Profound structural alterations of the extracellular collagen matrix in postischemic dysfunctional (“stunned”) but viable myocardium

Profound structural alterations of the extracellular collagen matrix in postischemic dysfunctional (“stunned”) but viable myocardium

1322 lACC Vol. 10. No.6 December 19R7:1322-34 EXPERIMENTAL STUDIES Profound Structural Alterations of the Extracellular Collagen Matrix in Postisch...

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lACC Vol. 10. No.6 December 19R7:1322-34

EXPERIMENTAL STUDIES

Profound Structural Alterations of the Extracellular Collagen Matrix in Postischemic Dysfunctional ("Stunned") but Viable Myocardium MENGJIA ZHAO, MD, HONG ZHANG, MD, THOMAS F. ROBINSON, PHD, STEPHEN M. FACTOR, MD, FACC, EDMUND H. SONNENBLICK, MD, FACC, CALVIN ENG, MD Bronx. New York

Ultrastructural studies of the extracellular collagen matrix were made on the "stunned" myocardium using scanning, conventional and. high voltage transmission electron microscopy and light microscopy. Regional myocardial dysfunction was produced by 12 sequential 5 minute occlusions of the left anterior descending coronary artery, separated by 10 minute intervals of reperfusion. A final 90 minute reperfusion period documented persistent myocardial dysfunction. At the end of the final reperfusion period, the percent systolic shortening, measured by sonomicrometers, was depressed significantly to 35 ± 9% of baseline. The heart was then perfusion fixed, and samples were taken from both control and stunned areas. No changes associated with irreversible cellular damage were noted in the stunned region. However, scanning electron microscopy of the stunned area showed that the extracellular collagen matrix underwent profound structural changes. Collagen cables were roughened, uncoiled and discontinuous. Linear grooves on the surface of the myocytes were frequently seen, indicating complete loss

Ischemic injuries that do not result in myocardial necrosis can cause persistent mechanical dysfunction (I) that may persist for as long as I week (2). This delayed recovery of cardiac contractile function, after transient myocardial ischemia, has been termed "stunned myocardium" (3). The adenosine triphosphate (ATP) content of stunned tissue has From the Cardiovascular Research Laboratories, Division of Cardiology, Department of Medicine, Alben Einstein College of Medicine, Bronx, New York. This study was supported by Grants HL23171, HL272l9. HL37412 and HL24336 from the National Institutes of Health, an Established Fellowship and a Grant-in-Aid from the New York Heart Association and a grant from NIH Biotechnology Resources to the University of Colorado High Voltage E.M. Laboratory. Mengjia Zhao, MD and Hong Zhang, MD are visiting scientists from Henan Medical University, Zhengzhou, Henan, People's Republic of China. Manuscript received March 3, 1987; revised manuscript received June 3. 1987, accepted June 26, 1987. Address for reprints: Calvin Eng, MD, Cardiovascular Research Laboratories, Albert Einstein College of Medicine, Forchheimer Room 715. 1300 Morris Park Avenue. Bronx, New York 10461. ©19R7 by the American College of Cardiology

of collagen cables. The usual dense collagen weave surrounding myocytes became patchy or absent. Myocyte to myocyte struts were sparse and frequently absent, with remnant nodular or nublike structures indicative of breakage. High voltage electron microscopy of the stunned area showed that the collagen struts were discontinuous and vacuolated with rounded tips. Light microscopy of silver-stained sections of the stunned tissue demonstrated large patchy areas that were devoid of silver, indicating absence of the collagen matrix. There was a progressive increase in percent systolic bulging during each sequential coronary occlusion, suggesting increasing myocardial compliance. These results indicate that the myocardial collagen matrix is severely damaged from reversible ischemic cell injury. The greater myocardial compliance and less effectivecontractile effort in the stunned myocardium might be explained on a structural basis: disruption of the mechanical coupling function provided by the extracellular collagen matrix. (J Am Coli Cardiol 1987;10:1322-34)

been demonstrated to be initially depressed by 50% with only partial recovery to 80% of normal within :3 days (4). The prolonged biochemical abnormality was suggested as an explanation for the prolonged mechanical recovery. Similarly. a variety of other biochemical abnormalities have been postulated as the basis for the stunned myocardium: oxygen-derived free radicals (5), abnormal calcium transport by the sarcoplasmic reticulum (6), interruption of the creatine phosphate shuttle (7) and abnormalities of the cardiac sympathetic nerves (8). However, several studies (9,10) have shown that inotropic agents such as dopamine or epinephrine can stimulate the stunned myocardium to perform in a sustained manner at a level considerably above the baseline stunned level. The function of the stunned myocardium can also be selectively enhanced by augmentation of coronary blood flow to levels greater than normal using certain vasodilators (II). It appears from these findings that the contractile machinery is basically intact in the stunned 0735-1097/87/$3.50

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myocardium, biochemical abnormalities notwithstanding. Thus, a different process may be operative, A highly complex and widely distributed collagen network exists in the mammalian heart ( 12-14). Collagen struts connect myocyte to myocyte, with collagen weaves and cables binding together groups of muscle fibers. Along with the intercalated discs, the collagen matrix provides a mechanical coupling mechanism for individual cells and muscle bundles (15-17). Sato et al. (18) noted irregularities of the collagen struts after 20 minutes of myocardial ischemia and breakage and disappearance of the entire collagen network after 2 hours of ischemia in the porcine heart, In the pig, tissue necrosis occurs as early as at 20 minutes of ischemia (19). Thus, the findings of Sato et al. (18) regarding the collagen matrix changes may be secondary to the necrotic process. Caulfield et al. (20) reported the loss of the collagen matrix after 2 hours of coronary ligation in the dog heart and breakdown of the extracellular matrix in experimental myocarditis of the mouse (21). In both studies, tissue necrosis was noted. The effects of myocardial ischemia without overt tissue necrosis on the extracellular collagen matrix are not known, Because the collagen matrix may provide a mechanical coupling function for the myocytes and muscle bundles, disruption of the connective tissue could adversely affect mechanical performance. This sequence of events could provide a basis for postischemic myocardial dysfunction, In this study, we examined the ultrastructural changes of the collagen matrix in stunned myocardium using scanning electron microscopy, conventional and high voltage transmission electron microscopy and light microscopy. These combined techniques revealed marked changes in the collagen matrix of the stunned myocardium in the absence of tissue necrosis.

Methods Animal preparation. Seven mongrel dogs of both sexes, weighing 20 to 25 kg, were anesthetized with intravenous sodium pentobarbital, 30 mg/kg body weight. Additional pentobarbital was given as required. After intubation, ventilation was provided by an intermittent positive pressure respirator with 100% oxygen supplementation. A left thoracotomy was performed in the fourth left intercostal space and the heart exposed in a pericardial cradle. After administration of heparin, 10,000 U, a polyvinyl catheter was inserted into the femoral artery and passed retrograde into the thoracic aorta to measure systemic pressure. Left ventricular pressure and its first derivative (dP/dt) were measured with a catheter inserted into the left ventricular cavity through the left atrial appendage. The left carotid artery was cannulated with a stainless steel cannula attached to perfusion tubing. The left anterior descending coronary artery was dissected free, cannulated

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with a 13 gauge metal cannula, and perfused from the carotid artery. Coronary flow was measured with an extracorporeal flow transducer (model BLC-2064-FI5, Biotronex Laboratories) and electromagnetic flow meter (model BL41O, Biotronex Laboratories). A bypass circuit around the flow transducer permitted frequent mechanical zero flow reference without interrupting coronary perfusion, A port at the tip of the coronary artery cannula measured the perfusion pressure, Pressures were measured with Statham P23Db transducers with zero reference at the level of the right atrium. Pressure and flow measurements were recorded on a Beckman type SII multichannel recorder. The electromagnetic flowmeter was calibrated at the end of each experiment using a Harvard pump and autologous blood, Regional myocardial function was assessed by sonomicrometers implanted in both the left circumflex and anterior descending coronary artery perfusion regions, and connected to an ultrasonic dimension system (model 401, Schuessler and Associates). The ultrasonic crystals, 1.5 mm in diameter, were implanted into the subendocardial layer through stab wounds approximately 10 mm apart and perpendicular to the major axis of the left ventricle. The sites of the crystals were confirmed to be in the inner one third of the left ventricular wall at the time of tissue sampling. A 5 minute occlusion of the coronary perfusion line was followed by 10 minutes of reperfusion. This was repeated 12 times. The final reperfusion period was 90 minutes. Rapid recordings of hemodynamics (100 mm/s) were made at the end of each occlusion and reperfusion period, after zero references for flow, pressures and calibration of segment length measurements were made. Prolonged myocardial dysfunction using this protocol was confirmed by segment length measurements at the end of the final 90 minute reperfusion period. One dog developed ventricular fibrillation during the fourth and sixth reperfusion periods, and the signals from the ultrasonic crystals were altered by defibrillation, This dog was excluded from the results, giving a total of six dogs analyzed. Tissue sample processing. After the final 90 minute period of reperfusion, the heart was stopped with 20 ml of concentrated potassium chloride given intravenously. The heart was rapidly removed, along with a segment of ascending aorta, for coronary perfusion of fixatives. After infusion of 300 ml of phosphate-buffered saline solution (pH 7.3 to 7.4) into both the aorta and the left anterior descending coronary artery perfusion tubing, the heart was perfused with 500 ml of 2.5% phosphate-buffered glutaraldehyde (pH 7.3 to 7.4) for 10 minutes to ensure a uniform fixation. The entire heart was then transferred into fresh 5% phosphate-buffered glutaraldehyde (pH 7.3 to 7.4) and sliced from apex to base into transverse rings. Using topical landmarks of the perfusion territories, tissue samples from the center of the stunned and control regions were obtained while still immersed. Transmural samples were then divided

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into inner, mid and outer layers. Care was taken to avoid sampling near the sites of the ultrasonic crystals. While immersed, the samples were cut parallel to the muscle fiber orientation under a dissecting microscope (Olympus). Samples for conventional and high voltage transmission electron microscopy were about I mm in thickness, and samples for scanning electron microscopy were about 0.5 to I crrr' in area and 1 to 2 mm in thickness. Samples were then immersed in 5% phosphate-buffered glutaraldehyde (pH 7.3 to 7.4) at 4°C overnight and postfixed with I % osmium tetroxide (OS04) for I hour at room temperature. Another set of transmural samples from both stunned and normal areas was stored in 5% glutaraldehyde for light microscopy. Conventional transmission electron microscopy. Samples were stained en bloc with I% uranyl acetate for I hour at room temperature, dehydrated in a graded ethanol series and then cleared with propylene oxide. The samples were embedded in Epon-Araldite using flat silicone rubber molds with reference to the muscle fiber orientation. The embedded samples were placed in a 60°C oven for polymerization. At least four blocks from each tissue sample were trimmed and sectioned both longitudinally and transversely to the muscle fiber orientation into I /-Lm thick sections, stained with toluidine blue and examined by light microscopy. Ultrathin sections of 100 to 150 nm were made with a Reichert Ultracut microtome using glass knives, and picked up with 300 mesh naked copper grids. The sections were then stained with 4% uranyl acetate followed by I% lead citrate. Two or more grids of both longitudinally and transversely oriented sections from each sample were examined using a JEOL 100CXelectron microscope at an accelerating voltage of 80 to 100 kV.

Scanning electron microscopy. Samples were dehydrated in an ethanol series after postfixation with OS04' After critical point drying (Samdri 790) using liquid carbon dioxide as transitional fluid, samples were subjected to dry fracture, mounted onto stubs, sputter-coated with 180 A of gold in Denton (Desk-l-Cold-Sputter-Etch Unit) and examined using a JEOL JSM-25S scanning electron microscope at an accelerating voltage of 15 kY. In our experience, dry fracture results in significantly better extracellular structure exposure as compared with freeze fracture, which more frequently exposes the intracellular compartment. High voltage electron microscopy. The use of this technique permits the study of much thicker sections with high resolution than is possible in conventional transmissionelectron microscopy. This allows better sampling of the collagen struts. The de-embedding technique (22.23) was employed to enhance the contrast of the collagen struts without staining. Tissue from one dog was taken for high voltage electron microscopy. After being osmicated, samples were infiltrated with graded polyethylene glycol (mixture of 4,000 and 1,000 mol wt) at 60°C in an oven and embedded in pure poly-

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ethylene glycol in gelatin capsules. The polyethylene glycol blocks were then mounted on blank resin blocks for trimming and sectioning. The blocks were dry-sectioned into I /-Lm thick sections with glass knives and picked up with a loop containing 40% polyethylene glycol solution. The sections were then transferred onto poly-L-Iysinecoated grids that were coated with Formvar and carbon. Sections were de-embedded in double distilled water, dehydrated in an ethanol series, critical point dried and coated with carbon in an evaporator. Sections were examined using a JEOL 1000CXhigh voltage electron microscope at an accelerating voltage of 800 to 1000 kY. This microscope is located in the HYEM laboratory of the University of Colorado, Boulder. Light microscopy. Silver reactions. The silver stain employed in this study reacts with collagen fibers throughout the section thickness (17). This includes the epimysial and perimysial collagen fibers, collagen weaves surrounding individual myocytes, collagen struts between myocytes and the adventitia of the vessels. This technique is a modification of a method developed by Hasegawa and Ravens (24), which is in itself a modification of a silver carbonate impregnation technique described by Del Rio Hortcga (25). The glutaraldehyde-fixed tissue was rinsed in tap water, and cryosections approximately 60 to 80 /-Lm in thickness were cut and floated in distilled water for 5 to 10 minutes. Tissue slices were placed in absolute methyl alcohol, Folch's solution and absolute methyl alcohol. The sections were rinsed in distilled water and then immersed in 5% potassium hydroxide. The sections were washed in running tap water, placed in 11600 potassium permanganate, washed in tap water and bleached in 5% oxalic acid. Oxalic acid was removed by washing in running tap water and then distilled water. The sections were then impregnated with 50% Del Rio Hortega's lithium silver solution until they turned yellow-gray. They were rinsed in ammoniated distilled water and reduced with 0.25 to 0.5% neutral formalin, then rinsed in distilled water and toned in 1/500 gold chloride. Finally, they were placed in 5% sodium thiosulfate, rinsed in distilled water and mounted with water-soluble mounting medium.

Table 1. Hemodynamic Measurements in Six Dogs Control Heart rate (beat s/min) Aortic pressure (mm Hg) LVEDP (mm Hg) Coronary perfusion pressure (mm Hg) Coronary blood flow (ml/min)

135 103 5.3 95 27 .2

± 7 ± 6

± 0.7 ± 6 ± 7.1

Postischemic

119 ± 103 ± 5.7± 95 ± 20 .5 ±

8 5

1.1 5 3.7

Values are mean ± SEM ; p = NS for all comparisons between the control and the postischemic variables by paired Student's t tests. LVEDP = left ventricular end-diastolic pressure .

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Percent systolic shortening = (EDL - ESL)/EDL x 100 Percent systolic bulging = (Lm. , - EDL)/EDL x 100, where L ma , is the maximal systolic segment length during coronary occlusion. Results are expressed as mean ± SEM. Non-normalized segment length values are reported. Student's paired t tests were used for comparing hemodynamics between the control and postischemic states. One-way analysis of variance with repeated measures was employed for the comparison of regional myocardial function variables at different states. The Scheffe's test was used as a post-hoc test. A p value <0.05 was considered significant.

9 10 11 12

REPERFUSION NUMBER

Results

Figure 1. Percent systolic shortening of both ischemic and normal regions. Results of function during reperfusion periods are expressed as percent of the control baseline. The percent shortening of the ischemic region is significantly depressed after the third occlusion and reperfusion (p < 0.01 one way analysis of variance) and persists throughout the experiment. The 12th reperfusion (square) represents the percent shortening at the end of the final 90 minute reperfusion period. The percent systolic shortening in the normal zone during reperfusion intervals did not significantly change with time from the baseline (p = NS).

Hemodynamic measurements (Table 1). There were no statistical differences between control values and those obtained after the 90 minute postischemic period in all variables measured. Left anterior descending coronary artery blood flow during the final reperfusion period was slightly lower than the control value (p = 0.36), possibly owing to autoregulatory adjustment to decreased regional cardiac contractility. The heart rate was also lower during the postischemic period (p = 0.07). Regional myocardial dysfunction. Figure I shows the regional contractile function in the ischemic and normal regions. The percent systolic shortening in the ischemic region decreased to 56 ± 15% of baseline function during the first coronary reperfusion period. The percent systolic shortening was significantly depressed statistically at the third reperfusion period (28 ± 17% of baseline function, p <0.0 I, one-way analysis of variance) and this level of dysfunction persisted throughout the experiment. The percent systolic shortening after the final 90 minute reperfusion period was 35 ± 9% of baseline function (p < 0.0 I, oneway analysis of variance). There were no significant changes in myocardial function in the normal control region.

Hematoxylin-eosin stain. Transmural samples from both ischemic and normal regions were embedded in paraffin, sectioned and stained with hematoxylin-eosin for light microscopy. Observations were performed using a Zeiss Ultraphot microscope. Data analysis. Regional myocardial function was determined by measurement of the regional segment length changes in both control and ischemic regions. End-diastolic length (EDL) was measured at the time of initial rise of the first derivative of left ventricular pressure (dP/dt) at enddiastole, and the end-systolic length (ESL) at peak positive left ventricular dP/dt. Two indexes (26) were used to express regional myocardial function and were defined as follows:

Table 2 summarizes the data during sequential coronary

Table 2. Hemodynamic and Segment Length Measurements During 12 Coronary Occlusions in Six Dogs Occlusion I LVSP (mm Hg) LVEDP (mm Hg) EDL (mm) Lma> (mm) O/OSB

107 7 12.3 13.9 10.1

± ± ± ± ±

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± ± ± ± ±

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± ± ± ± ±

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*p < 0.001; tp = 0.01 when compared with first occlusion. One-way analysis of variance with repeated measures followed by Scheffes test. The analysis of variance was performed on data from all 12 occlusions. Data from the first, sixth and twelfth occlusions are summarized. Segment length data are non-normalized absolute values. EDL = end-diastolic segment length in stunned region; Lmax = maximal segment length in stunned region during coronary occlusion; LVEDP = left ventricular end-diastolic pressure; LVSP = peak left ventricular systolic pressure; O/OSB = percent systolic bulging.

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nificantly greater during the 12th occlusion (p < 0.001, one-way analysis of variance). Thus, the calculated percent systolic bulging during coronary occlusion showed a progressive increase with the cumulative number of coronary occlusions (Fig. 2). The percent systolic bulging at the 12th occlusion period was significantly greater than that during the first occlusion (p = 0.01, one-way analysis of variance). Because the left ventricular systolic pressure did not change significantly during the entire protocol, these data indicate a progressively increasing myocardial compliance in the ischemic region with cumulative occlusions.

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OCCLUSION NUMBER Figure 2. The cumulative effect of brief repetitive coronary occlusions on systolic bulging of theischemic region during coronary occlusion. The systolic bulging is progressively more severe as the number of ischemic insults increases. Because the left ventricular systolic pressure did not change significantly during the protocol (Table 2), this suggests increased myocardial compliance.

occlusions. Peak left ventricular systolic pressure, end-diastolic pressure and ischemic end-diastolic segment length were not significantly different during the experimental period. The maximal ischemic segment length (Lmax ) was sig-

Conventional transmission electron microscopy. The ultrastructural criteria used for judging irreversible ischemic injury included the presence of any of the following: amorphous densities in the matrix space of mitochondria, subsarcolemmal blebs, disrupted sarcolemmal membranes or myocardial contraction bands (27-29). No evidence of such irreversible ischemic changes was found in the six dogs. Ultrastructural features present in the stunned myocardium were typical of reversible ischemic changes observed by other groups (27-31). These included nuclear chromatin clumping and margination, cytoplasmic glycogen depletion, extracellular and intracellular edema and wide I bands. In most of the sections from the stunned area the majority of the mitochondria were still densely packed with cristae and were virtually indistinguishable from those in the control region. Variable degrees of swelling with disorganization of the cristae were observed in a few mitochondria. Myo-

M

Figure 3. A representative scanning electron micrograph from normal tissue demonstrating theusual dense and florid collagen weave enveloping the individual myocytes. Also note abundant collagen struts (S) that interconnect myocyte (M) to myocyte, and myocyte tocapillary (C). The collagen struts are also connected to the collagen weave. Original magnification x 3,000; reduced by 32%.

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fibrils remained in register even though relaxed. The degree of the intercellular edema varied from heart to heart. There were no apparent morphologic changes in the capillary endothelium (32). Scanning electron microscopy. Representative views of the ultrastructure in the normal myocardial collagen matrix are shown in Figures 3 and 4. Smooth a~d coiled large collagencables enveloped groups of musclefibers. Collagen cables frequently branched into smaller cables and connected with the extensively distributed collagen weave that enveloped individual myocytes. Some segments of the cables were directly attached to the surface of the myocyte by collagen struts. In addition, a weave of collagen surrounded groups of muscle fibers and attached to collagen cables (Fig. SA). Numerous collagen struts interconnected the lateral surfaces of adjacent myocytes. The struts were anchored just lateral to the Z line at the cell surface and were also attached to the collagen weaves. Lateral struts connectingmyocytesand capillaries (33) were also abundant (Fig. 3). Most of these observations have been described previously (12-14,16,17). The scanning electron micrographs of the stunned myocardium revealed a remarkable structural transformation of the collagen matrix. Collagen cables were rough and irregular and had a notchedappearance (Fig. 6). In some samples the collagen cables were severely uncoiled with exposed linear grooves or indentations on the surface of the myocytes, suggesting a previously continuous cable overlying the indentation (Fig. 7). Residual fragments of cables were frequently seen in the grooves on the surface of the myocyte, indicatingcomplete degradation of collagen cable segments

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(Fig. 8). The usually dense arid florid collagen weave surrounding myocytes was patchy or absent in the stunned myocardium. In many areas the weave had a degraded appearance (Fig. 6 and 7). The degraded or remnant pattern of the weave was granular in appearance (Fig. 6 and 8). The perimysial collagen weave between muscle groups was almost completely absent (Fig. SB). The normally ubiquitous myocyte to myocyte struts were minimal or absent, with nodular or nublike structures indicative of breakage (Fig. 8). Changes in myocyte to capillary collagen struts were not as extensive. These degenerative features in the collagen matrix were consistently observed in all stunned myocardial samples in all layers of the ventricularwall. The linear grooves on the surface of the myocyteswere observed more often in the midwall samples. Using scanningelectron microscopy, Sage and Gavin (34) described a characteristic "open" appearance of the cytoplasm of necrotic tissue; this was not observed in the tissue samples. High voltage transmission electron microscopy. The thick sections of polyethelene glycol embedded tissue allowed for better resolutionand depth of field for the analysis of fine collagen structures. Tissue from only one dog was processed for high voltage transmission electron microscopy. In the normal myocardium, the extracellular structures were abundant and collagen strut connections between the lateral surfaces of adjacent cells were densely and uniformly distributed(Fig. 9). The latticelike collagen structures could be better appreciatedin stereo paired micrographs. At higher magnification, these struts were smooth with occasional nodular structures; an interwoven pattern was discerned. Some struts with sharp free ends were observed, indicating

Figure 4. A scanning electron micro-

graph from a nonischemic region showing normal extensive strut connections between the lateral surfaces of adjacent myocytes and regular pattern ofthe cell surface collagen weave. Collagen cables (arrowhead) are smooth and continuous. Cables branch off into smaller cables and also connect with the underlying collagen weave on the surface of the myocyte. A fibroblast (arrow) is seen to be in the actual process of laying down the collagen structures. Original magnification x 5.700; reduced by 30%.

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Figure 5. Comparison of the perimysium in normal and stunned myocardium from the same heart, A, From normal tissue showing the normal dense array of collagen fibers connecting two muscle layers, B, From the stunned myocardium demonstrating near absence of collagen fibers between muscle layers, The muscle layers appear to be completely dissociated from each other. Some remnant collagen fibers lie slack on the surface of the myocytes. Original magnification x 450; reduced by 31%,

sectioned ends of struts, In the stunned region, both the intercellular and intracellular spaces were markedly enlarged, Collagen struts between the myocytes were relatively sparse and not as evenly distributed as those in the normal region, A majority of struts in stunned areas were discontinuous and had rounded free tips (Fig. 10), Multiple electron lucent vacuoles on the intermyocyte collagen struts

were seen at high magnification. However, collagen struts between myocytes and capillaries were still densely distributed and were indistinguishable from those in normal control tissue. Light microscopy. A majority of the hematoxylineosin-stained sections of the ischemic tissue had normal or nearly normal light microscopic features, However, in a few

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Figure 6. Scanning electron micrograph from stunned myocardium. Several collagen cables are present; they are characterized by a rough, irregular and notched appearance, contrasting sharply with the normal cable appearance in Figure 4. The collagen weave is generally matted. There is a beaded and granular appearance of the weave suggestive of degeneration. Original magnification x 9,000; reduced by 30%.

sections from three dogs, rare foci of hypereosinophilia were noted. These areas involved no more than two to three myocytes and were mainly located in the subendocardial layers. The silver impregnation of normal tissue gave uniform staining of the collagen fibers in all of the sections examined. Collagen cables were extensively distributed with a smooth and frequently branching appearance. Weaves of collagen

fibers that encased large regions of the myocyte were clearly seen. Optical focusing through the entire thickness of the sections revealed that the weaves and wrapping fibers were pericellular. The silver-stained sections of the stunned tissue revealed widely distributed and clearly delineated patchy areas that were devoid of silver. These patchy areas were predominantly located in the subendocardial and midwall layers

Figure 7. Scanning electron micrograph from the stunned region demonstrating severe uncoiling of the collagen cables with distal degeneration. Not so apparent in this figure are the exposed grooves on the surface of myocytes just beneath degenerated segments of the cables. Original magnification x 630; reduced by 30%.

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Figure 8. Scanning electron micrograph of stunned myocardium reveals almost complete absence of the perimysial weave. Nublike structures (arrowhead) may represent remnants of broken collagen struts. Note the absence of myocyte to myocyte struts as compared to their abundance in Figures 3 and 4. Also of note on the surfaceof the myocyte bundles aregrooves (*) that represent the location of degenerated collagen cables. The arrows indicate discontinuous remnants of degraded cables. Original magnification x 2, 100: reduced by 30%.

(Fig. II). Although the changes in the subepicardial layers were less obvious, subtlechanges werealso observed. These included relatively fewer and smaller patchy areas of absent stainingand a clumpedor nodularappearanceof the collagen fibers. The patchy areasdevoid of silver presumably indicate a lack of collagen in these regions.

Figure 9. High voltage electron micrograph of I JLm thick polyethylene glycol de-embeddedsection from normal myocardium showingdensely and uniformly distributed strut connections between the lateral surfaces of adjacent myocytes. Note struts with sharp free ends indicating sectioned ends of the struts (arrows). The intracellular organelles are also densely packed. Original magnification x 75,000; reduced by 31%.

Discussion The major finding of this study is thai in the stunned but viable myocardium, the extracellular collagen matrix is markedly and profoundly altered. We employed a multiple coronary occlusion protocol to produce prolonged myo-

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Figure 10. High voltage electron micrograph from the stunned myocardium of the same dog demonstrates markedly enlarged inter- and intracellular spaces. The collagen struts are clustered in some areas. Also note that the free ends of the strutshaverounded tips (arrows), in contrast with those in Figure 9, suggesting a pattern of retracted collagen struts. Under higher magnification the collagen fibers have multiple vacuoles (not shown). The enlarged intercellularspaces are more easily appreciated in lower magnifications of transverse sections. Original magnification x 75,000; reduced by 31%.

cardial dysfunction. This procedure has been demonstrated by other investigators (10,35,36) to produce myocardial stunning without irreversible tissue damage. Persistent myocardial dysfunction was apparent at the end of the final 90 minute reperfusion period; percent systolic shortening was only 35% of baseline control function. Myocardial contraction was not completely lost, but was reduced substantially. The passive properties of the myocardium were also changed. The progressively more severe systolic bulging during coronary occlusion suggests that the stunned myo-

,

cardium became more compliant as the cumulative number of ischemic injuries increased. A possible explanation for this observation is that the connective tissue structures in the stunned myocardium were progressively degraded during the experimental protocol as evidenced by the results.

Structural Alterations in the Stunned Myocardium Myocardial viability. The viability of the ischemic myocardium was verified by conventional transmission elec-

Figure 11. Light micrograph of the silver-stained section from the stunned tissue showswell defined areasof patchiness which are devoid of silver. Note the clumped appearance of silver staining in other areas. Original magnification x 60. reduced by 29%.

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tron microscopy and light microscopy. The stunned tissue had reversible intracellular changes: swelling of the mitochondria, wide I bands, peripheral aggregation of the nuclear chromatin and variable degrees of myocardial edema. Features indicating irreversible cell damage (27-31,37) were not observed. Although hypereosinophilic staining within a period of::; 4 hours after ischemia is of unclear significance, we did find rare areas of hypereosinophilia in three of the six dogs. However, these areas were much smaller than the areas of patchiness in the silver stain and were unrelated to each other. Structural changes in the collagen matrix. The scanning electron microscopy results demonstrated profound structural changes of collagen matrix in the postischemic but viable myocardium. Collagen cables were roughened, uncoiled and sometimes discontinuous. Linear indentations on the surface of the myocytes were frequently observed, indicating complete loss of collagen cables. The usual dense collagen weave enveloping myocytes became patchy or absent. Myocyte to myocyte collagen struts were sparse and frequently absent, with remnant nodular or nublike structures indicative of breakage. These findings were supported by the high voltage transmission electron microscopy and silver stain techniques. The results showed that extensive structural changes in the extracellular collagen network occur before irreversible cellular damage. The loss of the collagen structures probably contributes to the myocardial "wavy fibers" as described by Bouchardy and Majno (38), which, as they concluded, does not necessarily imply irreversible ischemic injury. Thus, the wavy fibers may be a morphologic feature of myocardial fiber uncoupling at the light microscopic level. However, we did not observe any overt waviness on light microscopy in the stunned myocardium. The different fixation methods (perfusion versus immersion) probably contributed to the differences in results. Etiology of the collagen changes. The extracellular collagen matrix underwent severe degradation of the magnitude reported by Sato et al. (18) for necrotic conditions. These investigators studied the effects of coronary occlusion on the collagen network in the porcine heart. They noted irregularities of collagen struts at 20 minutes of ischemia, breakage and disappearance of collagen and elastin at 40 minutes of ischemia and near absence of the entire collagen network at 120 minutes of ischemia. In the pig, there is evidence of tissue necrosis at 20 minutes of coronary occlusion (19). Thus, the findings of Sato et al. (18) regarding the collagen matrix may be secondary to the necrotic process as such. Cannon et al. (39) demonstrated collagen degradation at 24 hours after myocardial infarction, which was hypothesized to be mediated by infiltrating inflammatory cells in the necrotic tissue. In addition, with tissue necrosis, intracellular enzymes are activated and released into the interstitium and may cause the degradation of collagen fi-

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bers. However, in our study, there was no evidence of tissue necrosis. Importantly, no inflammatory infiltration was observed in the stunned region by light microscopy. Thus, the collagen changes found in the stunned myocardium are not likely to be explained by either mechanism. The normal myocardium is continuously deformed throughout the cardiac cycle (40). The various layers of the ventricular wall contract differently, and a shearing strain occurs in the wall during contraction (41). Occlusion of a coronary artery results in not only the loss of contraction, but actual systolic bulging of the myocardium (26,42). The resulting abnormal stress and shearing forces (26,43) are likely to impact on the underlying fine structure of the collagen network. Although pure mechanical stress forces could potentially break fine strut structures as well as large cables, the general appearance of the collagen matrix loss suggests additional factors. The loss of the collagen weaves that surround myocytes cannot be readily explained by pure mechanical stress and shearing. The observation of roughening and nicking of the collagen fibers also supports a degradation process. The ischemic process may activate endogenous collagenase, perhaps of extracellular origin. Alternatively, electrolyte abnormalities during ischemia might also contribute to the loss of structure of the collagen matrix (44). Potential effects of structural changes in the collagen network on myocardial performance. Structural degradation of the extracellular collagen matrix could be an important contributing factor in the functional abnormalities of the "stunned myocardium." The heart consists of a population of force-generating cells organized for the production of tension during systole, and an orderly reversal of geometric changes during relaxation. To accomplish this, the individual contracting cells must be coordinated with each other in a well controlled manner. It has been generally assumed that the major structures involved in the transmission of force from myocyte to myocyte are the intercalated discs. However, Winegrad and Robinson (15) demonstrated that in a ventricular muscle bundle with intercalated discs opened by the calcium chelator ethyleneglycol tetraacetic acid (EGTA), force generation was not impaired. This study suggested that the extracellular collagen matrix could be a major force-bearing structure during the coordinated contraction of individual myocytes. The heterogeneous loss of these collagenous force-bearing structures may reduce optimal myocardial performance during systole by uncoupling cells and muscle fibers. Myocytes in isolation cannot generate force without a coupling mechanism. The augmentation of contractile performance with inotropic agents in the stunned myocardium (9,10) is not inconsistent with this coupling hypothesis. As long as complete extracellular decoupling does not occur and individual myocytes can respond appropriately to inotropic stimulation, better overall performance can result. The microscopic findings in this study indicate spatially heterogeneous collagen loss.

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The collagen matrix could also affect diastolic events, Borg et al. (16) correlated the density of the collagen bundles with ventricular stiffness as a function of age and species, They found that the collagen matrix in newborn hamsters and rats was virtually absent and developed to normal form in 15 days. Myocardial stiffness changed along with the structural changes, Caulfield (45) and Weber et al. (46) recently showed a marked increase of the collagen matrix in hypertrophied human and primate hearts, This alteration in the collagen network could contribute to the increased ventricular stiffness in hypertrophied hearts, In contrast, a marked decrease of the collagen network in stunned myocardium should then result in increased myocardial compliance. Several studies have demonstrated that there is an early increase in myocardial compliance during ischemia (47,48). The progressive increase in systolic bulging found in our study suggests such an increase in myocardial compliance in the ischemic region, The compliance changes are presumably due to a mechanical uncoupling of passive muscle properties. The collagen matrix serves a coupling function at all levels of the myocardium: between myocytes, between muscle fibers and between layers of muscle cells, A partial uncoupling of the force-generating structures from the force-bearing structures would be expected to impact on both passive and active changes in mechanical properties and performance. In addition, the prolonged time course (measured in days) required for functional recovery of stunned myocardium may reflect the time course of resynthesis or remodeling of these collagenous components. The selective destruction of the extracellular collagen matrix could be a primary mechanism involved in ventricular dilation during ischemic cardiomyopathy, Substantial damage to the collagen matrix could be followed by "remodeling" of the ventricular geometry into a larger heart size. This scenario may result in a load on the normal region by means of Laplace's law, with further positive feedback volume changes. We thank Herbert Parker and Walter Leon for excellent technical assistance in animal preparations and Regina Dominitz for light microscopic preparation. We are grateful to Jane Fant, Frank Macaluso and Leona CohenGould for advice and use of facilities. We would like to thank Mircea Fotino, George Wray, Mary Morphew of the HVEM laboratory of the University of Colorado, Boulder, for guidance and assistance in use of high voltage electron microscopy technique, and John Wolosewick, PhD, of the Department of Anatomy of the University of Illinois, Chicago, for advice in the polyethylene glycol embedding technique.

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