Impact of Delayed Reperfusion of Myocardial Hibernation on Myocardial Ultrastructure and Function and Their Recoveries After Reperfusion in a Pig Model of Myocardial Hibernation

Impact of Delayed Reperfusion of Myocardial Hibernation on Myocardial Ultrastructure and Function and Their Recoveries After Reperfusion in a Pig Model of Myocardial Hibernation Chunguang Chen, MD,*,†,§ Jing Liu, MD,*,‡ Dongping Hua, MD,*,‡ Lijie Ma, MD,*,† Tianjie Lai, MD,* John T. Fallon, MD, PhD,储 David Knibbs, PhD,*,† Linda Gillam, MD,*,† Judy Mangion, MD,*,† Delvin R. Knight, PhD,** and David Waters, MD*,† *Division of Cardiology, Hartford Hospital, Hartford, Connecticut; †University of Connecticut School of Medicine, Farmington, Connecticut; ‡Division of Cardiology, Department of Medicine, University of Medicine and Dentistry New Jersey, Newark, New Jersey; §Non-Invasive Cardiac Laboratory, Newark Beth Israel Medical Center, Newark, New Jersey; 储Department of Pathology, The Mount Sinai Medical Center, Mount Sinai School of Medicine, New York, New York; **Pfizer Central Research, Groton, Connecticut

ⴙⴙ This study examined the effect of delayed reperfusion of myocardial hibernation from 24 hours to 7 days on myocardial ultrastructural and functional changes and their recoveries after reperfusion. Background: We have previously shown in pigs that after reperfusion the functional and structural alterations in short-term myocardial hibernation which was reperfused in 24 hours can recover in 7 days. The effect of delayed reperfusion of hibernating myocardium on the extent and severity of cellular and extracellular structural changes of hibernating myocardium, and their recoveries after reperfusion is not known. Methods and Results: A severe LAD stenosis was created in 27 pigs, reducing resting flow by 30–40% immediately after placement of the stenosis and producing acute ischemia as evidenced by regional lactate production, a decrease in regional coronary venous pH, reduced regional wall thickening (from 38.5 ⫾ 5.1% to 10.4 ⫾ 8.0%) and a 33% reduction of regional oxygen consumption. The stenosis was maintained either for 24 hours in 9 pigs (group 1) with LAD flow of 0.65 ⫾ 0.13 ml/min/g (38% reduction), or for 7 days in 17 pigs (group 2) with LAD flow of 0.67 ⫾ 0.14 ml/min/g (36% reduction). There were no differences (p ⫽ NS) in the reduction of wall thickening, rate-pressure product, lactate production, or regional oxygen consumption between group 1 and group 2. Quantitative morphometric evaluation of the ultrastructure on electromicrographs revealed a greater decrease in sarcomere volume and a higher incidence of myocytes with reduced sarcomere volume in 7-day than in 24-hour hibernating regions (53 ⫾ 19% versus 33 ⫾ 14%, p ⬍ 0.05). Patchy myocardial necrosis with replacement fibrosis was common, but 6 of the 18 pigs had no myocardial necrosis or replacement fibrosis in the 7-day hibernating group, and 4 of 9 pigs had no patchy myocyte necrosis in the 24 hour hibernating group. In 6 pigs in group 1 in which the stenosis was then released and hibernating myocardium reperfused in 24 hours, regional wall thickening recovered to 30 ⫾ 6% (p ⫽ NS compared to baseline) after one week of reperfusion. In 12 pigs in group 2 in which the stenosis was released and hibernating myocardium reperfused in 7 days, regional wall thickening recovered slowly, from 10.1 ⫾ 7.2% to 18.1 ⫾ 8.3% at one week (n ⫽ 5) and to 28.0 ⫾ 3.6% at 3–4 weeks of reperfusion (n ⫽ 7, p ⬍ 0.05 compared to baseline). Simi-

Manuscript received October 15, 1999; revised January 18, 2000; accepted February 1, 2000. Address for correspondence: Chunguang Chen, MD, Cardiac NonInvasive Laboratory, Newark Beth Israel Medical Center, 201 Lyons Avenue, Newark, NJ 07112. Tel: (973) 926 6535; Fax: (973) 318 7207; E-mail: cchenSBHCS.com. This study was presented in part at the 68th Annual Scientific Session Cardiovascular Pathology Vol. 9, No. 2, March/April 2000:67–84  2000 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

of American Heart Association, November, 1995, and was supported in part by a grant from the National Heart, Lung and Blood Institute (RO1HL63688-01), a Grant-in-Aid Award from American Heart Association, Connecticut Affiliate with fund contributed by the New Jersey Affiliate, the Hartford Hospital Research Fund and Foundation of University of Medicine and Dentistry New Jersey.

1054-8807/00/$–see front matter PII S1054-8807(00)00029-6

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larly, the sarcomere volume or myofilament recovered significantly (p ⬍ 0.01) and was not different compared to the normal region (p ⫽ NS) in the 24-hour hibernating region of group 1, but the recovery was much slower and was incomplete at 4 weeks (p ⬍ 0.01) compared to baseline in the 7-day hibernating region of group 2. Recovery of regional wall thickening correlated with ultrstructural recovery (p ⬍ 0.01). By multivariate stepwise regression analysis, the degree of LAD flow reduction, the extent of fibrosis, and myofilament loss were independent predictors of the extent of functional recovery. Conclusions: In a porcine model of myocardial hibernation with myocardial hypoperfusion, systolic dysfunction, and metabolic adaptations, a longer period of myocardial hibernation with delayed reperfusion was associated with more severe abnormalities of myocytes. an increasing interstitial fibrosis, and more protracted myofibrillar and functional recoveries after reperfusion. The extent of functional recovery is related to the degree of coronary flow reduction, the severity of the ultrastructural changes, and the extent of interstitial fibrosis. Cardiovasc Pathol 2000;9:67–84 © 2000 by Elsevier Science Inc.

Recent studies (1–10) in experimental animals and in patients with severe coronary disease have shown that regional left ventricular (LV) dysfunction as a consequence of hibernation can recover after successful revascularization. However, the time required for recovery differs substantially among reports from immediate recovery to a late recovery in several months (4,8,9). Furthermore, the extent of recovery also varied significantly from near complete recovery to modest recovery in regional LV systolic function (4,8,9). The mechanism of the recovery and the factors related to differences in recovery have not been well understood (11,12). We have demonstrated previously that reperfusion of hibernating myocardium within 24 hours after creating coronary stenosis has led to almost complete recoveries of function and ultrastructure within a week (10). However, clinically, in patients with chronic ischemic heart disease, myocardial contractile functional recovery often may take weeks or months after reperfusion of hibernating myocardial region, and ultrastructural damages of myocytes in the hibernating region appear more severe compared to short-term myocardial hibernation in dogs of the study of Matsuzaki et al. or in pigs of our previous study (6–8,10). Furthermore, the improvement of LV ejection fraction indicating myocardial contractile functional recovery is often modest and incomplete in patients. Whether these differences are related to the duration of hypoperfusion, the severity of hypoperfusion or interspecies differences is not known (6–12). This study was, therefore, designed to test the hypothesis that more extensive ultrastructural abnormalities develop in a longer period of myocardial hibernation than in the short-term myocardial hibernation with a similar degree of hypoperfusion or severity of coronary stenosis. As the severity of the abnormality of the contractile ultrastructure increases, the time required to regenerate the structure may also increase, and thus, the time needed for recovery will also lengthen. Additionally, the structural abnormalities may not recover completely, resulting in incomplete recovery of contractile function. This might be particularly true if it is associated with an increasing degree of fibrosis in the prolonged hibernating region.

We tested the above hypotheses in a previously established porcine model of myocardial hibernation in which myocardial hibernation was maintained for either 24 hours or 7 days with an identical degree of reduction in myocardial blood flow, and was followed by either 1 week or 3–4 weeks reperfusion to evaluate differences in functional and structural abnormalities and their recoveries with various durations of hibernation and subsequent reperfusion. By continuous monitoring of coronary flow and hemodynamics, this study should also shed light on two controversial issues: whether experimentally produced short-term myocardial hibernation can be sustained for a prolonged period, and whether persistent or episodical hypoperfusion leading to myocardial hibernation or repeated stunning is the cause of sustained myocardial dysfunction in this pig model.

Methods Animal Preparation The study was approved by the Committees on Animal Care at Hartford Hospital, Newark Beth Israel Medical Center and New Jersey Medical School. The animal care guidelines of the American Heart Association were followed. Twenty–seven Yorkshire pigs weighing 28–48 kg were studied using techniques previously described in detail (9,10). General anesthesia and normal arterial blood gases were maintained with isoflurane (0.5–1.5%) and a 40:60% oxygen-nitrous oxide mixture. The isoflurane concentration was titrated to suppress the pain reflex but without deeper anesthesia to minimize the dose-dependent cardiodepressive and ventilatory effects of isoflurane and to retain dynamic coronary autoregulation. A midline thoracotomy was performed and the heart was suspended in a pericardial cradle. Left ventricular, left atrial, and arterial blood pressures were monitored with fluid filled catheters. Full anticoagulation was achieved with heparin 200 IU/kg i.v. and was maintained by 30 IU/kg i.v. every hour. A 3–4 F coronary perfusion catheter was advanced retrograde via the coronary sinus to the proximal interventricular vein running parallel to the LAD to monitor oxygen content, lactate, and pH.

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The LAD was carefully dissected free over 1 cm to accept a flow probe (Transonic Inc., Ithaca, NY) to measure coronary flow. The cuff of the flow probe was carefully aligned parallel to the vessel to ensure accurate measurement. To prevent local coronary spasm due to surgical manipulation, 3% lidocaine drops were intermittently applied at the proximal LAD site of manipulation. The LAD stenosis was created either by gradually filling a hydraulic occluder (total n ⫽ 12, n ⫽ 4 in group 1, n ⫽ 8 in group 2) around the LAD with saline or by silk ties (total n ⫽ 14, n ⫽ 5 in group 1 and n ⫽ 9 in group 2) with a 22 gauge arterial needle to create a graded stenosis to reduce flow by 30–40% as described previously. In pigs in which the hydraulic occluder was applied for creating the stenosis, coronary flow at times returned to baseline after an initial inflation of the hydraulic occluder. To maintain a relatively stable flow reduction of 30–40% in those pigs in which coronary flow was continuously monitored by an implanted flowmeter, the hydraulic occluder was adjusted hourly in group 1 and daily in group 2.

Experimental Protocol Two groups of 27 pigs were studied. Baseline measurements of wall thickening by echocardiography, heart rate, LV and aortic pressures, regional coronary flow and coronary venous lactate, pH and oxygen content were obtained under stable conditions as described previously (10). LAD flow was reduced by 30–40% from baseline and the reduction was maintained for 24 hours in 9 pigs (group 1) or 7 days in 18 pigs (Group 2) as shown in Figure 1. The stability of the stenosis was verified by hourly flow monitoring for 5 pigs in group 1 and for 8 pigs in group 2. In the remaining pigs, measurements of coronary flow were obtained at baseline and at 15 minutes, 30–60 minutes, 24 hours and 7 days under the same conditions of anesthesia. Morphine of 10 mg was given intravenously immediately after placement of the LAD stenosis with the assumption that pigs may suffer from ischemic chest pain. After verification that the LAD stenosis was stable for more than 30–60

Figure 1. Study design and protocol diagram.

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minutes, the chest was closed in layers with the pericardium left open. The animals were then allowed to recover in their cages. Aspirin (325 mg) and intravenous heparin (20,000 IU/24 hours) were given postoperatively to prevent thrombotic coronary occlusion. The LAD coronary flow, blood pressure, and heart rate were monitored hourly in 5⁄9 pigs of group 1 and daily for at least 1 hour/each day for 7 days in 8⁄17 pigs in group 2 under conscious sedation achieved by intravenous Versed of 0.05–0.3 mg/kg or Valium 0.5–3.0 mg/kg. After 24 hours in group 1 or 7 days in group 2, the animals were restudied. The catheters and flow probe were checked or reinstalled in the same positions as before, and all measurements were repeated under the same anesthesia. In group 1, after 2 transmural myocardial biopsies each from the hibernating and normal regions were obtained in 6 pigs, the stenosis was released to reperfuse the hibernating region. Three pigs in group 1 were sacrificed and myocardial samples were obtained at the autopsy at 24 hours without reperfusion. In group 2, six pigs were sacrificed and myocardial samples in the anterior and inferior regions at the mid-papillary muscle level were obtained. In the 12 remaining pigs, myocardial biopsies were taken from the same regions using a Tru-Cut biopsy needle (20-mm 14-gauge, Travenol Laboratories). The LAD stenosis was then released to reperfuse the hibernating region. In all pigs with reperfusion, the chest was closed in layers with the pericardium left open. Daily monitoring of regional wall motion was performed with echocardiography. In group 1, pigs were kept only for an additional 7 days because daily echocardiographic monitoring showed that the wall thickening in the hibernating region recovered almost to a baseline level by 7 days after reperfusion. In group 2, 5 pigs were kept alive for an additional 7 days after reperfusion and the other 7 pigs for 3–4 weeks to document various recoveries of regional myocardial function and ultrastructure after reperfusion. In these 12 animals regional LV wall thickening was monitored daily for the first week and weekly thereafter by transthoracic echocardiography. Echocardiograms were repeated at the end of the experiment to obtain high quality

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echocardiographic images. The heart was harvested for morphological evaluations with gross inspection after triphenyl tetrazolium chloride staining, light and electron microscopic examinations.

Echocardiographic Measurements Two-dimensional echocardiography was performed from the epicardial surface of the right ventricle or conventional transthoracic windows to evaluate regional wall thickening in the short-axis view at the LV mid-papillary muscle level. Identical echocardiographic views were obtained before and after creation of the LAD stenosis, at 7 days, after release of the stenosis, and before sacrifice (Figure 2). Wall thickness was measured at the mid-papillary muscle level (9,10). Regional LV wall thickening was calculated as end-systolic minus end-diastolic wall thickness divided by end-diastolic wall thickness, expressed as a percentage. LV volumes were measured using the area-length method and LV ejection fraction was calculated (13). All echocardiographic measurements were performed independently by 2 observers and the mean values of their measurements are presented.

Regional Myocardial Blood Flow LAD flow was measured using a cuff flow probe connected to a transonic flowmeter. At the conclusion of each experiment the flowmeter was calibrated against a known flow rate to ensure accuracy. Methylene blue was injected into the LAD and the stained tissue was dissected and weighed to determine the regional myocardial mass perfused by the stenotic LAD. Coronary blood flow is expressed as ml/min/g of wet tissue (10). In 11 pigs myocardial blood flow was also measured in the LAD region using a reference sample colored microsphere technique. Yellow, red, or blue 15-␮m Dye-trak microspheres (Triton Technology, Inc., La Jolla, CA) were randomly selected and infused over 30 seconds at baseline, 15 minutes, and 24 hours or 7 days after placement of the LAD stenosis. Reference blood samples were withdrawn via the arterial catheter starting 20 seconds prior to, and continuing for 120 seconds after microsphere injection. At the end of the experiment, myocardial tissues were harvested and weighed and all samples were placed in coded vials that were analyzed by observers unaware of the experimental protocol. The colored microspheres were recovered from digested tissues, quantified spectrophotometrically, and used to calculate myocardial blood flow (ml/min/g of wet tissue) via a previously described dye extraction microsphere technique (14).

Myocardial Metabolic Measurements Arterial and coronary venous blood samples were obtained anaerobically in cold, dry syringes containing heparin fluoride to inhibit glycolysis. Samples were divided for

Figure 2. Two-dimensional echocardiograms show an example of LV cross-sections at mid-papillary muscle level at baseline, immediately after placement of the stenosis and 7 days with stenosis and 3 weeks after release of the stenosis. Abbreviations: ED ⫽ enddiastole; ES ⫽ endsystole. Note that there was severe LV dysfunction after LAD stenosis with akinesia in the anterior and anteroseptal walls (arrows). The severe LV dysfunction and the akinetic walls were not changed at 7 days with LAD stenosis. By 3 weeks of reperfusion, regional wall thickening in the anterior wall and anteroseptum improved although not to normal.

blood gases, glucose, and lactate content, stored in ice and processed immediately after the experiment. Blood gases were measured in duplicate and the values were averaged. Plasma for lactate content was measured using the enzymatic method. Regional myocardial oxygen consumption and lactate production/consumption were calculated as described previously (10).

Pathological and Histochemical Morphology Gross and light microscopic examinations. After the animals were sacrificed and methylene blue was injected into the LAD distal to the stenosis to delineate the area at risk, the LV was cross-sectioned at 0.5-cm intervals from apex to base. The area at risk was dissected and weighed. The LV sections, including both at risk and non-risk re-

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gions, were then immersed in a 0.09-M sodium phosphate buffer (pH 7.4) containing 1.0% triphenyl tetrazolium chloride (TTC) for 15 minutes at 37⬚C to identify myocardial necrosis. Myocardium with deep red staining by TTC was considered viable, and unstained myocardium by TTC was deemed necrotic. In pigs with patchy necrosis (not stained by TTC), total surface area, necrotic area and normal area of each LV section in regions supplied by the LAD (stained by blue due) were traced on a transparent paper. The infarct size for each pig was calculated by integrating necrotic areas from all LV sections and expressing them as a percent of the total area at risk. All LV sections, including areas with patchy necrosis by TTC, were then fixed with 10% formalin, embedded in paraffin, sliced into 5-␮m sections and stained with hematoxylin and eosin (H&E) and trichrome for light microscopic examination. On a commercially available computer integrated digital microscopic system (Image-Pro Plus, Silver Spring, MD), with a magnification of 100 times the extent of myocardial necrosis with granular tissue and replacement fibrosis was quantitatively evaluated by tracing the area of necrosis/fibrosis and total area at risk in all consecutive LV sections from the apex to base and expressed as % of area at risk. Electron microscopic examinations. The transmural myocardial samples which measured 3 mm ⫻ 7–10 mm obtained by biopsy or autopsy from the hibernating and control regions were evaluated. The biopsy site was sealed by a suture which also served to identify the site for obtaining later samples at autopsy to evaluate serial changes. The specimens were immediately prepared for examinations on electron microscopy as described previously (10). Assuming the myocardial structure to be partially anisotropic, special care has to be taken to generate isotropic conditions for the sectioning of biopsy samples that allow the application of common rules of stereology. Therefore, each transmural sample was divided into 6 tissue blocks which include subendocardial, middle, and subepicardial layers. Each tissue block was embedded into a sphere and rolled in random section, which allows measurement of the myocardial structure without bias. All tissue blocks and sections were evaluated. Transmural myocardial samples of 3–4 mm ⫻ 6–10 mm from the region where myocardial biopsies were taken after 24 hours or 7 days of LAD stenosis were also taken and fixed with glutaraldehyde and prepared as described above for electron microscopy to evaluate recovery of the ultrastructure after reperfusion. To evaluate quantitative changes of cellular organelles in myocytes, a morphometric technique for quantitative electron microscopy was used according to a previously described technique with minor modifications for this study (15,16). Myocytes for morphometry were selected according to the arbitrary sampling method advocated by Weibel (15). From each grid, 8–10 sequential squares were selected. From each of these, all myocytes with nuclei were selected from each of the selected sequential squares along

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the grid on electron microscopy, photographed and printed with ⫻3700 magnification. For each heart, 120–150 myocytes were randomly selected and evaluated in each normal and LAD-perfused region from all available blocks of the transmural biopsy samples. The principle of a box-counting planimetry system was applied as described previously (7,10). Briefly, a grid system consisting of vertical and horizontal lines, providing 360 mini-squares (1 ⫻ 1 mm) on a transparent paper was used. The grid system was then superimposed on a selected myocyte on an electron micrographic print. The number of squares enclosed in a certain structure was counted. The squares intersected by the profile border were counted as fractions in proportion to the fraction covered by the structure. Counting of the number of points overlying a certain structure (organelle) in a cell resulted in quantitative determination of volume density of the structure under investigation in relation to total volume of a cell, which was estimated by the total squares a cell was overlying excluding nuclei. The volume density (fraction) of myofibrils, mitochondria, cytoplasma were calculated according to the established principle (Delesse’s) (15,16). Percentages were used to express the volume density (fraction) or the quantitative relation between entire cell volumes (excluding nuclei) and volumes of intracellular structures. Because myocytes in the hibernating region were not uniformly affected, the percentages of affected cells were also counted. Cells where myofibrils comprised less than 55% of the cell’s total volume excluding nuclei were classified as affected cells; the selection of 55% as the cut-off point was based on data from the normal control region where myofibrils occupied 63.8 ⫾ 4.9% of total cell volume in our previous study (10). Interstitial content or connective tissue was defined as tissues in between myocytes including collagen, interstitial cells, vascular structures, and edema in acute ischemia. In this study, epicardial or intramural vessels with their perivascular space were excluded from the measurement. Interstitial tissue volume in the hibernating and normal myocardium was determined by quantitative morphometry as described previously with the same commercially available computer-integrated digital microscopic system with a magnification of ⫻400 (6,7,9,10). For each pig, more than 120–150 fields were studied on 5–6 pathohistological slides of hibernating and normal regions from the apex to base of the LV. With the system, all myocytes and interstitial space were traced and area of interstitial or myocyte spaces on each microscopic field were integrated. Interstitial tissue volume density (fraction) was calculated as % of area occupied by interstitial tissue to the total area of interest including myocyte and interstitial spaces. Values from multiple measurements by the same observer in the hibernating or control region of each animal were averaged for analysis. Interobserver’s variability was evaluated by two observers who were not aware of each other’s results in 30 myocytes in the hibernating region, 10 myocytes in the normal region, and 30 microscopic fields for intersti-

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tial tissue content. There was no difference in myofilament volume (45 ⫾ 6% versus 46 ⫾ 7%, p ⬎ 0.05), mitochondria volume (22 ⫾ 5% versus 23 ⫾ 6%, p ⬎ 0.05), cytoplasmal volume (23 ⫾ 7% versus 21 ⫾ 6%, p ⬎ 0.05), or interstitial volume (16 ⫾ 4% versus 17 ⫾ 5%, p ⬎ 0.05) between two observers.

Statistical Analysis All parametric data are expressed as mean ⫾ 1SD. Parametric data among different stages was compared by ANOVA using a commercially available statistical software package (RS1, BBN Software Co., Cambridge, MA). If ANOVA revealed a signficiant difference, two stages were compared using a paired t test. A paired t test was also used to examine parametric data between normal control and hibernating regions. Corrections for multiple comparisons were applied using Tukey-Honestly significant difference test where applicable. Non-parametric data in normal and hibernating regions were compared by Chi-Square or Fisher’s test. Linear regression was used to assess correlations among parametric data. Multiple stepwise (forward) analysis was used to determine independent factors related to functional recovery. A p value of 0.05 or less was considered significant.

Results The hemodynamic, functional, and metabolic measurements at baseline, during the 24 hours or the 7 days of LAD stenosis and after reperfusion are depicted in Table 1. The small changes in heart rate and blood pressure that occurred during the study were not statistically significant.

Coronary Flow Reduction and Myocardial Hypoperfusion in Hibernating Regions In group 1, by flowmeter, the stenosis reduced LAD flow by 39% at 15 minutes while by color microsphere (n ⫽ 4), myocardial flow in the hibernating region decreased from 1.2 ⫾ 0.18 to 0.76 ⫾ 0.14 ml/min/g, a 37% reduction at 15 minutes. In group 2, by flowmeter, the stenosis reduced LAD flow by 39% (range 31–49%) at 15 minutes. At 7 days this reduction was maintained at 0.65 ⫾ 0.15 ml/min/g, a 36% decrease from baseline. Blood flow to the subendocardial LAD region was reduced from 1.27 ⫾ 0.42 ml/min/g to 0.73 ⫾ 0.30 ml/min/g 15 minutes after stenosis placement, a 43% reduction, to 0.75 ⫾ 0.34 ml/min/g at 24 hours, a 41% reduction in group 1, and from 1.25 ⫾ 0.32 ml/min/g at baseline to 0.70 ⫾ 0.41 ml/min/g at 7 days, a 44% reduction in group 2. The reduction in subepicardial blood flow was slightly less, from 1.08 ⫾ 0.34 to 0.80 ⫾ 0.45 ml/min/g immediately, to 0.89 ⫾ 0.51 ml/min/g at 24 hours, and to 0.90 ⫾ 0.43 ml/min/g at 7 days after stenosis placement (p ⬍ 0.05), a 17% reduction. There was no difference (p ⫽ NS) in flow reduction between group 1 and group 2.

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Stability and Fluctuation of the Coronary Flow Reduction Coronary flow variations during the 24 hour to 7 day period of stenosis were assessed in 13 pigs with an implanted LAD flowmeter. Hourly or daily measurements of LAD flow are depicted in Figures 3A and 3B. LAD flow in the 5 animals in group 1 (Figure 3A) was 1.17 ⫾ 0.18 ml/min/g at baseline and 0.74 ⫾ 12 ml/min/g after placement of the stenosis (37% reduction of flow). Hourly monitoring showed a consistent reduction of the LAD coronary flow during the 24-hour period although there was a substantial flow variation (23–37% reduction of flow). In group 2 (Figure 3B), the LAD flow was reduced from 1.19 ⫾ 0.15 ml/ min/g at baseline to 0.77 ⫾ 0.12 ml/min/g (35% reduction of flow) after the LAD stenosis was created. On days 1 to 6 after stenosis placement, LAD flow was consistently reduced, ranging from 0.77 ⫾ 014 to 0.87 ⫾ 0.15 ml/min/g (27–35% reduction of flow). Averaged over 6 days, LAD flow in the 8 animals was 0.80 ⫾ 0.18 ml/min/g, 33% lower than baseline flow. LAD flow at 20 minutes after reperfusion was 1.40 ⫾ 0.24 ml/min/g for group 1 and 1.36 ⫾ 0.16 ml/min/g for group 2. These were slightly higher than at baseline (p ⬍ 0.05).

Systolic Myocardial Dysfunction in Hibernating Mycardium In group 1, anterior LV wall thickening decreased significantly at 15 minutes and at 24 hours (both p ⬍ 0.01 versus baseline). The reduction of wall thickening was not different between group 1 and group 2 (Table 1). LV wall thickening in the inferior region did not change immediately after stenosis placement but increased slightly by 7 days, as shown in Table 1. There was no difference (p ⫽ NS) in left ventricular ejection fraction between group 1 and group 2 before and after the stenosis (decreased from 56 ⫾ 3% at baseline to 38 ⫾ 6% immediately after stenosis placement, to 36 ⫾ 6 at 24 hours in group 1, and from 55 ⫾ 4% to 37 ⫾ 7% at 7 days in group 2 (both p ⬍ 0.01 versus baseline). The ratio of LAD flow/heat rate (x) correlated reasonably well with regional wall thickening (y) immediately after stenosis placement (y ⫽ 37.7x ⫺9.1, r ⫽ 0.70, p ⬍ 0.01), but correlated less well at 24 hours or at 7 days (y ⫽ 24.9x ⫺3.75, r ⫽ 0.49, p ⫽ 0.05).

Metabolic Changes and Oxygen Consumption in Hibernating Myocardium Arterial-coronary venous lactate balance shifted from extraction (30 ⫾ 14%) in all animals at baseline to production (⫺7.8 ⫽ 12%, p ⬍ 0.01) at 15 minutes after stenosis placement (Table 1); by 24 hours or 7 days lactate balance had reverted to extraction (10 ⫾ 4% at 24 hours and 19 ⫾ 8% at 7 days, p ⬍ 0.05 each versus baseline, p ⬍ 0.01 versus 15 minutes post-stenosis placement) despite the continuing

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Table 1. Hemodynamic, Functional, and Metabolic Measurements During LAD Stenosis and Reperfusion LAD Stenosis Baseline

Heart rate (bpm) Mean BP (mmHg) LAD coronary flow (ml/min/g) Anterior WT (%) Inferior WT (%) Regional Lac (␮M/min/g) Regional MVO2 (ml/min/100g) Regional pH

15 minutes

Group 1

Group 2

Group 1

Group 2

108 ⫾ 19 79 ⫾ 10

109 ⫾ 17 80 ⫾ 11

119 ⫾ 10* 78 ⫾ 13

118 ⫾ 11* 77 ⫾ 10

1.02 ⫾ 0.20 39 ⫾ 5 38 ⫾ 6%

1.01 ⫾ 0.24 39 ⫾ 4 37 ⫾ 8

0.62 ⫾ 0.12* 11 ⫾ 8* 40 ⫾ 4*

0.61 ⫾ 0.14* 11 ⫾ 9* 39 ⫾ 7*

0.76 ⫾ 0.42

0.80 ⫾ 0.39

⫺0.04 ⫾ 0.25*

5.5 ⫾ 1.8 7.41 ⫾ 0.05

5.6 ⫾ 1.7 7.40 ⫾ 0.04

3.7 ⫾ 1.0* 7.29 ⫾ 0.04*

⫺0.05 ⫾ 0.3* 3.8 ⫾ 1.1* 7.30 ⫾ 0.05*

24 hours:

7 days:

Group 1

Group 2

Reperfusion Group 1

Group 2

118 ⫾ 15 80 ⫾ 10

110 ⫾ 17 79 ⫾ 11

123 ⫾ 19 83 ⫾ 13

117 ⫾ 19 82 ⫾ 11

0.63 ⫾ 0.14* 12 ⫾ 9* 42 ⫾ 8*

0.65 ⫾ 0.15* 10 ⫾ 8* 43 ⫾ 8*

1.27 ⫾ 0.28* 19 ⫾ 10* 39 ⫾ 7

1.31 ⫾ 0.32* 14 ⫾ 10* 38 ⫾ 8

0.38 ⫾ 0.29†

0.45 ⫾ 0.41†

NA

NA

3.4 ⫾ 0.9* 7.38 ⫾ 0.05†

2.9 ⫾ 1.2*,† 7.40 ⫾ 0.04†

NA NA

NA NA

Abbreviations: BP ⫽ blood pressure; WT ⫽ wall thickening; NA ⫽ not assessed; MVO ⫽ myocardial oxygen consumption; Lac ⫽ Lactate consumption, negative value indicates lactate production. *p ⬍ 0.01 compared to baseline. †p ⬍ 0.05 compared to 15 minute left descending coronary artery (LAD) stenosis.

flow reduction. Similarly, calculated lactate consumption (0.76 ⫾ 0.42 ␮M/min/g) shifted to production 15 minutes after stenosis placement (⫺0.024 ⫾ 0.25 ␮M/min/g, p ⬍ 0.01), but returned to consumption of 0.38 ⫾ 0.29 at 24 hours and 0.45 ⫾ 0.41 ␮M/min/g at 7 days. Similar changes were observed in regional coronary venous pH, as listed in Table 1. The decrease in regional oxygen consumption from

baseline to 15 minutes after stenosis placement was 33% (p ⬍ 0.01), from baseline to 24 hours after the stenosis was 38% (p ⫽ NS versus 15 minutes), and from baseline to 7 days was 48% (p ⫽ 0.01 versus 15 minutes). This additional decrease occurred even though regional wall thickening, heart rate, blood pressure and regional coronary flow were unchanged. Regional oxygen consumption at 24 hours or 7

Figure 3. Data (mean value ⫾ SD) from continuous monitoring of LAD coronary flow in 5 pigs of group 1 (A) during 24-hour hibernation and in 8 pigs of group 2 (B) during 7-day hibernation, and 20 minutes after reperfusion. Abbreviations: B or Bas ⫽ baseline; ST ⫽ placement of LAD stenosis; h ⫽ hour; 1–7 D ⫽ 1–7 days; REP ⫽ reperfusion.

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days correlated with regional wall thickening (r ⫽ 0.81, p ⬍ 0.001), coronary flow (r ⫽ 0.83, p ⬍ 0.001) and myofilament volume (r ⫽ 0.89, p ⬍ 0.001). Multi-variate stepwise forward analysis revealed that only coronary flow (x1) and myofilament volume (x2) were independently related to the regional oxygen consumption (y): (y ⫽ 3.15x1 ⫹ 0.25x2 ⫺ 8.4, r ⫽ 0.94, p ⬍ 0.0001) in the hibernating region.

Morphology at 24 Hours or 7 Days After Stenosis Placement In group 1 of 24-hour hibernation which was reperfused at 24 hours after placement of the LAD stenosis and hypoperfusion, 5 pigs were noted to have patchy subendocardial necrosis which occupied 3.3 ⫾ 2.4% (2.2–7.1%) of the area at risk determined by TTC staining and confirmed on slides of 5 ␮m thickness and trichrome staining. Four pigs in group 1 were free from patchy necrosis or replacement fibrosis. In group 2 of prolonged 7-day hibernating myocardium which was reperfused at 7 days after the stenosis and reduced perfusion, 12/18 pigs had patchy myocardial necrosis with granular tissue and replacement fibrosis (Figure 4). The necrosis with granular tissue and replacement fibrosis involved 2.5 to 13.1% (5.6 ⫾ 4.1%) of the area perfused by the LAD (area at risk) in these pigs of group 2. This patchy necrosis was confined to the subendocardium, as illustrated in Figure 4B. No myocardial necrosis or replacement fibrosis was present in the other 6 pigs in group 2 (Figure 4A). Electron microscopic examination of tissue from the LAD region revealed loss of myofilaments or sarcomeres to varying degrees in all animals in both groups of hibernating myocardium with a duration of 24 hours or 7 days, as illustrated in Figure 5. In comparison to normal myocyte (panel A), abnormal myocyte (panel B) in the hibernating myocardium showed the depletion of myofibrils was most obvious in the perinuclear areas also in the cell peripheries. Scattered groups of myocytes were affected and adjacent cells often appeared normal. The empty myofilament areas were replaced with glycogen deposits and mitochondria. Mitochondria in these spaces varied in size but were usually smaller than mitochondria in normal regions. Glycogen deposition varied in different animals and in different myocytes from the same animals. Damage to myocyte was patchy, such that other regions of the same myocyte appeared to have a normal density of sarcomere, mitochondria and nuclei. Patchy disarrayed and partial loss of myofibrils with a partially dissolved sarcomere Z band was observed in both groups of 24-hour or 7-day hibernating myocardium (panel C). These changes may represent evolving loss of myofibrils. By quantitative electronmicroscopic evaluation (Table 2), there were more abnormal myocytes in the hibernating region in group 2 than in group 1 (p ⬍ 0.01) while 8 ⫾ 6% of myocytes in the control region of group 1 and 13 ⫾ 9% of myocytes in the control region of group 2 were abnormal (p ⫽ NS). Abnormal cells in the control region

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are not unexpected because LV dysfunction may affect myocardial ultrastructure. Other ultrstructural changes included deformed or irregularly shaped nuclei with dispersed heterochromatin. Lipid droplets were rare. The capillary vessel was intact and no hemorrhage or thrombus was observed. By quantitative evaluation, myofibril or sarcomere volume comprised 43.5 ⫾ 5.6% of the myocytes in the hibernating region in group 2 and was significantly less than sarcomere volume (48.8 ⫾ 6.7%) of the hibernating myocytes in group 1 (p ⬍ 0.01). Sarcomere volume correlated significantly (r ⫽ 0.71, p ⬍ 0.05) with LAD flow. Cytoplasmal volume and mitochondria were increased in the hibernating myocytes compared to the control cells in group 2. Similar but slightly less severe changes in mitochondria and cytoplasm were observed in group 1 (Table 2). Interstitial tissue volume (content) was significantly greater in the 7day hibernating myocardium than in the 24-hour hibernating myocardium (p ⬍ 0.01, Table 2), as shown in Figure 6. Difference in cellular changes between pigs with and without patchy necrosis. Decrease in sarcomere volume in pigs with patchy necrosis was slightly more severe (sarcomere volume ⫽ 46.9 ⫾ 3.4%) than in pigs without patchy necrosis (sarcomere volume ⫽ 50.4 ⫾ 4.2%) in group 1 of 24-hour hibernating myocardium although this did not reach a statistical significance (p ⫽ 0.07) due to a small number of pigs studied. In group 2 of 7-day hibernating myocardium, pigs with patchy necrosis (sarcomere volume ⫽ 39.8 ⫾ 6.4%) had a more severe reduction in the sarcomere volume than pigs without patchy necrosis (46.2 ⫾ 6.1%, p ⬍ 0.05). In pigs without patchy necrosis, the loss of sarcomere volume was also significantly greater in group 2 of 7-day hibernating myocardium than in group 1 of 24hour hibernating myocardium (p ⬍ 0.05).

Recovery of Function and Structure with Reperfusion in Hibernating Myocardium In group 1, LAD flow was 1.31 ⫾ 0.32 ml/min/g after 20 minutes of release of the stenosis while in group 2 LAD flow at 20 minutes after release of the stenosis was 1.27 ⫾ 0.28 ml/ min/g, significantly higher than during the stenosis (p ⬍ 0.01) or even at baseline (p ⬍ 0.05). Anterior wall thickening increased from 11 ⫾ 8% or 10 ⫾ 8% to 19 ⫾ 10% or 14 ⫾ 10% 20 minutes after release of the stenosis in group 1 (p ⬍ 0.05) or group 2 (p ⬍ 0.05), to 31 ⫾ 9% in group 1 (p ⬍ 0.01) or 18 ⫾ 9% in group 2 (p ⬍ 0.01) at one week, and to 28 ⫾ 3.6% at 3–4 weeks in group 2 (Figure 2). There was a significant difference in the anterior wall thickening between group 1 and group 2 at one week reperfusion (p ⬍ 0.01). The anterior wall thickening was not different from the wall thickening at baseline (p ⫽ 0.07) with a normal ejection fraction (53 ⫾ 4%) in group 1 one week after reperfusion. However, in group 2 the anterior wall thickening was significantly (p ⬍ 0.01) less than at baseline one week after reperfusion and was still significantly less than at baseline (38.8 ⫾ 5.1%, p ⫽ 0.05) 3–4

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Figure 4. Examples of trichrome staining of subendocardial hibernating myocardial region in 2 pigs (A and B, magnification ⫻40). (A) No evidence of patchy necrosis or replacement fibrosis was noted in the hibernating region; (B) small patchy necrosis and granulation tissue (solid arrows) are noted in the subendocardial hibernating myocardial region while the majority of myocardium are viable (red staining).

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Figure 5. (Continued)

weeks after reperfusion. LV ejection fraction improved from 35 ⫾ 7% to 51 ⫾ 9% (p ⬍ 0.05) over 4 weeks in group 2. In group 1, the majority of abnormal myocytes had recovered (Table 2) after one week of reperfusion. In the 5 pigs in group 2 sacrificed at 7 days after reperfusion, most of the affected myocytes had not recovered and 35 ⫾ 12% were counted as abnormal with a sarcomere volume of 46 ⫾

10% (p ⬍ 0.01 compared to group 1, Table 2). In the 7 pigs sacrificed after 3–4 weeks of reperfusion, 23 ⫾ 15% of myocytes in the hibernating region were still affected (Table 2). The content of myofibrils and sarcomeres in the hibernating cells was still reduced 51 ⫾ 9.6%, compared to 62 ⫾ 4% in the control region (p ⬍ 0.05). The ultrastructural changes had not recovered completely.

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Figure 5. Representative electromicrographs (⫻3700) of myocytes from normal and hibernating myocardial region. (A) Normal myocyte in the normal region: note that sarcomeres (S) are intact, mitochondria (M) in various sizes are noted around the nuclear (N) area and between sarcomeres; (B) Abnormal myocyte in the hibernating region: sarcomeres have lost (arrow) and numerous small mitochondria are seen in place of the lost sarcomeres. (C) Abnormal myocyte in the hibernating region: Disarrayed and partially lost myofibrils with partially dissolved sarcomere without Z band (arrows).

placement, sarcomere volume (p ⬍ 0.001) and the extent of patchy fibrosis/necrosis (p ⬍ 0.01) correlated with functional recovery.

Anterior wall thickening at 7 days or 3–4 weeks after reperfusion correlated by univariate analysis with sarcomere volumes (p ⬍ 0.001) with stenosis, the extent of fibrosis/necrosis (p ⬍ 0.001), LAD flow after stenosis placement (p ⬍ 0.01) and at 7 days (p ⬍ 0.01), and regional wall thickening after stenosis placement (p ⬍ 0.001) and at 7 days (p ⬍ 0.001). However, by stepwise multivariate analysis, only coronary flow (p ⬍ 0.001) at 7 days after stenosis

Discussion This study demonstrates that myocardial hibernation can be maintained in a pig model for 7 days without extensive

Table 2. Cellular and Extracellular Abnormalities in Hibernating Myocardium: 24 Hours versus 7 Days Hibernation LAD Stenosis

Sarcomere volume (%) Cytoplasmal volume (%) Mitochondria volume (%) Abnormal myocyte (%) Interstitial volume (%)

Reperfusion

24 Hours:

7 Days:

Control (n ⫽ 26)

Group 1 (n ⫽ 9)

Group 2 (n ⫽ 17)

Group 1 (n ⫽ 6)

Group 2 (n ⫽ 5)

Group 2 (n ⫽ 7)

62.4 ⫾ 4.1 17.0 ⫾ 3.5 20.6 ⫾ 4.6 10 ⫾ 2 12 ⫾ 3

48.8 ⫾ 6.7 27.3 ⫾ 3.8 23.9 ⫾ 4.6 33 ⫾ 16 15 ⫾ 4

43.5 ⫾ 5.6* 29.4 ⫾ 6.0* 27.1 ⫾ 7.0* 52 ⫾ 17* 18 ⫾ 4*

59.2 ⫾ 7.3 20.1 ⫾ 7.8 20.7 ⫾ 6.8 11 ⫾ 6 14 ⫾ 3

46.2 ⫾ 10.1* 30.2 ⫾ 4.0* 23.6 ⫾ 5.0* 35 ⫾ 12* 17 ⫾ 3*

51.8 ⫾ 7.2† 24.4 ⫾ 6.3† 23.8 ⫾ 7.0† 23 ⫾ 15† 17 ⫾ 4†

Values in the control region were pooled from group 1 and group 2. *p ⬍ 0.05 compared to group 1. †p ⬍ 0.05 compared to one week after reperfusion in group 2.

1 Week

3–4 Weeks:

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Figure 6. (Continued)

infarction but at the cost of more severe myocyte ultrastructural abnormalities and increasing fibrosis. The features of myocardial hibernation that are present in the model include a severe, fixed coronary stenosis, a persistent regional coro-

nary flow reduction, a sustained regional left ventricular dysfunction which is reversible after reperfusion, an initial ischemic lactate production with later recovery and a persistent reduction in regional oxygen consumption. These char-

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Figure 6. An example of increased interstitial content in proloned hibernating myocardium of 7 days. Note that the interstitial tissue was increased in the hibernating myocardium which was reperfused in 7 days (C), compared to the hibernating myocardium which was reperfused in 24 hours (B) or to the control normal myocardium (A).

acteristics indicate that functional, metabolic and structural adaptations have developed in the hibernating myocardium. The results of this study suggest that myocardial cellular ultrastructural and extracellular abnormalities worsen as the duration of hypoperfusion and hibernation increases and reperfusion is delayed. As shown in Table 2, more abnormal myocytes were found in the 7-day hibernating region than in the 24-hour hibernating region. No obvious loss of contractile materials were observed in the first 3 hours of myocardial hibernation in a previous study (17) whereas myofilament loss is obvious at 24 hours in this and previous studies in our laboratory (10). A degree of intrasarcomere edema might have contributed to the extended degree of the decreased myofilament volume fraction in the 24-hour hibernation group. However, as shown in our previous study the decreased myofilament fraction was associated with loss of sarcomere or myofilaments of 24-hour hibernating myocyte. This study confirmed that loss of myofilament in hibernating myocardium can occur as early as at 24 hours of hibernating myocardium. Various different stages of loss of myofilament were observed in the hibernating myocardium (Figure 5B and Figure 5C). In Figure 5C, partially lost and disarrayed myofilament with dissolved sarcomere which may represent an evolution or early stage of loss of myofibrils was observed in pigs with 24-hour hibernation al-

though a late stage of loss of myofilament with replacement of glycogen deposition and increased small mitochondria was also present at 24 hours (Figure 5B). The degree of loss of myofilaments increased with the prolongation of hibernation to 7 days. Mechanism of the loss of myofilaments in the hibernating myocardium is not clear. Since normal myocardial proteins have a variable half-time, ranging from a few hours to several days, one of the possible explanations for the loss of myofilament may be that the synthesis of new myofilament proteins had been slowed progressively in hibernating myocardium. However, the extent of loss of myofilament and the early stage of disarray or loss of myofilament at 24 hours of hypoperfusion in this study suggest that an active mechanism by an accelerated degradation of myofilament may also be involved. Further study is required to address the timing and mechanism of destruction or clearance of myofilaments with myocardial hibernation. Understanding of the regulative mechanism of myofibrillar and sarcomere loss and replacement during hibernation and recovery may help to develop interventions that could preserve myofilaments during myocardial hibernation and enhance regeneration of myofillament or sarcomere after revascularization. Myocyte abnormalities were associated with increases in myocardial interstitial tissues. The interstitial volume

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slightly increased in 24-hour hibernating myocardium but the increase was not statistically significant. Speculatively, a degree of interstitial edema might have been involved in the early stage of ischemia or hibernation. However, the fact that the increase in interstitial volume was more severe at 7 days than 24 hours in hibernating myocardium when the edema should have decreased at 7 days, and the fact that 3–4 weeks of reperfusion after 7-day myocardial hibernation did not significantly change the interstitial tissue volume in hibernating myocardium suggest not edema but an increase in connective tissue or interstitial fibrosis with prolonged hypoperfusion. This was further supported by preliminary data from a more recent study with hydroproline measurements of collagen content in this laboratory (18). In the recent preliminary study we further demonstrated a even more pronounced increase in interstitial fibrosis in 3–4 week hibernating myocardium with increased hydroproline contents in comparison to that in 7-day hibernating myocardium. Therefore, myocardial hibernation with down-regulation of mechanical function to reduced oxygen consumption which is considered a protective mechanism to prevent acute ischemic injury may be at cost of progressive myocyte structural abnormalities and myocardial fibrosis if the hypoperfused, hibernating myocardium is not reperfused or revascularized in a timely manner. It is not clear why the cellular and extracellular abnormalities worsen as the duration of hypoperfusion and hibernation increases. Speculatively, it may be related to the mechanical and hormonal factors that cause LV remodeling as a consequence of myocardial dysfunction (19), reduced myocardial blood flow, repetitive stress-induced ischemic injuries, or other unknown factors that lead to myocyte abnormalities (6,7,10,20).

Relations Between the Degree of Myocardial Hypoperfusion and Function or Structural Abnormalities During acute coronary flow reductions, a close linear relationship between coronary flow and regional wall thickening can be demonstrated in this and other studies (9,10). However, this relationship no longer predominates after 7 days of reduced coronary flow. Ultrastructural damage in the myocardium with depletion of contractile units may be partly responsible for the persistence of regional LV dysfunction. Reperfusion at 24 hours or 7 days after myocardial hibernation resulted in only a small degree of immediate early improvement in myocardial systolic function and ultrastructural abnormalities. The function and ultrastructures recovered later, almost completely by 7 days of reperfusion in the hibernating myocardium which was reperfused within 24 hours. In contrast, myocardial function and ultrastructure recovered only minimally at one week of reperfusion and did not recover completely by 3–4 weeks of reperfusion in the prolonged hibernating myocardium which was reperfused after 7-day hibernation. The rate of recovery correlated with sarco-

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mere volume at the time of reperfusion, the extent of fibrosis and the severity of flow reduction in this study. This supports the hypothesis that with the same degree of hypoperfusion the longer the duration of myocardial hibernation the more protracted recoveries of myocardial structures and function is likely to be after reperfusion. The delay in recovery of systolic function after reperfusion may be due to a more extensive ultrastructural damage to myocytes that requires a longer period of time for their repair. The main determinant of the regional dysfunction present at the time of reperfusion was not the patchy necrosis and fibrosis because most of the dysfunction subsequently disappeared when contractile materials (such as sarcomere) increased. But part of the residual regional dysfunction may be attributed to patchy necrosis and fibrosis, and this part is unlikely to recover. Whether enough necrosis and fibrosis was present in the animals in this study to prevent full recovery is not known. After 3–4 weeks of reperfusion, myofibrillar volume and regional wall thickening had still not returned to baseline; however, further recovery may have occurred if the follow-up period had been extended. Patchy necrosis limited to the subendocardium and involving only 2 to 13% of the area at risk was found in 5 of 9 pigs in group 1 and in 11 of 17 pigs in group 2 in this study. Although this difference is not statistically significant because of a relatively small number of animals that were studied, it seems likely that the risk of developing patchy necrosis and fibrosis in hibernating myocardium increases over time if reperfusion is delayed. Further studies with a larger number of animals and a longer period of hibernation are needed to clarify this issue.

Relationship Between Functional and Structural Recovery Hibernating myocardium reperfused after 5 or 24 hours recovered completely within 7 days in previous studies by our group (10) and by Matsuzaki et al (8). However, hibernating myocardium of 7-day duration in this study recovered only minimally by one week of reperfusion, and required 4 weeks for substantial recovery after reperfusion. The timing and extent of recovery of LV function varied greatly among the pigs in this study, similar to what has been observed in patients. Immediate recovery of regional LV wall motion abnormalities after coronary bypass surgery has been observed in the operating room (21), but recovery in most patients may take weeks to months after revascularization (22–25). The factors that correlated with the rate and extent of functional recovery of hibernating myocardium were the severity of hypoperfusion, the severity of the abnormality of regional wall thickening, the extent of ultrastructural damage to sarcomeres, and the presence of patchy myocardial necrosis. As noted above, a comparison between our results after 24 hours and 7 days of hibernation suggests that the

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longer this condition lasts, the more extensive the structural changes, the more delayed and more incomplete recovery is likely to be. The duration of hibernation may thus account for most of the variation in functional recovery observed clinically, but this variable cannot usually be accurately assessed in patients. In clinical studies where biopsies were taken from hibernating myocardium during coronary bypass surgery, a range of myocyte degeneration and fibrosis was found, and regional function recovered at 6 months. The severity of morphological changes correlated with the degree and rate of recovery in some studies (25–27), but not in the other (28).

Critique of the Model of Myocardial Hibernation This model is difficult to establish and maintain. Most of the difficulties are related to maintenance of a stable severe coronary stenosis. Full dose heparin and aspirin were used to prevent coronary thrombosis and the cyclic flow variations (29) caused by microthrombi. The end of the silk tie used to produce the stenosis was anchored to the adjacent RV myocardium to prevent migration. The hydraulic occluder was adjusted to maintain a relatively stable flow reduction. Resting LAD flow was similar under anesthesia immediately after placement of the stenosis, at 24 hours, at 7 days or during daily flow monitoring in both groups. This excludes the possibility of difference in the severity of flow reduction to cause a difference in functional recovery or the degree of myocyte abnormality between the 2 groups in this study. Under conscious sedation LAD flow varied somewhat over the 24 hours or the 7 days in these animals even though the stenoses were fixed. These variations were closely related to variations in arterial pressure and heart rate. In general, both arterial pressure and LAD flow were slightly higher under conscious sedation than anesthesia, but LAD flow was still reduced compared to baseline. These conditions probably reflect what happens in patients as heart rate and arterial pressure vary during daily activities and influence perfusion and myocardial oxygen balance in the hibernating region. In this study, we measured only transmural myocardial blood flow in most pigs. Subendocardial myocardial blood flow reduction would have been proportionally greater if it had been measured (30). In fact, in pigs in which color microsphere technique was used to measure transmural myocardial flow distribution in this study, subendocardial blood flow was substantially more reduced than the subepicardial myocardial blood flow.

Hibernation versus Stunning Most authors define hibernation as myocardial dysfunction resulting from a reduction of coronary flow at rest (1,2,4,24) and stunning as myocardial dysfunction persisting after restoration of flow (3,5,31,32). Thus, determining the level of myocardial perfusion associated with myocardial dysfunction is the key to differentiating hibernation

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from stunning (33–35). The definition of hibernation deals only with coronary flow at rest; as perfusion pressure and heart rate change during normal activities, coronary flow across a fixed stenosis would be expected to vary according to the principle that flow through a conduit is proportionally related to driving pressure and diameter of the conduit. Therefore, coronary flow varies with coronary perfusion pressure. If coronary flow is measured at only a few points, as in our previous short-term hibernation study (10) or in other animal studies (36–38), and in patients (6,7), the distinction between hibernation and stunning based only on a single or a few points of flow measurements may not always be obvious. One of the strengths of this study is that the coronary flow was continuously monitored in most pigs. This allowed us to provide data of flow variations in the hibernating myocardial region that is supplied by a fixed coronary stenosis. In spite of fluctuations of myocardial blood flow in the hibernating region, LAD flow was consistently reduced in this model; therefore, in our model myocardial dysfunction represents predominantly hibernation and not stunning. Restoration of normal flow does not immediately normalize function in either hibernating or stunned myocardium. Potential explanations for the slow recovery of hibernating myocardium may include not only ultrastructural damage to sarcomeres but also changes in adrenergic responses (39) or calcium responsiveness (40). A degree of ischemic or postischemic dysfunction (stunning) may be superimposed on the hibernating myocardium. Such a mixed situation may reflect what happens clinically, because flow and myocardial oxygen demands do not remain static (11,12).

Evidence of Myocardial Hibernation from Previous Studies The concept of myocardial hibernation (1–4) and most of the evidence for its existence have come from clinical studies (5–8,17–23). However, the interpretation of clinical data has been controversial because few studies have provided simultaneous myocardial perfusion and functional measurements (6,7,17–23,27,28,41–43). Acute animal studies support the hypothesis of hibernating myocardium (4,9,10,44– 47) but data from chronic animal studies have been limited (5,12–14,48). Acute animal models with severe coronary stenoses have been maintained for 60 minutes to 24 hours with reduced regional coronary flow and regional wall thickening, with minimal infarction in pigs and dogs (9,10,34–38). Metabolic recovery of the ischemic myocardial region with regeneration of creatinine phosphate and cessation of lactate production and regional acidosis occurs within 60 minutes to 3 hours (37–40). Inotropic stimulation of short-term hibernating myocardium induces ischemic metabolism with lactate production, leading to infarction if the stimulation is prolonged for 90 minutes (49). Since sympathetic stimulation commonly occurs during normal activi-

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ties in patients, whether hibernating myocardium can be maintained over days or weeks without significant myocardial necrosis has been a concern. A porcine model of coronary stenosis sustained for 4 to 7 days was reported by Bolukoglu et al. and Liedtke et al (34,35). Their coronary stenosis reduced the hyperemic response by 50% without a reduction in mean resting coronary flow. Wall thickening initially remained normal, confirming that resting coronary flow was not reduced, but was reduced after 7 days with the stenosis. Whether their model represents repeated myocardial stunning or hibernation is debatable. Shen and Vatner used an ameroid constrictor in minipigs to produce a severe coronary stenosis over several days, allowing collaterals to develop (38). Measurements obtained after 20 days revealed severe regional dysfunction, but normal resting coronary flow, including collateral flow, a finding more consistent with stunning than with hibernation. A similar pig model was used by Mills et al. (50) to study the coronary vascular response to a 4 to 32 week reduction in perfusion pressure and flow. A direct comparison of their study to other studies is difficult because coronary flow was not measured early after placement of the stenosis, no continuous monitoring of the degree of flow reduction was performed, and no serial functional and myocardial ultrastructural assessments were conducted in their study. Our model provided important serial data of degree of coronary flow reduction, myocardial functional and structural abnormalities and their recoveries that are similar to the abnormalities seen in patients with hibernating myocardium (23–26).

Relation Between Metabolic Adaptations and Morphological Changes The metabolic adaptations of myocardial hibernation, recovery of ischemic lactate production and the recovery from regional acidosis, persisted for the 7 days of this study. Regional oxygen consumption decreased by 32% immediately and by a further 16% at 7 days, without a further change in wall thickening, heart rate, or blood pressure. In a previous report oxygen consumption of stunned myocardium was higher than at baseline (51). The mechanism for the further reduction in oxygen consumption at 7 days is not clear, although the extent of myofilament loss, but not the presence of patchy necrosis, correlated with the reduction of oxygen consumption. The reduction in oxygen consumption was also related to the reduced wall thickening. Regional wall stress, which was not measured in this study, should theoretically be related to oxygen consumption. Further study is required to determine whether the cardiac energetics of hibernating myocardium is altered.

Clinical Implications and Future Research The loss of sarcomeres in hibernating myocardium at 7 days was greater than at 24 hours. This may occur either be-

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cause the appearance of structural abnormalities peaks later after the initial ischemic insult or because the structural alterations or adaptations continue to develop over time. The decrease in sarcomere volume may represent either an accelerated loss or a depressed synthesis of contractile proteins. Further studies are necessary to determine the stability and fate of chronic hibernating myocardium of a longer duration, to investigate whether progressive necrosis and fibrosis gradually develops in hibernating myocardium and to assess interventions that could prevent this progression, to define the parameters that regulate myofibrillar and sarcomere loss and replacement during hibernation and recovery, and to identify other unknown factors that may influence systolic dysfunction and its recovery. The common clinical situation where severe coronary disease and severe irreversible left ventricular dysfunction coexist in the absence of previous myocardial infarction may represent the adverse outcome of unsalvaged hibernating myocardium. Early reperfusion or revascularization to the hibernating myocardial region may prevent development of advanced, end-stage ischemic cardiomyopathy in this condition. The authors wish to thank William Dyckman and Eddie Hall for their technical expertise in the preparation of the animal model, Andrew H Smith for his expertise in the colored microsphere technique and Jeffrey Mather, BS, for his statistical advise.

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