylin- and eosin-stained sectio)s. longitudinallk oriented cells w ere classified into four groups on the criteria of being nons\s ollen or s\s ollen. and by the presence or absence or contraction bands. The percent of each cell ty pe ws-as estimated b\- direct counts of 1000 successix-e cells from each heart.
Results Gross Observations
The conditions used in the present study are similar to those reported previously.'5 with the exception that a small poly ethylene catheter was placed through the tip of the left ventricle to prev-ent dilation of the heart due to intraventricular pressure and, w-e hoped, to allow perfusion of the subendocardium which. in prev ious experiments, had not been perfused. During perfusion, when the hearts no longer underwent spontaneous contractions, a v-ariable but usually significant flows ensued from the catheter, demonstrating incompetence of the aortic \-alv-e. In anoxic hearts, with their high perfusion flo- rates. much of the flo\- came through the catheter. Thus. coronary- flosw rates could not be accuratel\measured. Hearts sectioned at the completion of the experiments shoswed that a variable but consistently present area of the subendocardium swas pink and appeared nonperfused. as w-as the case in uncatheterized hearts. This area was not included in samples. In accordance with previous obser-ations. hearts releasing CPK or shoswing morphologic e-idence of irrev-ersible injury also changed from a reddish to a pale. opaque appearance. Enzyme Release Studies Cyanide-inhibited Controls
To show that 5 m\M cyanide is a biologically effective inhibitor of rat hearts in Litro. perfusion was conducted -ith either oxy genated or nitrogen-equilibrated, substrate-deficient media for 240 minutes. Released creatine phosphokinase activ-ity w-as an indicator of cellular injury. Results are shown in Text-figure 1. Enz\-me release occurred under both aerobic and anaerobic conditions, but release of CPK began later from oxy genated hearts than from anoxic hearts. The results show that cy-anide is an effectixe metabolic inhibitor under the experimental conditions but that its action is delayed, it did not significantly inhibit CPK acti-ity released
332
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GANOTE ET AL
3 3
CP
r-
.E
ID
0D en 0 0 6,
a,
C)
Minutes
TEXT-1I-Rt-E 1-\lxocardial eniz'me release dtiringz anoxic or ox%genated perfusion in the presenlCe of 5 m\l c\ anide Enzsme release from oxwzenated hearts solid line \-.as dela\ed compared to that from anoxic hearts dashled linie The difference in enzx me release patterns is thought to be due to the tinme required to effect metablic inhibition bh cx anide ;Double asterisk. P < 0.01. sinyle asterisk. P < 0 05
from injured hearts, and it did not impart superimposed injury beyond that caused by anoxia alone. Effect of Cyanide on Reoxygenation
After 43 or 60 minutes of substrate-free anoxic perfusion, oxy-gen induced the release of CPK from injured hearts (Text-figures 2 and :3). Cyanide added 13 minutes before reox}-genation reduced the initial peak of CPK release but did not prevent initiation of a slo-ver release of this enzyme bx- oxy-gen (Text-figure 2). The cyanide present in anoxic media perfused for 60 minutes totallx- inhibited oxy-gen-induced CPK release. Subsequently. a slo-wly rising CPK release followed a pattern similar to that seen X ith continuous anoxic perfusion (Text-figure 3). Our own unpublished obser-ations using dog heart slices.32 as well as reports by- others using toad urinary bladders 26 and perfused hearts in uitro.7 have shown that cy-anide requires 30 to 40 minutes for maximal inhibition of respiration. The results obtained here suggest that a similar period of time is required for cyanide inhibition of perfused rat hearts. Effects of Calcium-Free Perfusion
The calcium-paradox is a 'well-documented phenomenon33-37 -here, after 3 to 7 minutes of calcium-free perfusion, readmission of calcium causes cardiac muscle cells to suddenly contract, forming single large contraction bands and releasing intracellular materials. The ultrastruc-
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tural changes in these irrev-ersibl- injured cells resemble those seen after anoxic injury and reoxx-genation.33-37 The rapidity- of these events was used to study the effect of sudden contracture on CPK release and to estimate the time required to release CPK from perfused rat hearts. Results are sho-n in Text-figure 4. Little or no CPK w as released during 20 minutes of calcium-free perfusion. but after 2.3 m\l calcium. CPK (203.7 IU g heart) Xvas released, and the release Xwas nearly completed after 13 minutes. Mopooi Studies Light Microscopy
Table 1 indicates the relative proportions of cell ty-pes and total CPK released from experimental hearts. Nonswollen cells. swollen cells, and cells -with contraction bands mvere counted in hematoxvlin- and eosinstained sections. Percentage values are estimates. since only longitudinally oriented fibers could be evaluated, and it was difficult to kno'w -hether or not the entire cell was present in the section. Anoxia and Reoxygenatio
Cells from hearts perfused wvith anoxic medium for 60 minutes contained few contraction bands and had correspondingly little CPK release. RRoxygematloe,
Asoxic
Perfmsson
9.0
TEXT-F ICIRE 2-N\I-ocar-
the anoxic medium after .30 minutes and s%as continuousIv present during reoxygenation The sudden large peak of enznme release from control hearts solid line .,-as inihibited in cxanide-treated hearts dashed line. Some ox-xgeninduced enz-vme release %sas obser%ed %-hen canide s%as present onlls 13 minutes before reox' genatioll.
>
8.0
dial enzx-me release after 45 minlutes of anoxic perfusion
andl subsequent reoxx genation Cx-anide %sas added t o
SamM KCN r,
0
3
7.0
6.0
F
5.0
-
4.0
-
C
30.-
\vControl
(-) 0 ..0
0 0 4)
a-
3.0
KCN Added
2.0
5mM
IKCN
/
1.0 - B- -
0
cry
0
20
40
60
80
Minutes
100
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.^
--30
6 o
3
S
Ij
39 0 1-r 0 0
D C0
2
I
2
0
20
40
60
80
100
120
140
160
180 200
220 240
Minutes TEX;T-HI( tiE '3-\Iocardial enz\ me release after 60 minutes of anoxia and subsequent reox\ enation. 0\x\1en inidtced a large peak of enz\ me release fronm control hearts solid linte Hearts
perfused sith mediuim icontainini c\ anide from the beeinnnltaiv of anoxic perfusion shosted total inhibition of ox\\Ieninduced enz\ me release dashed lirt' suhsebktquent enz\ me release \% as similar to that s-een dtirinaz continuous anoxic perfusion 1
Reoxygenation after 45 or 60 minutes of anoxia resulted in rapid release of CPK and most injured cells contained contraction bands (Figure 2). Reversibly injured cells Nvere protected by reoxy genation and resembled normal mvocardial cells (Figure 1). A small number of nonswollen cells w ith contractures or contraction bands w as present in these hearts (Figure 3). Cyanide Inhibition
After 60 minutes of anoxia. cvanide inhibited contraction band formation upon reoxy genation (Figure 4 and Table 1). The late CPK release and morphologic ev idence of cellular degeneration were similar to that seen with long periods of continuous anoxic perfusion. Hearts made anoxic for 43 minutes with cyanide added 15 minutes before reoxygenation had small to moderate numbers of sm -ollen cells w ith contraction bands. Death of these cells probably accounts for the lov- level of oxy gen-induced enzvrme release allowed by partial inhibition of respiration. Calcium-Free Media
Follo'wing calcium-free perfusion and readdition of calcium (calciium paradox), nearly every cell had hypercontractures. was separated from
335
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Vol. 84, No. 2 August 1976
..*
.
.
-4
>
150
TEXT-F IGIURE 4-\I%-ocardial enz-xme release from hearts perfused mvith calcium-free medium for 21 minutes w-ith subsequent perfusion swith 2.5 m\1 calcium media calcium paradox A massiv e and rapid onset of enzv me release occurred immediately upon reperfusion of hearts w-ith calcium. Notice difference in time and enz%-me release scales from prev ious figures.
.-
3
130
0
3C
h0
CP cn
E
90
70
.0 50
1;
cL
30 10
20
24
28
32
36
Minutes
adjacent cells, and had clumps of mitochondria at either pole (Figure Oectron
--
3).
scopy
Hearts subjected to 45 or 60 minutes of anoxia and reoxx-genation contw-o cell types: a) reversibly injured cells resembling normal myo-
tained
Table 1-Percentage of Cell Types in Hearts After the Indicated Perfusion Conditions Percent of 1000 cells of left ventricle* Nonswollen Swollen cells Swollen Normal cells with with no cells with Total CPK Experimental appearing contraction contraction contraction released condition cells bands bands bands (IU/g heart) Anoxia (60 minutes) 4.2 i 5.3 0 90.4 ± 10.7 5.5 ± 5.4 8.76 ± 6.1
(N = 3)
Anoxia (45 minutes) followed by 15 minutes reoxygenation
(N = 6)
Anoxia (60 minutes) followed by 120 minutes reoxygenation
(N = 6)
Anoxia (60 minutes) followed by 180 minutes reoxygenation with 5 mM KCN
47.9 ± 8.1 (P
The effects of 5 mMf potassium cyanide (KC-N) on creatine phosphokinase (CPK) release and cellular morphology were studied. Rat hearts were perfused with substratedeficient media gassed with 02 or N2 (02 medium. N2 medium) at 37 C, and effluent was collected for creatine phosphokinase analysis. Tissue fixation was with glutaraldehyde for light and electron microscopy. Experiments included the follow-ing: a) continuous perfusion with 02- or N2-medium in the presence of KCN: b) 45 or 60 minutes of perfusion with N2-medium followed by 02-medium for 15 or 180 minutes, respectively: c) 45 minutes of perfusion with N2-medium with KCN- added 15 minutes before reoxygenation with 02-medium plus KCN: (4) 60 minutes of N2-medium plus KCN- followed by 02-medium plus KCN- for 180 minutes: d) as a control for irreversible injury. 21 minutes of perfusion with calcium-free 02-medium followed by 2.5 mMI calcium-02-medium ("calcium paradox"). The following results were seen: a) Initial CPK release occurred about 30 minutes later from hearts perfused with 02-medium plus KCN- than from hearts perfused with N2-medium plus KCN. b) Upon reoxygenation after either 45 or 60 minutes of anoxia. hearts had a sudden peak of oxygen-induced CPK release. NMost irreversibly injured cells were massively swollen and had sarcolemmal defects and contraction bands. Reversibly injured cells in the same hearts resembled normal my-ocardium. A previously unrecognized third population of cells is described. These cells were characterized by contraction bands but were not swollen, had intact sarcolemma. and contained both normal and damaged mitochondria with intramatrical calcium accumulation granules. It could not be determined if these cells were rev ersibly injured or in an early stage of irreversible injury. c) KCN- added 15 minutes before reoxygenation of hearts after 45 minutes of anoxia inhibited the sudden peak of oxygen-induced CPK release but not a slow sustained release. Small to moderate numbers of cells in these hearts contained contraction bands. d) After 60 minutes. KC_N completely inhibited both oxygen-induced CPK release and contraction band formation. e) Addition of calcium to calcium-free hearts caused both massive CPK release and contraction band formation. It is concluded that: the beginning of CPK release from oxygenated KCNinhibited hearts requires about 30 minutes longer than from anoxic hearts: KCN- can inhibit both oxy gen-induced CPK release and contraction bands in irreversibly injured rat myocardial cells: sudden contracture of myocardial cells as occurs in the calcium paradox can result in massive CPK release: contraction bands occur in nonswollen cells, hence contraction bands can occur independently of massive cell swelling or membrane rupture. It is postulated that there may be two stages of irreversible myocardial injurn-: a) loss of control of contraction and b) progressive loss of mitochondrial and membrane integrity. (Am J Pathol 84:327-350, 1976) From the Department of Pathohlogz. N rth%ettern 1-nlit -rlitx \ledical sch-l (icO()I 111111i Supp,)rted in part h! (;rant HL-19000 from the National Ilnstitiutet of Health Accepted for publicatmn April 1 19:6 F anote. Dtepartmetrit 4 Pathowloiz. N()rth%%eterT Address reprint requests to Dr (Chalres E. t1ni-ersitx \ledical Scho(l. 303 East Chicaizo A'enue. Chicaco. IL 606i11 327
328
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American Journal of Pathology
STUDIES OF THE PATHOGENESIS of cell injury have shown that brief ischemic or anoxic stress causes cells to pass into a state of reversible injury. The term anoxia is used here to mean a reduction in oxygen tension such that mitochondria can no longer function but the cells remain exposed to constantly renewed extracellular fluid. Ischemia is a reduction of blood flow to an area of tissue to a level below that necessary to meet normal oxygen demand. Total ischemia is absence of arterial flow. During anoxia, mitochondria of reversibly injured cells stop functioning and cells accelerate anaerobic metabolism, with stores of intracellular glycogen being the principal substrate.1'2 With this inefficient metabolic pathwav and depletion of glycogen, adenosine triphosphate (ATP) levels fall,3 and the cells swell,"5 lose potassium,6 and accumulate sodium, water,"5 and perhaps calcium. If blood flow or oxygen is restored, reversiblv injured cells recover.5 However, when anoxia or ischemia is prolonged, there is a transition from reversible to irreversible injury. This transition has been called the "point of no return,"8 and once it has been passed the cell is considered dead. Reoxygenation or reflow of blood to irreversibly injured cells does not affect survival. In studies of nonmyocardial cells 4.5s8-12 the onset of irreversibility has been at or about the time of nonspecific increases in cell membrane permeability. Prior events seem at least potentially reparable.'3 Increased membrane permeabilitv allows influx of calcium into the cells, causing uncoupling of mitochondria and escape of vital intracellular materials. 1'2 The subsequent events constitute necrosis, which is degeneration of cellular components usually aided by release of Iysosomal enzymes.5'1o Anoxic injury to isolated perfused hearts can result in irreversible injurv with changes strikingly similar to those occurring in vivo following ischemic injur of dog myocardium.'5-18 In both experimental models, reversiblv injured myocardial cells show minimal structural changes of cellular swelling, mitochondrial swelling, and loss of normal matrix granules. Reflow of blood or reoxygenation results in rapid functional recovery and survival of these cells.15'18 With continued injury, myocardial cells develop amorphous densities in mitochondrial matrix spaces, clumping of nuclear chromatin, and terminal increases in cell membrane permeability which indicate cell death.18 With either reflow of blood or reoxygenation, changes in irreversibly injured myocardial cells are rapid: heat production increases abruptly,'9 contraction bands form,16 2 cells swell,'9'2' and sodium and calcium accumulate.' Large blebs of edema fluid occur beneath the sarcolemma,15'17'2' and defects in cell membranes are demonstrable bv electron microscopy.8 15 Enzymes, including creatine phosphokinase (CPK),
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are released from the irreversiblv injured cells and mitochondria accumulate calcium in the form of granular dense bodies."156'20.22 In injured myocardium, defects have been shoxN-n in mitochondrial function, calcium metabolism, cell volume control, and membrane permeability. To define the primary cause of cell death, -e have sought the earliest event or events that dictate the onset of irreversibility. In the present study, the effects of cy anide on ex ents occurring after reoxy genation of irreversibly injured, in vitro perfused rat hearts are reported. Cyanide inhibits the acute rapid changes occurring upon reoxy genation. Assuming that irreversible myocardial injury and increased membrane permeability are related events and that cvanide acts as an inhibitor of mitochondrial function,23 27 the data suggest that there are two stages of irreversible myocardial injury. The first stage is perhaps unique to contractile cells and consists of conditions causing uncontrolled hypercontracture upon reoxygenation and rapid increases of cell membrane permeability. The second stage appears to be a more slox lv developing cellular degeneration e-entuating in cell death. Materials and Methods Hearts Xvere obtained from male, specific pathogen-free Sprague-Da.-ley rats wseighing 200 to 400 g that svere fed ad libitutn. Hearts mvere rapidly remox-ed from animals anesthetized by intraperitoneal injection of 40 mg kg sodium pentobarbital iDiabutal. Diamond Laboratories, Inc.. Des Moines. Io va) and immersed in ice-cold perfusion fluid. After dissection, hearts 'sere blotted and sseighed, and the aortas 'vere cannulated on the perfusion apparatus.
Experimental Design All hearts svere perfused for an initial 13-minute period mvith an oxygenated medium. The follosving experiments mvere performed a) Perfusion of hearts mvith substrate-free media containing 3 mMn KCN' gassed 'vith either 953% 02-3% C02 or 953c \-2-5c CO2 for 240 minutes b) Anoxic perfusion for 43 minutes follom -ed by reoxx-genation for either 13 minutes without KCN.- or 73 minutes in the presence of 3 m\M KC\ X hich "-as added 13 minutes before reoxy genation. c Anoxic perfusion for 60 minutes folloNved bx- reox -genation for 120 minutes ssithout KCN or I80 minutes in the presence of 3 m\I KC\ wshich "vas added at the beginning of the anoxic perfusion period. d Perfusion of hearts for 21 minutes Xwith oxygenated calcium-free medium containing glucose and 0. I mn EDTA follomved b\ a similar medium but X ith 2.3 mMn calcium for an additional 13 minutes. e Anoxic perfusion of hearts for 60 minutes. Experimental '-alues are expressed as mean ± SE. where N = 6. except ,-here so noted. At the end of each experiment hearts Nvere fixed bx- retrograde perfusion of glutaraldehyde for morphologic studies. Perfusion Apparatus The perfusion X"as accomplished on a modified double-resen-oir. double-heart. nionrecirculating Langendorf 2s apparatus. The apparatus and techniques used "vere the same
330
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American Journal of Pathology
as previously described 15 Both reservoirs vvere maintained at 37 C in a water bath. Fluid w as pumped through tx-gon tubing to glass condensing tubes. Temperature as controlled by a thermal probe placed in an aortic cannula. The probe, connected to a Yjellomv Springs Instruments (Mlodel 73) temperature controller. maintained the fluid passing through the aortic cannula at 37 ± 0.3 C. Pressure -as recorded on a Grass Model 3 polygraph Perfusion could be rapidly s -itched from either reservoir. In experiments requiring a third medium, the unused reserxoir was emptied, rinsed, and refilled mvith the new medium. Flov- rates vvere individuallb measured b- timed collection of effluent in a graduated cx-linder. After the 13-minute control perfusion period. flow rates averaged 19.6 ± 3.6 ml min ± SD. After 13 minutes of anoxic perfusion. the flov rates averaged 14.9 ± 1.3. Perfusion Fluid
Perfusion fluid for the control period of perfusion "vas a Krebs-Henseleit-bicarbonate medium 2' containing 2.3 m\I calcium and 10 m\1 D-glucose gassed with 93% 02-5% CO2. The medium for anoxic perfusion Xvas equilibrated Xvith 93% N3-3% CO2 and had the same ionic composition except that 10 m\M mannitol replaced glucose. The medium for reoxxgenation also contained 10 m\M mannitol in place of glucose. Five millimolar KCN was added to either the anoxic or reoxygenation medium. Osmolalit! "as 290 to 293 mOsmoles liter, and pH "as 7.3 to 7.4. All media "vere gas equilibrated for at least 43 minutes prior to beginning the experimentt Creatine Phosphokinase Analysis
One- to t%vo-milliliter samples of effluent "vere collected serially in glass or plastic vials and stored in crushed ice until the end of the experiment. Creatine phosphokinase activity -as measured on 0. I-ml aliquots bx- the method of Oliver 3" in a Beckman DU spectrometer using reagents obtained from Boehringer-NMannheim GMBH (\annheim. German-). The results of CPK activity expressed in international units per minute per gram initial heart vveight "ere obtained from the follom-ing formula: IU min g=
CPK acti'-it'-
IU liter) X flomv (ml
min) X
1000 ml
Initial heart %veight (g)
Total CPK release "as obtained bx- meighing enzx-me release curves cut from standard graph paper
Morphologic Studies At the end of each experiment, hearts "vere fixed bx- perfusion of I % glutaraldehx-de in m(odified Tvrode's solution.3' Folloving fixation, the apex and atria were discarded. Four equally spaced horizontal sections of the heart "vere taken and processed for light microscopy. In addition, 0 3-mm sections v"ere cut from tissue adjacent to the light microscope sections and "vere placed in glutaraldehx-de for an additional 1 hour. Following postfixation in I % osmium tetroxide. one group of blocks "vas soaked in aqueous uranyl acetate. a second group "as left unstained Follo' ing dehx-dration and treatment Xvith propylene oxide, blocks "vere flat-embedded in Epon S12 (Shell Chemical Co., Nemv York. N.Y.. Semi-thin sections were stained with toluidine blue for light microscopy. and areas were selected for thin sectioning using diamond knives. Thin sections of tissue stained en bloc Xvere mounted on plain copper grids and examined directly. Tissue not stained before
Vol. 84, No. 2 August 1976
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embedding s-as stained wsith 7,.7` aqueous uran\ l acetate prior to exaamination in an Hitachi HU-12 electron microscope. Estimation of Cell Damage
U-sing light microscopy of hematoxylin- and eosin-stained sectio)s. longitudinallk oriented cells w ere classified into four groups on the criteria of being nons\s ollen or s\s ollen. and by the presence or absence or contraction bands. The percent of each cell ty pe ws-as estimated b\- direct counts of 1000 successix-e cells from each heart.
Results Gross Observations
The conditions used in the present study are similar to those reported previously.'5 with the exception that a small poly ethylene catheter was placed through the tip of the left ventricle to prev-ent dilation of the heart due to intraventricular pressure and, w-e hoped, to allow perfusion of the subendocardium which. in prev ious experiments, had not been perfused. During perfusion, when the hearts no longer underwent spontaneous contractions, a v-ariable but usually significant flows ensued from the catheter, demonstrating incompetence of the aortic \-alv-e. In anoxic hearts, with their high perfusion flo- rates. much of the flo\- came through the catheter. Thus. coronary- flosw rates could not be accuratel\measured. Hearts sectioned at the completion of the experiments shoswed that a variable but consistently present area of the subendocardium swas pink and appeared nonperfused. as w-as the case in uncatheterized hearts. This area was not included in samples. In accordance with previous obser-ations. hearts releasing CPK or shoswing morphologic e-idence of irrev-ersible injury also changed from a reddish to a pale. opaque appearance. Enzyme Release Studies Cyanide-inhibited Controls
To show that 5 m\M cyanide is a biologically effective inhibitor of rat hearts in Litro. perfusion was conducted -ith either oxy genated or nitrogen-equilibrated, substrate-deficient media for 240 minutes. Released creatine phosphokinase activ-ity w-as an indicator of cellular injury. Results are shown in Text-figure 1. Enz\-me release occurred under both aerobic and anaerobic conditions, but release of CPK began later from oxy genated hearts than from anoxic hearts. The results show that cy-anide is an effectixe metabolic inhibitor under the experimental conditions but that its action is delayed, it did not significantly inhibit CPK acti-ity released
332
American Journal of Pathology
GANOTE ET AL
3 3
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ID
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Minutes
TEXT-1I-Rt-E 1-\lxocardial eniz'me release dtiringz anoxic or ox%genated perfusion in the presenlCe of 5 m\l c\ anide Enzsme release from oxwzenated hearts solid line \-.as dela\ed compared to that from anoxic hearts dashled linie The difference in enzx me release patterns is thought to be due to the tinme required to effect metablic inhibition bh cx anide ;Double asterisk. P < 0.01. sinyle asterisk. P < 0 05
from injured hearts, and it did not impart superimposed injury beyond that caused by anoxia alone. Effect of Cyanide on Reoxygenation
After 43 or 60 minutes of substrate-free anoxic perfusion, oxy-gen induced the release of CPK from injured hearts (Text-figures 2 and :3). Cyanide added 13 minutes before reox}-genation reduced the initial peak of CPK release but did not prevent initiation of a slo-ver release of this enzyme bx- oxy-gen (Text-figure 2). The cyanide present in anoxic media perfused for 60 minutes totallx- inhibited oxy-gen-induced CPK release. Subsequently. a slo-wly rising CPK release followed a pattern similar to that seen X ith continuous anoxic perfusion (Text-figure 3). Our own unpublished obser-ations using dog heart slices.32 as well as reports by- others using toad urinary bladders 26 and perfused hearts in uitro.7 have shown that cy-anide requires 30 to 40 minutes for maximal inhibition of respiration. The results obtained here suggest that a similar period of time is required for cyanide inhibition of perfused rat hearts. Effects of Calcium-Free Perfusion
The calcium-paradox is a 'well-documented phenomenon33-37 -here, after 3 to 7 minutes of calcium-free perfusion, readmission of calcium causes cardiac muscle cells to suddenly contract, forming single large contraction bands and releasing intracellular materials. The ultrastruc-
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tural changes in these irrev-ersibl- injured cells resemble those seen after anoxic injury and reoxx-genation.33-37 The rapidity- of these events was used to study the effect of sudden contracture on CPK release and to estimate the time required to release CPK from perfused rat hearts. Results are sho-n in Text-figure 4. Little or no CPK w as released during 20 minutes of calcium-free perfusion. but after 2.3 m\l calcium. CPK (203.7 IU g heart) Xvas released, and the release Xwas nearly completed after 13 minutes. Mopooi Studies Light Microscopy
Table 1 indicates the relative proportions of cell ty-pes and total CPK released from experimental hearts. Nonswollen cells. swollen cells, and cells -with contraction bands mvere counted in hematoxvlin- and eosinstained sections. Percentage values are estimates. since only longitudinally oriented fibers could be evaluated, and it was difficult to kno'w -hether or not the entire cell was present in the section. Anoxia and Reoxygenatio
Cells from hearts perfused wvith anoxic medium for 60 minutes contained few contraction bands and had correspondingly little CPK release. RRoxygematloe,
Asoxic
Perfmsson
9.0
TEXT-F ICIRE 2-N\I-ocar-
the anoxic medium after .30 minutes and s%as continuousIv present during reoxygenation The sudden large peak of enznme release from control hearts solid line .,-as inihibited in cxanide-treated hearts dashed line. Some ox-xgeninduced enz-vme release %sas obser%ed %-hen canide s%as present onlls 13 minutes before reox' genatioll.
>
8.0
dial enzx-me release after 45 minlutes of anoxic perfusion
andl subsequent reoxx genation Cx-anide %sas added t o
SamM KCN r,
0
3
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5.0
-
4.0
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30.-
\vControl
(-) 0 ..0
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3.0
KCN Added
2.0
5mM
IKCN
/
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20
40
60
80
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100
120
American Journal of Pathology
GANOTE ET AL
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.^
--30
6 o
3
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39 0 1-r 0 0
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2
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2
0
20
40
60
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100
120
140
160
180 200
220 240
Minutes TEX;T-HI( tiE '3-\Iocardial enz\ me release after 60 minutes of anoxia and subsequent reox\ enation. 0\x\1en inidtced a large peak of enz\ me release fronm control hearts solid linte Hearts
perfused sith mediuim icontainini c\ anide from the beeinnnltaiv of anoxic perfusion shosted total inhibition of ox\\Ieninduced enz\ me release dashed lirt' suhsebktquent enz\ me release \% as similar to that s-een dtirinaz continuous anoxic perfusion 1
Reoxygenation after 45 or 60 minutes of anoxia resulted in rapid release of CPK and most injured cells contained contraction bands (Figure 2). Reversibly injured cells Nvere protected by reoxy genation and resembled normal mvocardial cells (Figure 1). A small number of nonswollen cells w ith contractures or contraction bands w as present in these hearts (Figure 3). Cyanide Inhibition
After 60 minutes of anoxia. cvanide inhibited contraction band formation upon reoxy genation (Figure 4 and Table 1). The late CPK release and morphologic ev idence of cellular degeneration were similar to that seen with long periods of continuous anoxic perfusion. Hearts made anoxic for 43 minutes with cyanide added 15 minutes before reoxygenation had small to moderate numbers of sm -ollen cells w ith contraction bands. Death of these cells probably accounts for the lov- level of oxy gen-induced enzvrme release allowed by partial inhibition of respiration. Calcium-Free Media
Follo'wing calcium-free perfusion and readdition of calcium (calciium paradox), nearly every cell had hypercontractures. was separated from
335
ENZYMES IN MYOCARDIAL INJURY
Vol. 84, No. 2 August 1976
..*
.
.
-4
>
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TEXT-F IGIURE 4-\I%-ocardial enz-xme release from hearts perfused mvith calcium-free medium for 21 minutes w-ith subsequent perfusion swith 2.5 m\1 calcium media calcium paradox A massiv e and rapid onset of enzv me release occurred immediately upon reperfusion of hearts w-ith calcium. Notice difference in time and enz%-me release scales from prev ious figures.
.-
3
130
0
3C
h0
CP cn
E
90
70
.0 50
1;
cL
30 10
20
24
28
32
36
Minutes
adjacent cells, and had clumps of mitochondria at either pole (Figure Oectron
--
3).
scopy
Hearts subjected to 45 or 60 minutes of anoxia and reoxx-genation contw-o cell types: a) reversibly injured cells resembling normal myo-
tained
Table 1-Percentage of Cell Types in Hearts After the Indicated Perfusion Conditions Percent of 1000 cells of left ventricle* Nonswollen Swollen cells Swollen Normal cells with with no cells with Total CPK Experimental appearing contraction contraction contraction released condition cells bands bands bands (IU/g heart) Anoxia (60 minutes) 4.2 i 5.3 0 90.4 ± 10.7 5.5 ± 5.4 8.76 ± 6.1
(N = 3)
Anoxia (45 minutes) followed by 15 minutes reoxygenation
(N = 6)
Anoxia (60 minutes) followed by 120 minutes reoxygenation
(N = 6)
Anoxia (60 minutes) followed by 180 minutes reoxygenation with 5 mM KCN
47.9 ± 8.1 (P
