Early Morphologic Changes in Cat Heart Artery Occlusion - Europe PMC

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Feb 9, 1990 - ture of2400 heart muscle cells and nuclei was stud- .... The hearts were arrested by ... with a magnification of 9600 were obtained at random. A.
American Journal ofPathology, Vol. 136, No. 2, February 1990 Copyright © American Association ofPathologists

Early Morphologic Changes in Cat Heart Muscle Cells After Acute Coronary Artery Occlusion Gottfried Greve,* Svein Rotevatn,* Knut Svendby,t and Ketil Grongt From the Department ofAnatomy* and the Surgical Research Laboratory,t University ofBergen, Bergen, Norway

The left descending coronary artery (LAD) was occluded in 16 open-chest cats for 10, 20, 40, or 60 minutes (four cats in each group). In addition, four sham-operated cats served as controls. Specimens for electron microscopy were obtained from the normal and ischemic zones, guided by in vivo injection offluorescein, and verified by bloodflow measurements with microspheres. The ultrastructure of2400 heart muscle cells and nuclei was studied. Fractional volumes of main cell components, mitochondrial surface density, and mitochondrial surface:volume ratio were calculated in 480 micrographs. After 10 minutes of ischemia we observed signs of sarcolemmal fragility, mitochondrial swelling, and lipid droplet accumulation. After 20 minutes of ischemia sarcolemmalfragmentation, chromatin clumping or margination and a maximal cytoplasmic edema were evident. The fractional volume of mitochondria was equally increased in ischemic zones ofall groups. In both normal and ischemic zones there was a tendency toward smallerfractional volumes of lipid droplets during ischemia. In the normal zone there was mild cytoplasmic edema and slight mitochondrial swelling 10 minutes after occlusion as compared with the sham group. The present study demonstrates that a large proportion ofcardiac myocytes undergoes severe damage within 20 minutes of coronary occlusion. (Am J Pathol 1990, 136: 273-283)

In the last two decades ischemic myocardial cell damage has been thoroughly described biochemically, physiologically, and morphologically.1-6 Signs of irreversible ischemic injury such as sarcolemmal fragmentation, mitochondrial disintegration, mitochondrial dense bodies, cy-

toplasmic edema, and nuclear disintegration are well established in the literature. In the ischemic process, a point can be reached at which the cells will not be able to recover their normal function. Accurate information of cellular events leading to cell death is of primary interest because it might enable us to retard, stop, or even reverse the cell damage. In a recent article Spinale et a17 reported irreversible injuries of right ventricular myocardium 15 to 30 minutes after the onset of ischemia. To discover early ultrastructural changes exhibited by the cells before the irreversible damage is sustained, it is necessary to study more sensitive variables than those listed above. We have shown previously that studies of lipid droplet accumulation, mitochondrial swelling, and fragility of the sarcolemma are well suited to reveal moderate cellular alterations during ischemia.5-10 The aim of this study was to give more precise information on the sequence of the ultrastructural alterations at early stages of the ischemic process and, if possible, to identify the point at which cell damage becomes irreversible. Also of major interest is the relationship between sarcolemmal damage and sarcoplasmic edema, and the succession of events during ischemia.

Materials and Methods Twenty male outbred, specific-pathogen-free cats (Iffa Credo, L'Arbresle, France), whose mean weight was 4.2 ± 0.7 kg (SD), were anesthetized with sodium pentobarbital i.v. (25 mg/kg body weight) and allocated into five groups, each with four cats. One sham-operated group served as controls, while the remaining groups were subjected to 10, 20, 40, or 60 minutes of regional ischemia, respectively. All animals were tracheotomized and ventilated with 50% N20, 47.5% O2, and 2.5% C02 was added through a positive-pressure ventilator for maintaining anAccepted for publication September 18, 1989. Supported by grant from the Norwegian Research Council on Cardiovascular Diseases. Address correspondence and reprint requests to Gottfried Greve, MD, Department of Anatomy, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.

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esthesia (LOOSCO Infant Ventilator MK2, Amsterdam). Arterial blood gas analyses were undertaken to assure stable oxygenation and metabolic conditions throughout the experiment. Body temperature was kept constant at 37 C by an adjustable heat pad connected to a rectal thermistor. A midline thoracotomy and a longitudinal pericardiotomy was performed. Heart rate, left ventricular systolic (LVSP) and end-diastolic (LVEDP) pressures, and cardiac contractility (dP/dt) were continuously recorded with a short catheter through the apex connected to a Statham P23De pressure transducer (Hato Ray, Puerto Rico). The left atrium was cannulated with a short polyethylene catheter for injection of radiolabeled microspheres and fluorescein. The abdominal aorta was cannulated via the left femoral artery for collection of reference blood. The left anterior descending coronary artery (LAD) was dissected free at a proximal site and was loosely encircled by a 4-0 silk thread just distal to the branching of the left main stem. After registration of baseline hemodynamic variables, LAD was permanently occluded by tightening the ligature. In the sham group the ligature was not tightened, and a 1 0-minute period was allowed before termination of experiments. Hemodynamic variables were recorded throughout the experiments. Three minutes before sacrifice, approximately 1 X 106 microspheres (average diameter of 15.6 ± 0.1 ,m (SD)) were injected for measurements of regional blood flow rate."1 The spheres were labelled with 46Sc, 81Sr, or 153Gd selected at random. Reference blood sample was collected from the abdominal aorta via a catheter in the left femoral artery by a constant-rate extraction pump (Sage Instruments 351, Cambridge MA). Injection of spheres lasted for 60 seconds and the withdrawal of the reference blood sample started 10 seconds before and terminated 50 seconds after delivery. Exact withdrawal rates were calculated from the weight of blood samples. Immediately before sacrifice 0.8 ml of 10% sodium fluorescein in distilled water with 4.06% sodium bicarbonate (Fluorescite, Alcon Laboratories Inc., Fort Worth, TX) was injected into the left atrium for demarcation of normally perfused myocardium. The hearts were arrested by perfusion fixation. Ice cold fixative was injected directly into the left ventricle during partial occlusion of the aorta. The fixative solution contained 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer with 0.1 M sucrose and 1.25 mM CaCI2 (pH 7.4; vehicle osmolality - 300 mosmol/kg) prepared according to the recommendations of Ericsson et al.12 The hearts were then rapidly removed from the animal, the left ventricle dissected free, and the heart submerged in a bath of the same ice cold fixative.

Tissue preparation While still in fixative, and guided by ultraviolet light, three transmural ischemic samples (with no fluorescence of the

cut surfaces) and three nonischemic samples (with bright fluorescence of the cut surfaces) were collected. Three corresponding transmural samples with bright fluorescence of the cut surfaces were collected from the sham group. All samples were subdivided into two samples of 100 to 200 mg, left and right. From the cut surface between the paired subsamples a midmyocardial specimen was obtained for electron microscopy (Figure 1). All subsamples were weighed and together with the reference blood sample, the residual in the syringe and isotope standards, were counted for gamma emission (CompuGamma 1282, LKB-Wallac Co., Turku, Finland). Regional tissue blood flow rate and cardiac output were calculated according to Heyman et al.11 After counting all tissue subsamples were dried at 73 C for 3 days, reweighed, and the water content calculated. Specimens for electron microscopy were fixed by immersion for 3 hours or longer, washed in cacodylate buffer (vehicle osmolality = 300 mosmol/kg), and postfixed in 1 % OS04 solution of the same buffer before they were dehydrated in ethanol and embedded in Epon. Ultrathin (50 to 75 nm) sections stained for 1 hour with uranyl acetate and 15 minutes with lead citrate were prepared.

Data Acquisition Ultrastructure of cardiac muscle cells were studied in randomly selected sections from all specimens. From each section 20 myocytes were studied (ie, a total number of 2400 myocytes), and proportions of cells with sarcolemmal disruption (strictly focal or extensive) were calculated (Figure 2). Disruptions were considered strictly focal when they were restricted to single holes in the cell membrane. Such focal disruptions were usually accompanied by a restricted cytoplasmic edema localized close to the sarcolemma. In addition, 20 nuclei were investigated in every specimen, and the proportion of cells with chromatin clumping or margination (Figure 3) as well as nuclear membrane fragmentation was calculated; altogether 2400 nuclei were studied. The ultrastructure was studied at a magnification of 12,000 or more. When necessary sections were tilted ±60 degrees to verify that disruptions of the sarcolemma or nuclear membrane were real. From all specimens, four micrographs (18 X 24 cm) with a magnification of 9600 were obtained at random. A total of 480 micrographs were studied. Standard pointcounting technique was used for measuring fractional volumes of mitochondria (Vvmit), myofibrils (Vvmyo), lipid droplets (Vvlip), and remaining cytoplasm, including the lipid droplets (Vvcyt) according to the Delesse principle.8'13 Except for the fractional volume of lipid droplets, a grid lattice with a line distance of 25 mm was superimposed. Approximately 63 intersections of grid lines fell within each graph.

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ISCHEMIC

FL

NORMAL

10 MIN

m

0.10

0.16

0t.08

[0.08

20 MIN 0.05 ±0.03

[ 0.09 100.04

40 MIN 0.04

0.03

Figure 1. The left ventricle was cut in long transmural slices across the fluorescein demarcation line (FL). Samplesfor bloodflow measurements, were obtained from the central part of the normal (hatched) and ischemic zones (open). Each sample was cut into left atnd right subsamples (broken lines). Bloodflow rate (ml/min. per g) ± SEM was calculatedfor both the 3 left and the 3 right subsamples in all groups (10, 20, 40, and 60 minutes). Specimens (- )for electron microscopy (EM) were taken from the midmyocardial cut surface of the two paired subsamples.

tO.02 ,,+0.01' 60 MIN 0.04 ±0.01

Data on the mitochondrial surface were obtained by counting intercepts of the outer mitochondrial membrane with grid lines, correcting for magnification and the distance between lines in the grid lattice. Mitochondrial surface density, defined as the ratio of mitochondrial surface to total cytoplasmic volume, and mitochondrial surface-tovolume ratio were calculated. Lipid droplets are spherical, vaguely opaque structures that are not membrane bound.10 They are usually localized between myofibrils close to mitochondria (Figure 4). In a previous study we have shown that these droplets contain lipid material.10 For the calculation of fractional volumes of lipid droplets, we used another grid lattice with a distance of 5 mm between the lines. To minimize the effects of anisotropy in the plane, all morphometric measurements were performed with the grid lines both at 0 degrees and 15 degrees to the graph edges. Parallel counting was performed by two independent observers (interobserver variability less than 5%). In the further calculations the mean value obtained by the two observers at both grid angles in each micrograph was used. Counting errors in each zone in each group should be less than 10%, ie, expected biologic variability among animals in a given experimental group. For cytoplasm (the least frequent structure) we counted 176 points (sham group) or more within one zone in each group, yielding a sampling error of 7.5% or less.14 Corresponding sampling errors for lipid droplets were 5.6% or less, for mitochondria, 2.8% or less, and for myofibrils, 2% or less. Sarcomere length was measured at the same magnification in micrographs in which myocytes were cut strictly

! 0.06 t ±0.03

.i

EM longitudinally. It was possible to measure the length of 118 sarcomeres in 27 graphs from sham-operated cats, 701 sarcomeres in 99 graphs from the normal zones, and 582 sarcomeres in 92 graphs from the ischemic zones.

Statistics The effects of coronary artery occlusion on hemodynamic variables in the LAD-occluded cats were statistically analyzed by paired sample t-tests, whereas postocclusion hemodynamic values were compared by a one-way analysis of variance (ANOVA) by the Minitab statistical package (Minitab Project, The Pennsylvania State University, University Park, PA).15 Variables recorded at a certain time point after coronary artery occlusion were pooled. Mean blood flow rates of the three left and three right subsamples from the same zones (ischemic and nonischemic) were calculated for all cats with LAD artery occlusion. In addition, blood flow rates were calculated for the corresponding subsamples obtained from each sham-operated heart. Tissue blood flow values and water content were statistically analyzed by a two-way ANOVA by Minitab.15 Morphometric data from the sham group were compared with corresponding data from normal and ischemic myocardium in cats with 10 minutes of LAD occlusion by a one-way, four-level nested ANOVA by the statistical package BMDP85 (P8V) (BMDP Statistical Software, Los Angeles, CA).16 Significant differences between groups were tested individually by a Newman-Keuls multiple comparison procedure.17

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2a

.,q

,,F FX

r.P.

Figure 2. a: Intact sarcolemma in normal zone of the 60-minute group. b: Focal disruption (arrow) of the sarcolemma associated witb a submembranous cytoplasmic edema in ischemic zone of the 60-minute group. c: Fragmentation of sarcolemma (arrowheads) in the ischemic zone of the 10-minute group. d: Severe sarcolemmal fragmentation (arrows) and a prominent sarcoplasmic edema (asterisk) in iscbemic zone of the 60-minute group. Erythrocyte (Ery) in a capillary in the upper part of micrograpb. Lipid droplet (Lip) seen in the lower part. Magnifications: a and b X 13,680; c X 31,920; d X 16,188. All sections stained by uranyl acetate and lead citrate.

For statistical analysis of the morphometric data obtained from the four time groups with LAD occlusion, we used a similar two-way, four-level nested ANOVA. In the case of significant interaction effect (noted for P < 0.1), an individual one-way, four-level nested ANOVA was performed between the two zones of each group and be-

tween the four groups of each zone. A significant zone effect was explored by a Newman-Keuls multiple comparison procedure. In the case of no significant interaction effect combined with a significant time effect, the group means were compared by a Newman-Keuls multiple comparison procedure directly. In addition, the time de-

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pendence of Vvlip was tested by a one-way analysis of covariance by BMDP85 (P2V),16 with time as covariate and zones as grouping factors.18 All results from measurements of hemodynamics, blood flow, water content, and morphometry are given as mean values ± SEM. Significant zone or time effects were noted for P < 0.05. The results of the quantitation of cell injury in the electron microscope are represented by median values.

Results Body weights did not differ significantly among the five groups. Judged from the preocclusion measurements of heart rate, LVSP, LVEDP, and dP/dt, no significant differences was observed between the groups. The results from the four groups subjected to LAD occlusion were therefore pooled (Table 1). In the sham group there were no significant changes over time, and average values were: heart rate, 210 ± 14 beats/minute, LVSP, 120 ± 5 mmHg, LVEDP, 4 ± 1 mmHg, and dP/dt, 3250 ± 395 mmHg/s. LAD occlusion resulted in a sudden fall in dP/ dt and LVSP, and 10 minutes after occlusion they were significantly lower than before occlusion (Table 1). After

the initial reductions both tended toward increased levels, and 60 minutes after occlusion they even exceeded the preocclusion values. However, the increase could not be confirmed statistically because of the small number of cats within each group. Furthermore, LVEDP increased rapidly after LAD occlusion and remained high throughout the experiments. Cardiac output did not differ between the sham group and the cats with 10, 20, 40, or 60 minutes of LAD occlusion (238 ± 47, 222 ± 42, 209 ± 27, 204 ± 20, and 281 ± 23 ml/minute, respectively). There was a significant reduction of blood flow rate in ischemic subsamples as compared to normally perfused subsamples (Figure 1), and ischemic samples were only accepted if there was >85% reduction in blood flow rate in both the left and right subsamples. There was no difference between paired subsamples of either zones, demonstrating that specimens were taken from clearly ischemic or clearly normally perfused myocardium. Furthermore, myocardial tissue blood flow (neither in ischemic nor normally perfused myocardium) did not differ in cats killed after 10, 20, 40, or 60 minutes. In sham-operated cats mean blood flow was 1.36 ± 0.12 ml/min/g in the left subsamples and 1.33 ± 0.13 ml/min/g in the right subsamples. In nonischemic myocardium, mean tissue water content was 74.39% ± 0.55%, and there was no difference

C7~~~~~~~~~~~~~~~~ Figure 3. a: Intact heart-cell nucleus in normal zone ofthe 60-minute group. b: Nucleus with chromatin clumping and margination (arrow) in a myocyte from the ischemic zone of the 10-minute group. Magnifications: a X 20,520, b X 13,680. Stained by uranyl acetate and lead citrate.

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Figure 4. Disruption of the sarcolemma associated withsubmembranous edema (arrow) in an otherwise normal-appearing heart muscle cell (upper part). Lipid droplet (arrowhead) in an edematous cell (lower part). The cells are connected by intact intercalated disc. Magnification X 13,680. Stained by uranyl acetate and lead citrate.

tissue water content in the sham 0.34%. In ischemic myocardium, however, there was a significantly larger water content (79.18 ± 0.23%) as compared with the nonischemic myocardium (P < 0.0001), but there was no difference among

among groups. Mean group was 76.37% ±

groups.

Both in the sham group and normal zones of all four with LAD occlusion, the sarcolemma was mainly intact (Figure 2a). Less than 5% of the cells in the normal zones had strictly focal disruptions of the sarcolemma, appearing as small isolated holes in the membrane. The basal lamina outside the holes was intact. In this zone we never observed cells with nuclear chromatin clumping or margination (Figure 3a), or nuclear membrane disruption. In ischemic myocardium the proportion of cells with sarcolemmal disruption increased, and after 10 minutes of ischemia 20% of the myocytes (median value) had small isolated holes in the sarcolemma (Figure 2b), and 5% revealed more extensive disruption (Figure 2c and d). In groups with prolonged LAD occlusion (.20 minutes), 60% or more of the cells had extensively fragmented sarcolemma, whereas less than 10% of the cells had only focal sarcolemmal disruptions. After 10 minutes of LAD occlusion, 23% of nuclei had chromatin clumping or margination, increasing to more than 85% in animals with progroups

longed ischemia (Figure 3b). The nuclear membrane was disrupted only in a few cells in the ischemic zones. There was no sign of ischemic contracture. The sarcomere length in ischemic myocardium of all four groups was 2.17 ± 0.32 ,m, and did not differ significantly from the sarcomere length found in nonischemic myocardium (2.26 ± 0.18 Am) and the sham group (2.47 ± 0.09,um). Within each zone there was no significant difference between time groups with respect to sarcomere length, and the values are in accordance with normal sarcomere length in cat hearts as reported by Fawcett and McNutt"9 and by us in previous works.ee After 10 minutes of ischemia, Vvmk was significantly larger in the ischemic (32.41% ± 1.19%) than in the normal zone (27.64% ± 1.13%), and the sham group (26.39% ± 0.66%) (Table 2 and Figure 5). There was no further increase in Vvmft in the ischemic zone with prolonged periods of LAD occlusion. Within 60 minutes there was, however, no sign of mitochondrial loss, but there was an increasing fraction of mitochondria with damaged cnstae. Vvmyo was significantly lower in the ischemic zones as compared to the normal zones in all groups. The reduction of Vvmyowas modest in the 10-minute group and most pronounced after 20 minutes, with intermediate values in the two other groups (Table 2).

Table 1. Hemodynamic Registrations Before and 10, 20, 40, or 60 MinutesAfter LAD Occlusion in Cat Postocclusion 10 minutes 20 minutes

Preocclusion

(n = 16) HR(beats/min) LVSP (mmHg) LVEDP (mmHg) dP/dt* (mmHg/s)

188±6 140 ± 6 5.8 ± 0.4 3734 ± 298

NS t t t

40 minutes

60 minutes

(n = 16)

(n = 12)

(n =8)

(n =4)

180±9

194±9 131 ± 9

192±14

212±16

144 11 10.5 1.2 4125 ± 482

147 19 10.5 2.2 4000 + 700

127 ± 7 13.3 ± 1.8 3227 ± 261

13.3 ± 2.5 3458 ± 330

Mean values ± SEM. * dP/dt is the first derivative of left ventricular pressure. t Denotes significant difference between preocclusion values and values obtained 10 minutes after LAD occlusion by paired sample t-tests. NS denotes not significant. No statistical difference was observed between the postocclusion values by one-way analysis of variance.

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Table 2. Fractional Volumes ofMitochondria (Vv,,it ), Myofibrils (Vvm,o ), and Remaining Cytoplasm (Vvc,,t ) in Normal and Ischemic Zones of Cat Hearts Subjected to 10, 20, 40, or 60 Minutes ofLAD Occlusion Time (minutes) Statistics Normal Ischemic Vvmit (%)

VVmyo (%)

VvcA (%)

10 20 40 60 10 20 40 60

27.64± 1.13 26.22 ± 0.87 24.66 ± 1.89 26.34 ± 0.92 66.50 ± 0.86 67.29 ± 0.45 69.58 ± 1.78 68.46 ± 1.37

10 20 40 60

Pnorm = 0.3481 5.34 ± 0.65 6.48 ± 0.87 5.76 ± 0.69 5.20 ± 1.16

++

++

++

++

++ +

Pno,m = 0.7270

32.41 ± 1.19 31.41 ± 0.65 31.74 ± 2.91 30.89 ± 1.24 60.15 ± 1.70 51.47 + 1.33* 55.30 + 1.08t 56.69 + 1.39t Pisch = 0.0069 7.44 ± 1.25 17.49± 1.11* 12.97 + 2.69t 12.42 + 1.76t Pisch - 0.0163

P,ime = 0.6533 Pzone = 0.0003 Pint =- 0.8189

Ptime = 0.0838 Pzone < 0.0001 Pint 0.0067

Ptime = 0.0237 Pzone < 0.0001 Pint = 0.0375

Mean values ± SEM. Statistics by two-way, four-level nested analysis of variance. P values denote probabilities between zones (Pzme), over time (P) and for interaction between zone and time (Pij). When P,, < 0.1, statistics by one-way, four level nested ANOVA for each of the four groups separately: + denotes P < 0.05, ++ denotes P < 0.001 and statistics by one-way, four-level nested ANOVA for each zone separately. P-values denotes probability for difference between the normal zones (P,,',m), and between the ischemic zones (Pi,,). When P,,,,, or Pi,,h < 0.05, individual comparisons were performed between means from each group by a Newman-Keuls multiple comparison procedure: * significantly different from the 1 0-minute group, t significantly different from the 10- and 20-minute groups, t significantly different from the 20-minute group.

There was no significant difference in Vvcyt between the ischemic (7.44% ± 1.25%) and normal zones (5.35% ± 0.65%) of the 10-minute group. After 20 minutes of ischemia, Vvcyt increased significantly in the ischemic zone (17.49% ± 1.11%) and even exceeded the values found after 40 and 60 minutes of ischemia. There was a significantly higher Vvmyo and lower Vvcyt in the sham group than in the normal and ischemic zones of the 10minute group (Figure 5), indicating a slight cytoplasmic edema also in the normal zone 10 minutes after LAD occlusion. There was an increased Vvlip in ischemic zones as compared to their corresponding normal zones (Table 3, Figure 4). There was a clear time trend for Vvlip in both normal and ischemic zones with prolonged ischemia, with a regression coefficient of -0.005% per minute18 (Table 3). Vvlip in the sham group (0.55% ± 0.07%) was not different from Vvlip in the normal zone of the 10-minute group (0.53% ± 0.1 1%). Both mitochondrial surface-to-volume ratio and surface density were lower in ischemic zones as compared to the normal zones of all groups (Table 4). Furthermore, they had significantly larger numerical values in the sham group (1.86 ± 0.03 ,um2/llm3 and 7.29 ± 0.20 4m2/Mm3, respectively) than in the normal zone of the 10-minute group (1.60 ± 0.11 gm2/MUm3 and 6.07 ± 0.43 gm2/,Am3, respectively) (Figure 5, Table 4). Despite no difference in Vvmit between the sham group and the normal zone of the

10-minute group, these findings indicate chondrial swelling in the normal zone.

a

slight mito-

Discussion After coronary artery occlusion there is a time-dependent development of ischemic injury in hypoperfused tissue. In the present study we observed after 10 minutes of ischemia an increased fragility of the sarcolemma, swelling of mitochondria, and accumulation of lipid droplets. These features are followed by increased cytoplasmic edema and sarcolemmal fragmentation with prolonged ischemia, whereas the fractional volume of mitochondria does not increase further. Regional ischemia induces morphometric alterations also in normal zones, already appearing after 10 minutes of ischemia. Scant cytoplasmic edema and swelling of mitochondria here might be due to the added stress on functional cells. There is no reason to believe that normally perfused tissue in the other time groups is different from the 10-minute group as evident from the statistical analyses in Tables 2 and 4. We have observed similar differences between sham-operated cats and normal zone in cat hearts with 3 hours of LAD occlusion.8 The early alterations in nonischemic parts of hearts with regional ischemia need further investigation as the mechanisms involved and their significance are still unknown.

280 Greve et al Al/9February 1990, VoL

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5a 0/0

p =0.0008

100 r

t *- -

80 I

60 F

4OF

p=0.0058 i-- * -4

M1+11-*-l

p = 0.0282

20

-*+NSV

Vvmit

vvcyt

VVmyo

pm2/ pm3 10

8r_

.b

P;0:0024 *+*-

6F

4k

p = 0.0342

-.

*

-I

211/ Vmit Surface density In the ischemic zone, even 60 minutes after LAD occlusion, there were no signs of contracture and the myofibrillar structure remained unchanged. Thus reduced Vvmyo and increased Vvcyt indicate that the myocytes themselves were swollen, resulting in a reduction of the fractional volume of myofibrils. Within the initial 15 to 20 seconds of ischemia, there is a shift in metabolism from mainly oxidation of fatty acids to glycolysis because all steps in the use of lipids, especially beta-oxidation, are depressed.5 20 However, the glycolysis is unable to meet the demands for high-energy phosphates21-22 and a number of metabolic and ionic changes lead to increased intracellular osmolarity, which explains the cellular edema.23-27 Depending on osmotic pressure within various organelles, swelling may also occur in specific cell compartments to dissimilar degrees.

Figure 5. a: Fractional volumes of mitochondria (Vvm,i,) myofibrils (Vvm,o), and cytoplasm (Vvct,,) in sham-operated cat hearts 0, normal 0, and ischemic 0 zones of cat hearts with 10 minutes of LAD occlusion. b: Mitochondrial surface-to-volume ratio (I/Vmit) and surface density in sham-operated cat heartsE0, normal 03, and ischemic 0 zones of cat hearts with 10 minutes ofLAD occlusion. Mean values± SEM. Statistics by one-way, four-level nested analysis of variance. P values denote propability between zones. Individual comparisons between group means by a Newman-Keuls multiple comparison procedure. * Significant differences notedfor P < 0. 05.

Increased Vvmit and reduced mitochondrial surface-tovolume ratio in the ischemic zones are consistent with earlier qualitative descriptions of mitochondrial swelling during ischemia.28 We found that swelling starts early, and is evident after 10 minutes of ischemia. Later, there was no further increase in Vvmit, ie, the absolute volume of mitochondria changed roughly in parallel to total cell volume. Spinale et a17 have reported mitochondrial swelling in the right ventricle after 10 minutes of ischemia, and the proportion of damaged mitochondria continued to increase for 180 minutes, but no morphometric measurements were offered. The stabilization of the VVmit found in the present study after 10 minutes of ischemia could not be explained by loss of mitochondria because degenerative structural changes were not sufficiently extensive within the 60-minute period.

Morphologic Changes in Cat Hearts 281 AJP February 1990,

During ischemia, metabolic intermediates, including neutral lipids, accumulate and the lipid droplets seen in ischemic as well as in normal tissues probably include triglycerides.29 Vvlip is larger in ischemic tissue, especially along its lateral border, than in normally perfused tissue.9 10 20o30 In ischemia, there is a shift in the rate-limiting step from transport of fatty acids into the cell to mitochondrial oxidation,31 and intracellular accumulation may aggravate the ischemic injury.32 Vvlip increased in the ischemic zone 10 minutes after occlusion of the LAD, but there was a falling trend of Vvlip in both zones with prolonged ischemia. Although covariance analysis gives a significant regression coefficient, a strong conclusion is not warranted because of the ex post facto choice of hypothesis. An increase in terms of absolute values in the ischemic zone might be concealed by the more extensive cytoplasmic edema occurring 20 minutes after LAD occlusion. There are, as previously reported, also substantial variations in lipid content between cats and even myocytes, making interpretation of these findings difficult.9'10 Sarcolemmal damage is less pronounced in the ischemic zone of the 10-minute group than in groups with longer periods of ischemia, and the discontinuities are, to a great extent, strictly focal (Figure 2b). Such disruptions are accompanied by restricted subsarcolemmal cytoplasmic edema. With prolonged ischemia the sarcolemmal damage is more severe and is extensive rather than focal. Widespread fragmentation of the sarcolemma might, therefore, represent discontinuities occurring in vivo. Even though the swelling imposes added stress on the sarcolemma,33 cell swelling per se is, as suggested by Steenbergen et al,34 not capable of rupturing the membrane, but the prolonged energy deficiency injures the cytoskeleton and sarcolemma, which may predispose the membrane to rupture.35 37 Such a hypothesis is consistent with our results because the maximal cytoplasmic edema coincided with the largest increase in sarcolemmal damage. We may, therefore, speculate that as long as the sarcolemma remains intact, cell swelling continues. After sarcolemmal disruption, the electrolyte distribution across Table 3. Fractional Volumes ofLipid Droplets (%) in Normal and Ischemic Zones of Cat Hearts Subjected to 10, 20, 40, or 60fMinutes ofLAD Occlusion Time (minutes) Normal Ischemic Statistics 10

0.53 ±0.11

0.76 ±0.03

20 40 60

0.46 ± 0.07 0.31 ± 0.02 0.26 ± 0.04

0.53 ± 0.11 0.42 ± 0.07 0.50 ± 0.11

rt"

Ptr

ine int

006

0.006

0.01686 -

Statistics by two-way, four-level nested analysis of variance. P values denote probabilities between zones (Pz,,,,) with time (Pw,,,,) and for interaction between zone and time (P,t). From the analysis of covariance the regression equation may be written: Y = 0.638% 0.005 (X)% ± 0.309%, where Y is V\,p, X is duration of ischemia. The common regression coefficient is -0.005%/min, and ±0.309 is the standard error of estimate.17 The coefficient of determination is 87% (P = 0.02). -

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Table 4. Mitochondrial Surface-to-Volume Ratio (I/Vmi,) and Surface Density in Normal and Ischemic Zones of Cats Subjected to 10, 20, 40, or 60 Minutes ofLAD Occlusion Time (minutes) Normal Ischemic Statistics

I/Vm,t (.UM2/AlM3) 10 20 40 60

6.07 ± 0.43 6.56 ± 0.52 6.89 ± 0.22 6.93 ± 0.52

4.96 ± 0.35 Ptme - 0.7518 4.65±0.26 Pe