Chronic hibernating myocardium in sheep can occur without degenerating events and is reversed after revascularization

Cardiovascular Pathology 23 (2014) 160–168

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Cardiovascular Pathology

Original Article

Chronic hibernating myocardium in sheep can occur without degenerating events and is reversed after revascularization F. Verheyen a, b, c,⁎, R. Racz d, M. Borgers a, c, R.B. Driesen a, e, M.-H. Lenders a, b, W.J. Flameng d a b c d e

CARIM, Maastricht University, Maastricht, The Netherlands Electron Microscopy Unit at CRISP Department of Molecular Cell Biology Department of Cardiac Surgery, Katholieke Universiteit Leuven, Leuven, Belgium Department of Experimental Cardiology, KU Leuven, Leuven, Belgium

a r t i c l e

i n f o

Article history: Received 11 November 2013 Received in revised form 6 January 2014 Accepted 6 January 2014 Keywords: Coronary artery stenosis Hibernation Morphology Myocardial blood flow Cardiomyopathy

a b s t r a c t Introduction: Our goal was to show that blunting of myocardial flow reserve is mainly involved in adaptive chronic myocardial hibernation without apparent cardiomyocyte degeneration. Methods and results: Sheep chronically instrumented with critical multivessel stenosis and/or percutaneous transluminal coronary angioplasty (PTCA)-induced revascularization were allowed to run and feed in the open for 2 and 5 months, respectively. Regional myocardial blood flow (MBF) with colored microspheres, regional and global left ventricular function and dimensions (2D echocardiography), and myocardial structure were studied. In sheep with a critical stenosis, a progressive increase in left ventricular end-diastolic and end-systolic cavity area and a decrease in fractional area change were found. Fraction of wall thickness decreased in all left ventricular wall segments. MBF was slightly but not significantly decreased at rest at 2 months. Morphological quantification revealed a rather small but significant increase in diffusely distributed connective tissue, cardiomyocyte hypertrophy, and presence of viable myocardium of which almost 30 % of the myocytes showed depletion of sarcomeres and accumulation of glycogen. The extent of myolysis in the transmural layer correlated with the degree of left ventricular dilation. Structural degeneration of cardiomyocytes was not observed. Balloon dilatation (PTCA) of one of the coronary artery stenoses at 10 weeks revealed recovery of fraction of wall thickness and near normalization of global subcellular structure at 20 weeks. Conclusion: These data indicate that chronic reduction of coronary reserve by itself can induce ischemic cardiomyopathy characterized by left ventricular dilatation, depressed regional and global function, adaptive chronic myocardial hibernation, reactive fibrosis and cardiomyocyte hypertrophy in the absence of obvious degenerative phenomena. Summary: Reduction of myocardial flow reserve due to chronic coronary artery stenosis in sheep induces adaptive myocardial hibernation without involvement of degenerative phenomena. © 2014 Elsevier Inc. All rights reserved.

1. Introduction In the original concept of chronic myocardial hibernation a permanent state of myocardial hypoperfusion as the consequence of a severe coronary artery stenosis was accepted resulting in impairment of cardiac function [1–4]. Another concept postulated repetitive episodes of demand ischemia in patients with coronary artery disease This work was supported by CARIM, Maastricht University, Maastricht, The Netherlands, the Department of Cardiac Surgery, Katholieke Universiteit Leuven, Leuven, Belgium, the Electron Microscopy Unit at CRISP and the Department of Molecular Cell Biology. ⁎ Corresponding author at: Faculty of Health, Medicine and Life Sciences, Electron Microscopy Unit at CRISP and Department of Molecular Cell Biology, Universiteitssingel 50, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel.: +31 43 3881354; fax: +31 43 3884151. E-mail address: [email protected] (F. Verheyen). 1054-8807/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.carpath.2014.01.003

[5]. The latter concept was based upon the observation that proximal coronary artery occlusion compensated by collateral development provides normal myocardial perfusion at rest but relative underperfusion during periods of increased oxygen consumption. This concept was also observed in experimental setups in dogs and pigs and suggested that episodes of demand ischemia resulted in repetitive myocardial stunning which finally led to a chronic state of hibernation of the myocardium [6–13]. Before the phenomenon of hibernation was recognized, typical morphological alterations such as necrotic cell death of cardiomyocytes (CMs), intracellular myolysis resulting in disintegration and loss of contractile material and concomitant increased glycogen accumulation within the remaining CMs and increased interstitial fibrosis were described in human myocardial biopsies taken from areas that were dysfunctional and recovered after surgical revascularization [14]. Since then, these morphological phenomena have been

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described in a number of additional studies [reviewed in Ref. 13]. Borgers and colleagues mainly focused on the adaptive and the surviving, reversible nature of the dedifferentiated fetal phenotype of hibernating CMs and proposed dynamic passive stretch as the underlying mechanism [15–19]. Schaper and co-workers also indicated the adaptive nature of some morphological changes and their reversibility up to a certain degree but emphasized the degenerative nature of more severe CM alterations resulting in necrotic and/or apoptotic cell death and increased replacement fibrosis upon the necrotic cell death [20–23]. In pigs, reversible ischemia in an area of chronically reduced coronary flow reserve was described to induce regional myocyte loss via an apoptotic mechanism [24] and hibernating myocardium as a result of chronic stenosis or occlusion always showed various degrees of interstitial fibrosis [25–28]. Although the latter studies seem to indicate that the adaptation of myocardial cells to the hibernating state by chronic flow reduction is accompanied by obvious changes such as necrosis, apoptosis and replacement interstitial fibrosis, it would be interesting to know whether the adaptation could occur in the absence of pronounced degenerative phenomena—which lead per definition to irreversible cell death—as well. Moreover, the study of reversibility of the hibernating state would possibly be easier to approach. During our study on critical multivessel stenosis in sheep, it appeared that chronic reduction of coronary flow reserve could induce ischemic cardiomyopathy upon repetitive stress, i.e., repetitive episodes of critical shortage of blood supply. This critical, repetitive shortage of blood supply resulted in cardiomyopathy which was characterized by left ventricular dilatation, depressed regional and global function and subcellular evidence of myocardial hibernation with a small increase in connective tissue formation. Percutaneous transluminal coronary angioplasty (PTCA) of one of the two stenoses was used to verify the influence of revascularization on regional and remote areas. 2. Methods 2.1. Experimental preparation and protocol Experiments were carried out in compliance with the “Guide for the Care of Laboratory Animals” published by the National Institute of Health. Experimental protocols were approved by the Ethical Committee for Animal Experiments of the Katholieke Universiteit Leuven. 2.2. Acute experiments The five animals in this acute study were premedicated with ketamine 10–20 mg/kg intramuscularly and anesthesia was induced with a halothane-oxygen mixture. The sheep were ventilated and the chest was opened. Catheters were inserted in the left atrium and the descending aorta for pressure monitoring and the administration and withdrawal of colored microspheres. A high-fidelity micromanometer (Micro Transducer Catheter, Drager, Germany) was introduced in the left ventricle via the left atrium. The left circumflex coronary artery (LCX) was instrumented with a pulsed Doppler flow probe (Pulsed Doppler 20 MHz module Baylor College of Medicine, Houston, TX, USA). For induction of the coronary stenosis C-shaped plastic rings of appropriate size were placed around left anterior descending (LAD) and LCX. The experimental protocol was as follows: (1) All baseline measurements were recorded after stabilization including a first injection of colored microspheres. (2) The LAD was occluded for 1 minute and at peak reactive hyperemia (30 seconds) another injection of colored microspheres was given. (3) After stabilization (15 min) the LCX was occluded for 1 minute and reactive hyperemia was recorded. (4) After stabilization of LCX, flow baseline measurements were performed and dobutamine infusion was started at incremental doses from 5 μg kg −1 min −1 at 5-min steps up to 15 μg

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kg −1 min −1. During the infusion of 10 μg kg −1 min −1 of dobutamine, a third injection of microspheres was given. During a 30-min period, the recovery phase was studied after cessation of dobutamine administration. (5) Then the coronary stenoses were induced and the experimental protocol was repeated. 2.3. Eight-week chronic coronary artery stenosis Eight sheep (body weight of 60±13 kg) were premedicated with ketamine 10–20 mg/kg intramuscularly, whereafter a two-dimensional echocardiography (Sonotron Vingmed CFM Horten Norway with 2.5 Mhz transducer) was performed to evaluate the left ventricular function from the parasternal long- and short-axis view of the mid-papillary muscle level of the left ventricle (LV). The echocardiograms were analyzed by two independent observers. Enddiastolic (ED) and end-systolic (ES) wall thickness were measured at the mid-papillary level. LV fraction of wall thickness (FWT) was calculated as end-systolic minus end-diastolic wall thickness divided by end-diastolic wall thickness, expressed as a percentage. Global left ventricular systolic function was measured by calculating the fractional area change (FAC) in the parasternal short axis view. ED and ES areas were defined after manual delineation of the endocardium and were measured for three consecutive beats and averaged. FAC was calculated as ED minus ES area, divided be ED area times 100. Then induction of anesthesia was performed with a halothaneoxygen mixture. The sheep were intubated and ventilated. A sterile left thoracotomy was subsequently performed and 30 min before opening of the pericardium 100 mg lidocain was administered to avoid ventricular arrhythmias. Catheters were inserted in the left atrium and the descending aorta for pressure monitoring and the administration of colored microspheres. After stabilization, baseline hemodynamic parameters were measured. Then colored microspheres were administered before dissection of the coronary arteries as a baseline value. Plastic rings of appropriate size (b2.5 mm) were placed around the LAD and the LCX to induce coronary stenosis. Approximately forty minutes later hemodynamic measurements were repeated as well as microsphere injection. Then the thoracotomy was closed and the anesthesia was stopped. The animals remained sedated for thirty minutes to perform the last echocardiographic examination. At Day 2 postoperatively the animals were transported to the farm where they were allowed to run in the open. At 2-week intervals the animals were again sedated with Ketalar 10 mg/kg intramuscularly and the echocardiogram was repeated. Finally, the animals were again sedated and a last echocardiographic examination was performed. Then the sheep were anesthetized, hemodynamic variables were measured and another injection of colored microspheres was administered. After termination of the experiments, the hearts were prepared for light- and electron microscopic examination and postmortem angiography (see below). 2.4. Chronic multivessel coronary artery stenosis followed by PTCA In this group, six sheep (body weight of 64.5±4.8 kg) were used. The preparation and initial 8-week protocol of sedation, echocardiography, hemodynamic measurements and microsphere injections were nearly similar to that in the eight week chronic stenosis group except that after surgery the echocardiography was only performed at 4 week intervals. At ten weeks after induction of coronary stenosis the animals were again anaesthetized and PTCA was performed at random of one of the two stenotic vessels. An in vivo coronary angiography was done before and after the PTCA procedure. A shot of microspheres was also given before and within 5 minutes after the PTCA. Echography was performed under sedation at 2 week intervals up to 20 weeks. At 20 weeks the sheep were again anaesthetized, hemodynamic parameters were measured and a last shot of microspheres was given. The heart was then removed and prepared

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for light- and electron microscopic examination, postmortem angiography and measurement of regional myocardial blood flow (see below). 2.5. Postmortem and in vivo angiography After termination of the experiments, i.e., 8 weeks or 20 weeks after induction of the stenosis according to the protocols, the hearts were arrested by cold cardioplegic solution and perfusion fixed at a constant pressure of 100 mmHg for 5 min at 25°C with 3% glutaraldehyde in 0.09 M KH2PO4+1.4% sucrose. Then tissue blocks were cut out including the left coronary ostium, the main stem and the proximal parts of the LAD and LCX coronary arteries. The block contained the constricting ring and at least 3–5 cm of the coronary arteries distal from the stenosis. A mixture of barium-gelatine was injected by hand in the coronary ostium. Because the hearts were previously fixed at a constant pressure, the barium-gelatine injection could be done by hand without interfering with the morphology of the coronary stenosis. Then X rays were made from the blocks at two incidences. The prestenotic and stenotic diameter of the vessels were measured in the two directions. An average value of the diameter was calculated. The % reduction in luminal area was calculated as follows: % reduction of area=(πR 21−πR 22/πR 21)×100 where R1=reference diameter before stenosis and R2 the minimal diameter. The degree of the coronary stenosis in the in vivo angiographic pictures was measured in a similar way as described for the postmortem evaluation. Further, the heart was cut into five slices perpendicular to the apex-base axis and used for the measurement of regional myocardial blood flow and/or morphological evaluation.

2.8. Quantitative assessment of the myocardial structure The amount of connective tissue was evaluated according to the basic principles of morphometry using a point counting system. In this way, the number of points overlying connective tissue compared to the total number of points (=100%) covering the tissue was determined. To quantify the number of hibernating cells, 100 subsequent cells per specimen containing a transected nucleus were evaluated for the presence or absence of glycogen accumulation in periodic acid schiff (PAS)-stained light microscopic Sections. A CM was considered as hibernated when the amount of glycogen accumulation accounted for more than 10% of the volume fraction of the CM. The mean CM diameter was calculated by measurement of 100 cells per specimen in the region of the cell nucleus. 2.9. Statistical analysis Data are expressed as mean (S.D.). Analysis of variance (ANOVA) with repeated measures (for time) was used to compare the hemodynamic, echocardiographic and microsphere data. Intergroup comparisons were tested using the Fisher's LSD test and paired comparisons were performed with the paired Student's t test. Intergroup comparisons of morphometric data were carried out using Student’s t-test for data that did not deviate from a normal distribution and with the Wilcoxon–Mann–Whitney rank-sum test in the case of non-normally distributed data such as percent myolysis. Correlations between morphometric measurements were assessed using the Pearson product–moment correlation coefficient. Two-sided P values .05 or less were considered to indicate statistical significance. Since the present study is purely exploratory, all P values should be interpreted as such.

2.6. Measurement of regional myocardial blood flow

3. Results

Polystyrene microspheres [diameter 15±0.1 μm, mean (S.D.)] with a density of 1.09 g/ml (Dye-Trak microsphere, Triton Technology, San Diego, CA, USA) labeled with red, violet, white, blue and yellow were used. Samples from the first and third slices of the hearts were collected for measurement of regional myocardial blood flow. The transmural wall was divided into subepi- and subendocardial samples, identified, coded, weighed and processed for spectrophotometry [29].

3.1. Degree of coronary artery stenosis

2.7. Light- and electron microscopic evaluation For the morphological study, samples from the third slices of hearts derived from sheep with multivessel stenosis and multivessel stenosis+PTCA were compared with samples of similar locations derived from hearts of normal control sheep. The samples originated from the anterior and posterior segments and for each segment a subepicardial and subendocardial sample was processed. After the fixation period, each sample was rinsed with 0.09 M KH2PO4 supplemented with 7.5% sucrose, postfixed in 2% OsO4 solution buffered with veronal acetate at pH 7.4 for 1 h at 4°C. After a short rinse in veronal acetate buffer (pH 7.4) with 7% sucrose (5 min, 4 °C), the samples were routinely dehydrated in graded series of ethanol and embedded in Epon. For light microscopic evaluation 2-μm-thick sections were stained with periodic acid Schiff and 0.1% toluidine blue to quantify the percentage of hibernating cells, the amount of interstitial connective tissue and the diameter of the CMs. Ultrathin sections were briefly stained with uranium acetate and lead citrate and examined in a Philips CM100 electron microscope. The presence or absence of necrotic and/or condensed apoptotic nuclei in CMs in light and electron microscopic sections was evaluated by experienced microscopists.

Postmortem angiography performed in the 8-week chronic coronary artery stenosis group showed an average % reduction in the luminal area of 53.8±18.5% for the LAD coronary artery and 42.6± 16.2% for the LCX coronary artery. In the group of animals subjected to chronic multivessel coronary artery stenosis followed by PTCA of one of the two stenotic vessels, in vivo angiography showed that the average degree of stenosis was 83± 9% luminal area reduction. In the dilated vessels, the degree of coronary stenosis was decreased from 83.8±9 to 53±11% (Pb.02). Postmortem angiography performed after 20 weeks indicated that the degree of coronary stenosis in the vessels which were not dilated, was 68±20% luminal reduction. The degree of stenosis in the PTCA related vessels was significantly lower: 34±22% luminal area reduction (Pb.05). 3.2. Acute experiments: effect of coronary constriction on myocardial blood flow In five open chest sheep, the LAD and the LCX were narrowed using rings with a diameter of between 1.5 and 2.5 mm, according to the size of the native vessel. The induction of coronary artery stenosis did not alter resting myocardial blood flow significantly. This can be seen in Fig. 1A: at rest, baseline flow measured using Doppler flow probe around the LCX tended to decrease after induction of the two vessel coronary artery stenosis: 41±10 ml/min versus 33±6 ml/min, but the difference did not reach statistical significance (PN.05). On the other hand, coronary vasodilator reserve, estimated by peak reactive hyperemic response, was significantly reduced to 57% of its original value: peak flow was 196±32 ml/min before and only 90±28 ml/min after stenosis (Pb.05). During dobutamine infusion up to 15 μg kg −1 min −1 LCX flow increased significantly from 41±12 ml/min at

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Fig. 1. (A) Coronary blood flow measured in the LCX coronary artery using a Doppler flow probe before and after (open versus closed circles) induction of a severe stenosis on the LCX and LAD coronary arteries. Data are given (mean value±S.D.) at baseline (BL1), at 60 s of LCX occlusion (Occl), at peak reactive hyperemia (Hyp), after stabilization of resting flow (BL2), and during and after the infusion of incremental doses of dobutamine up to 15 μg kg−1 min−1. (B) Myocardial blood flow (ml min−1 g−1) in the LAD perfusion area, measured by colored microspheres. Data (mean value±S.D.) are presented for the subepicardial (left panel) and the subendocardial layer (right panel) before (open bars) and after (closed bars) two vessel coronary artery stenosis. Microspheres were injected at baseline, peak reactive hyperemia and at 10 μg kg−1 min−1 of dobutamine infusion.

baseline to 120±31 ml/min (Pb.05) before induction of stenosis and only to 70±30 ml/min after induction of LAD and LCX stenosis (Pb.01). Nevertheless, this last value is significantly higher than the value before starting dobutamine (40±10 ml/min; Pb.05; see Fig. 1A). Similar results were obtained using microspheres. Because the LAD was not equipped with a Doppler flow probe, microsphere data are presented in Fig. 1B. In the subendocardium (right panel), flow increased from 0.96±0.40 ml min −1 g −1 at baseline to 3.59±1.20 ml min −1 g −1 at peak reactive hyperemia before stenosis (Pb.05) and only to 2.17±1.51 ml min −1 g −1 after induction of the stenosis (PN.05). In the subepicardial layer (Fig. 1B left panel), flow increased from 0.94±0.44 ml min −1 g −1 to 2.84±1.48 ml min −1 g −1 before stenosis (Pb.05) and only to 1.70±0.95 ml min −1 g −1 after stenosis (PN.05). This means that coronary reserve is reduced to about 60% of its original value as well in the subepi- as in the subendocardium. During dobutamine infusion (10 μg kg −1 min −1), myocardial blood flow increased homogeneously in all areas of the left ventricle. Before stenosis, subendocardial LAD flow increased to 2.15±0.50 ml min −1 g −1 and only to 1.46±0.73 ml min −1 g −1 after stenosis (Pb.05). In the subepicardium, before coronary stenosis, LAD flow increased to 2.39± 0.89 ml min −1 g −1, which value is significantly different (Pb.05) from that after stenosis (1.73±0.89 ml min −1 g −1, Pb.05. Also in the perfusion area of the LCX coronary artery, a similar response to dobutamine infusion is found. The acute effect of dobutamine stress on global and regional cardiac function was also evaluated. Heart rate was quite similar

Fig. 2. Global and regional cardiac function in the acute experiment. Upper panel: the effect of incremental doses of dobutamine up to 15 μg kg−1 min−1 on heart rate. Middle panel: the effect of dobutamine infusion on left ventricular pressure (LVP). The increase in systolic (upper graphs) LVP during stress is significantly less after double coronary stenosis than before (*Pb.05). At the end of the recovery phase, recovery of systolic LVP remains incomplete (#Pb.05 versus baseline and *Pb.05 versus prestenosis). Diastolic (lower graphs) LVP remains grossly unaltered. Lower panel: the effect of dobutamine stress on LV dP/dt max and min is significantly less (*Pb.05 versus pre-stenosis). Mean values±S.D. are presented.

before and after multiple coronary artery stenosis (PN.05; see Fig. 2 upper panel). However, this was not the case for peak systolic left ventricular pressure and LV dP/dt max and min, which were significantly lower during dobutamine infusion after induction of the stenosis than before (Pb.05; see Fig. 2 middle and lower panel, respectively). 3.3. Chronic coronary artery stenosis 3.3.1. Regional myocardial blood flow In the 8-week chronic coronary artery stenosis study, myocardial regional blood flow was determined under baseline conditions,

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Table 1 Eight week chronic coronary artery stenosis; influence on general hemodynamics Variables

Heart rate (beats/min) SAP (mmHg) DAP (mmHg) LVEDP (mmHg) Cardiac output (L/min) max LV dP/dt (mmHg/s) min LV dP/dt (mmHg/s)

Baseline

81±17 93±7 68±7 10±7 4.99±0.98 1069±232 1500±329

After stenosis

Table 3 Chronic multivessel coronary artery stenosis followed by PTCA: general hemodynamics Variables

20 minutes

8 weeks

86±17 92±7 64±10 11±6 4.86±0.7 1249±419 1457±280

85±17 93±12 68±9 18±4* 4.13±0.99 819±226* 1236±123

Values are means±standard deviation; DAP, diastolic aortic pressure; max LV dP/dt and min LV dP/dt, positive and negative left ventricular dP/dt; LVEDP, left ventricular end-diastolic pressure; SAP, systolic aortic pressure. *Pb.01.

immediately after induction of coronary stenosis and 8 weeks later. Regional myocardial blood flow did not differ significantly (PN.05) between the experimental conditions (data not shown). In the group of sheep subjected to chronic multivessel coronary artery stenosis followed by PTCA, regional blood flow was determined under baseline condition, immediately after induction of the stenosis on the LAD and LCX coronary arteries, 10 weeks later before and after PTCA of one of the two vessels, and once more at 20 weeks. As in the 8 week group, regional myocardial blood flow was not influenced by the induction of coronary stenosis. The values at baseline after stenosis and 10 weeks later were not different from each other (PN.05; data not shown). Immediately after PTCA, regional myocardial blood flow was increased in the subendo- and subepicardial layer of the perfusion area of the LAD and LCX which was due to a reactive hyperemic response elicited by the PTCA procedure in 50% of the vessels. However, the standard deviation in the values was very large and the increases were not significant. Ten weeks after PTCA, regional myocardial blood flow normalized completely and was not statistically different from baseline values (PN.05; data not shown). In order to evaluate possible differences in regional myocardial blood flow between the perfusion areas of the PTCA-related vessels and the nonPTCA-related vessels and to analyze a potential effect of percutaneous transluminal coronary angioplasty on regional myocardial blood flow, myocardial blood flow in the areas perfused by these vessels were calculated separately. Nearly the same results were found as for the LAD and LCX. None of the values differed significantly from each other in time (PN.05, ANOVA) and there was also no significant difference in myocardial perfusion between the non-PTCA- and PTCA-related vessels (PN.05, ANOVA) (data not shown). 3.3.2. Global and regional myocardial function Hemodynamic data in the 8-week chronic coronary artery stenosis group were measured under baseline conditions, at 20 min after induction of the coronary artery stenosis and 8 weeks later. The data are presented in Table 1. Induction of the coronary artery stenosis did not influence hemodynamics for all variables. However, 8 weeks after induction of the coronary stenoses, left ventricular end-diastolic pressure was significantly increased from 10±7 mmHg at baseline to 18±4 mmHg

Baseline

Heart rate (beats/min) SAP (mmHg) DAP (mmHg) LVEDP (mmHg) Cardiac output (L/min) max LV dP/dt (mmHg/s) min LV dP/dt (mmHg/s)

85±16 105±6 79±4 19±4 4.1±0.88 1111±164 1597±207

After stenosis 20 minutes

20 weeks

87±18 98±13 75±11 22±5 4.08±1.08 999±293 1513±481

89±15 98±8 75±10 17±5* 3.57±0.67 912±175 1376±433

Values are means±standard deviation; DAP, diastolic aortic pressure; LVEDP, left ventricular end-diastolic pressure; max LV dP/dt and min LV dP/dt, positive and negative left ventricular dP/dt; SAP, systolic aortic pressure. *Pb.05.

at 8 weeks (Pb.01). Also LVP dP/dt maximum did not change shortly after induction of the stenoses but decreased significantly (Pb.01) 8 weeks later. Echocardiographic variables were measured during general anesthesia before the surgical induction of coronary artery stenosis, during sedation at one hour post surgery and at two weeks intervals up to 8 weeks. LV cavity area, i.e., ED and ES area, were slightly lower at one hour post-surgery as compared to the baseline data (see Table 2). This can be explained by the cardiodepressive effect of the halothane during general anesthesia at baseline. During follow-up, LVED cavity area as well as LVES cavity area increased progressively and significantly (Pb.01). The data are presented in Table 2. FAC was smaller during baseline measurement under general anesthesia as compared to the one hour after stenosis value, obtained under sedation (see Table 2). During follow-up, the FAC values measured under sedation were progressively decreasing with time (Pb.01). Regional myocardial function was assessed by fraction of wall thickness (FWT; see Table 2). For the measured anterior and posterior areas, baseline values under general anesthesia were lower than the corresponding values one hour after stenosis, which were measured during sedation. During follow-up, FWT decreased progressively and significantly (Pb.05). FWT at 8 weeks was decreased to about 50% of the value immediately after induction of the stenoses. In the chronic multivessel coronary artery stenosis followed by PTCA group, general hemodynamics were studied under baseline conditions, at 20 minutes following the induction of coronary stenosis and 20 weeks later at the end of the experiment. The results are presented in Table 3. It can be seen that most hemodynamics remained stable during the observation period. Most of the values did not change significantly, except for LVEDP, which was significantly lower at 20 weeks versus the value immediately after induction of the stenosis (Pb.05). Global and regional function was measured by echocardiography at different time intervals (see Table 4 and Fig. 3, respectively). LVED cavity area increased significantly during the first ten weeks of coronary stenosis (see Table 4) and resembled the increase found in the 8-week group. After percutaneous transluminal coronary angioplasty, performed at 10 weeks, LVED cavity area decreased again to nearly baseline values. LVES cavity area also increased significantly during the first 10 weeks of coronary artery

Table 2 The effect of chronic coronary stenosis on cardiac function Variables

Baseline

After stenosis

2 weeks

4 weeks

6 weeks

8 weeks

LVED cavity area (cm2) LVES cavity area (cm2) FAC % Anterior WTF FWT % Posterior WTF FWT %

19.1±1 11±3 43±10 34.0±5.2 32.7±7.2

16±4 8±3 54±8 43.5±14.7 35.1±9.7

19±3 9±1 52±5 39.8±14.6 34.1±10.1

18±4 9±3 51±6 28.8±7.9 32.0±11.4

20±3 10±2 47±4 29.4±12.4 21.0±10.8

21±3** 11±4** 46±3** 20.3±9.5**,* 23.1±6.6*

Values are shown as mean±standard deviation; WTF FWT=fraction of wall thickness wall thickening fraction; *Pb.05 versus baseline; **Pb.05 versus after stenosis.

F. Verheyen et al. / Cardiovascular Pathology 23 (2014) 160–168 Table 4 The effect of chronic coronary artery stenosis and PTCA on global cardiac function Time

BS 4 weeks 8 weeks 10 weeks 12 weeks 14 weeks 16 weeks 18 weeks 20 weeks

Cavity area (cm2)

FAC (%)

LVED

LVES

15.9±2.3 18.3±1.9* 19.3±2.3* 17.6±2.4* 16±1.3 18±2.1 15.5±2.3 17.2±2.4 16.9±2.7

7.9±1.7 8.9±0.7* 9.4±1.3* 8.8±1.2* 7.7±0.3 8.7±0.7 7.3±0.9 7.9±0.7 8.5±1.2

46.5±9.4 46±13.4 45.1±12.6 47±7.3 49.2±4.8 47.3±8.6 50±6.2 50.5±9.2 48.5±5.1

Mean values±standard deviation; BS, baseline; *Pb.05 versus baseline.

stenosis and decreased after PTCA. However, this decrease did not reach statistical significance (PN.05). Regional myocardial function was estimated by FWT. During chronic coronary artery stenosis, i.e., the first 10 weeks of the observation period, FWT decreased progressively and significantly in the perfusion area of both stenotic vessels. After PTCA, FWT increased in the PTCA-related area to values reaching baseline values. In the non-PTCA-related segments, the FWT remained depressed.

3.3.3. Effects of chronic coronary constriction and PTCA on myocardial microscopic morphology As compared with the morphology of normal control sheep (Fig. 4A), the following changes were found after 8 weeks of stenosis in the CMs: depletion of sarcomeres (=myolysis), accumulation of glycogen, altered size and shape changes of mitochondria and redistribution of heterochromatin in the nucleus (Fig. 4B). The degree of CM changes was quantitatively analyzed. The percentage of affected cells was calculated and showed a significant increase in number of hibernating CMs in subepi- and subendomyocardium and transmurally after 8 weeks of stenosis as compared with the control LV myocardium (Fig. 5, left panel; Pb.05 vs. control). In the group of sheep subjected to chronic multivessel coronary artery stenosis followed by PTCA of one of the two stenotic vessels a complete reversal of hibernation was observed at 20 weeks at both the PTCA and the stenosis (remote) site (Fig. 5, right panel; P=.002 vs. 8 weeks). The percentage of hibernating cells in the transmural layer of the left ventricle at 8 weeks poststenosis and the percentage change in LVED cavity area correlated well (r=.888; Pb.008).

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Morphometric evaluation of the amount of connective tissue showed a clear increase in connective tissue area in the LAD-epi (Pb.001), LAD-endo (P=.073), Cx-epi (Pb.001) and Cx-endo (P=.003) sites after 8 weeks of stenosis, compared to the comparative sites in the control animals (Fig. 6). In the animals subjected to PTCA, the amount of connective tissue in all investigated sites was not different from that calculated in the control animals and was significantly less as compared with that in the 8 week stenosis group (Fig. 6). To assess whether changes in size of CMs had occurred the diameter of transmural normal non-hibernating and of hibernating CMs were measured in control sheep, sheep with 8 week chronic coronary artery stenosis and sheep with chronic multivessel stenosis followed by PTCA. It occurred that the normal, non-hibernating CMs tended to be larger after 8 weeks of chronic coronary artery stenosis (P=.107) than in the control sheep (Fig. 7). In the animals subjected to chronic multiple stenosis followed by PTCA (20 weeks) the diameter of the transmural normal non-hibernating CMs was largely reduced (P=.051) as compared to those in the 8-week group (Fig. 7). In the control (P=.011) and 8-week (P=.000) group, the diameter of the hibernating CMs was significantly larger as compared with the normal, non-hibernating CMs (Fig. 7). 4. Discussion Most models of experimental chronic left ventricular dysfunction are models of single stenosis [7,25,30,31], showing a decrease in regional myocardial function in the area perfused by the stenotic coronary artery. However, most patients suffering from an ischemic cardiomyopathy have multivessel disease and dilated left ventricles. In our model, the two major coronary arteries supplying the left ventricle are critically stenotic and this results in a chronic, progressive dilatation of the left ventricle with an increase in endsystolic and end-diastolic chamber area and a decrease in fractional area change. These findings correspond with those of Firoozan et al. [32], who developed a model of chronic ischemic cardiomyopathy by placing ameroid constrictors on the proximal portions of the two major coronaries in dogs. Left ventricular end-systolic size nearly doubled and the percent change in left ventricular size from end diastole to end systole decreased by more than 50%. However, this model is fundamentally different from ours: the coronaries were chronically occluded and extensive collateral development occurred. Models using ameroid constrictors are not stable over time in terms of myocardial perfusion: resting flow drops at the time of ameroid occlusion and recovers variably according to the degree of coronary collateral development [33]. This might explain why Firoozan and coworkers [32] found a combination of infarcted and non-infarcted regions. 4.1. Coronary artery stenosis characteristics

Fig. 3. Effect of PTCA on regional myocardial FWT. Mean values±standard deviation; BS=baseline; *Pb.05 versus baseline; **Pb.04 versus non-PTCA area.

In this study, we show that multivessel coronary artery stenosis induces left ventricular hypokinesis, dilatation and structural abnormalities. Coronary artery stenosis was achieved by implantation of plastic rings having a diameter of less than 2.5 mm. As shown by angiography, this resulted in a reduction of vessel diameter of more than 42%. This reduction only resulted in a slight decrease in resting myocardial flow. Coronary dilatory reserve on the other hand, measured by peak reactive hyperemia, was significantly reduced to about 60% of its original value. These are the known characteristics of a hemodynamically significant stenosis [34,35]. The increase in myocardial oxygen consumption of the myocardium due to incremental infusions of dobutamine was not met adequately by an appropriate increase in blood supply to the post-stenotic perfusion area. This was clearly shown in the open chest experiments. Thus, the dobutamine stress-test elicited a “demand ischemia” in the post-

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Fig. 4. Light microscopy (inserts) and electron microscopy of subendocardial samples from the left ventricle of a normal control sheep (A) and 8 weeks after stenosis (B). The light microscopic pictures are stained with PAS (revealing glycogen) and toluidine blue. (A) With the exception of two Purkinje fibers (arrows) the myocardium is almost devoid of PAS stain for glycogen (insert). Electron microscopy shows CM cytoplasm which is normally filled with sarcomeres (sm) and mitochondria (m). n=nucleus. (B) A large proportion of the CM shows a PAS positive cytoplasm (red/dark stain) indicative of glycogen accumulation. Signs of degeneration and cell loss are not present and changes in the extracellular space are not obvious. The electron microscopic picture demonstrates myolysis as indicated by depletion of sarcomeres with only fragmented sarcomeres (sm) remaining. A large part of the cytoplasm is filled with glycogen. Remnants of sarcoplasmic reticulum (sr) are seen in one area. Note the presence of many small, but healthy looking mitochondria (m).

stenotic area which resulted in an inadequate increase in left ventricular contractility during the stress test. 4.2. Chronic myocardial dysfunction in the presence of normal resting blood flow Our results show that multiple chronic coronary artery stenosis allowing near-normal resting myocardial blood flow can lead to chronic hypocontractility, left ventricular chamber dilatation and changes in myocardial subcellular structure in otherwise viable myocytes. The only prerequisite seems to be the degree of coronary stenosis, which must be critical enough to reduce coronary vasodilatory reserve significantly. It has been argued that chronic myocardial hypocontractility in viable segments receiving normal or near normal myocardial blood flow is consistent with a state of chronic or perpetual stunning [5,7–9]. Although we did not prove stunning in

Fig. 5. Percentage of hibernating CMs. Left panel: as compared with LV myocardium of the control animals, a significant increase in number of hibernating CMs was present after 8 weeks of stenosis in subepi- (Pb.05 vs control) and subendomyocardium (*Pb.05 vs control). Right panel: at 20 weeks, a complete reversal of hibernation (myolysis) was seen at both the PTCA and the stenosis (remote) site (#Pb.05).

the strict sense in our study, repetitive stunning secondary to demand ischemia by regular running in the open is the most likely mechanism. Many clinical studies on reversible chronic myocardial dysfunction [3,4,17, and others reviewed recently in Ref. 36] have shown a decreased resting myocardial blood flow. It has been suggested that acute reductions in coronary flow can induce a “down regulation” of function reflecting a state of “acute hibernation”. Indeed, experimental evidence exists showing that a short period of hypoperfusion can be well tolerated [37–39]. On the other hand, experimental studies where coronary hypoperfusion was installed for a longer period of time, ended up either with some degree of myocardial necrosis [31,40] or without infarction [30,31,41].

Fig. 6. Connective tissue area. After 8 weeks of chronic coronary artery stenosis the amount of connective tissue was slightly but significantly increased in three of the myocardial sites as compared with the comparative sites in the control animals (*Pb.05). In the sheep subjected to chronic multiple stenosis followed by PTCA the connective tissue area in all sites was significantly less compared to the area in the 8-week group (#Pb.05).

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Fig. 7. Diameter of normal, non-hibernating and hibernating CMs. As compared with the mean transmural diameter of the CMs in control sheep, the mean transmural diameter of the normal, non-hibernating CMs in the 8-week animals tended to be larger. The mean transmural diameter was largely normalized after the PTCA procedure at both the PTCA side and stenosis side. In the control, 8 weeks stenosis and 20 weeksstenosis group, the mean transmural diameter of the hibernating CMs was significantly larger than that of the normal, non-hibernating CMs (*Pb.05).

Fallavollita and co-workers described that myocardial dysfunction precedes the reduction in resting perfusion in the presence of a coronary artery stenosis and that the reductions in flow are a result rather than a transition from chronic contractile dysfunction. They speculate that there is a transition from stunning (normal resting flow) to hibernation (decreased resting flow) and that this transition is related to the physiological significance of the stenosis. Once flow is critically compromised, hibernation occurs reflecting an adaptation to minimize ischemia during subsequent episodes of increased demand [10,36]. Our findings are in agreement with their observations: a severe stenosis which reduces coronary reserve more than 50% is associated with significant and consistent reductions in fraction of wall thickness. This is in contrast to milder degrees of coronary stenosis having no or only minor and delayed effect on left ventricular dimensions and contractility. 4.3. Myocardial structural adaptation after chronic coronary constriction and PTCA As compared with the low number found in the control sheep, an obvious increase in number of dedifferentiated, fetal type hibernating CMs was observed after 8 weeks of dual stenosis in the epi- and endocard of the LAD and Cx regions. These dedifferentiated cells showed the same morphological characteristics as described previously in man and experimental animals, i.e., depletion of contractile filaments, accumulation of glycogen, redistribution of nuclear chromatin and changes in size and distribution of mitochondria [15– 19,42]. After PTCA of one of the stenotic coronary arteries, the number of hibernating myocardial cells decreased to control levels. This decrease was not only observed in the heart region irrigated by the coronary artery subjected to PTCA but also in the heart region which remained irrigated by the stenotic artery. Dedifferentiation of CMs has been described in various pathological situations such as volume and pressure overload [43], chronic atrial fibrillation [42] and coronary artery disease [36]. Dynamic passive stretch was regarded as the underlying mechanism for the occurrence of hibernating myocardium [15–19]. Particularly, this assumption is reinforced by the present study showing that, upon recovery of the increase in cavity area and decrease of FWT resulting from PTCA of only one of the stenosed coronary arteries, near normalization of cell structure was seen in myocardial areas of both the PTCA and the stenosed site. A similar observation was done in swine in which myolysis was seen to the same extent in the stenosed LAD region as well as in the normally perfused remote regions [28].

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Therefore, this global nature of myolysis suggests that the induction of hibernating myocardium is dissociated from intrinsic adaptation to ischemia. Related to comparative sites in the control sheep, a relatively small but significant increase in volume fraction of diffuse connective tissue was observed in LAD and Cx sites after 8 weeks of dual stenosis. In these sites, signs of degeneration of CMs were completely absent suggesting that the increase in connective tissue is not due to replacement fibrosis related to died CMs but rather to reactive fibrosis. Such a diffuse, reactive fibrosis is increasingly recognized in a variety of pathological conditions unrelated to ischemia [44]. Quite often, extracellular matrix remodeling is accompanied by CM hypertrophy and have been related to elevations in myocardial stress [45]. Also in our model a tendency to an increase in diameter of the normal nonhibernating CMs was observed after 8 weeks of stenosis. Most likely, this CM hypertrophy and extracellular matrix remodeling are compensatory phenomena that are related to the stress imposed by the alteration in ventricular dimension and hypocontractility as a result of myocardial hibernation. Upon PTCA treatment, the structural remodeling, including the amount of hibernating cells as well as the hypertrophy and increase in extracellular matrix components, was almost fully reversed. This indicates that dedifferentiated, viable hibernating myocardium has much potential for reverse remodeling to near normal contractile myocardial tissue resulting in important functional improvement. This is also suggested by clinical studies. Previous data on dobutamine stress echocardiography in patients identified hibernating myocardium and predicted recovery of left ventricular function after coronary revascularization [46]. Additionally, hibernating myocardium in patients with multivessel coronary artery disease was found to be characterized by impaired coronary vasodilator reserve which improved significantly after coronary revascularization [47]. Furthermore, increasing benefit from revascularization has been found to be associated with increasing amounts of myocardial hibernation [48], and among patients with ischemic cardiopathy, hibernating, but not ischemic, myocardium identified patients which might accrue a survival benefit with revascularization versus medical therapy [49]. In conclusion, we believe that the disease state of the heart in this sheep model with two critical coronary artery stenoses can be regarded as an uncomplicated pure hibernative state. In this way, it might be used to better understand the various processes in chronic myocyte remodeling, on the one hand, and reverse remodeling upon therapeutic interventions on the other. Acknowledgments We thank Luc Wouters and Frits Prinzen for valuable suggestions. This work was supported by CARIM, Maastricht University, Maastricht, The Netherlands. References [1] Rahimtoola SH. A perspective on the three multicenter randomized clinical trials of coronary bypass surgery for chronic stable angina. Circulation 1985;72(Suppl V):V123–35. [2] Rahimtoola SH. The hibernating myocardium. Am Heart J 1989;117:211–2. [3] Rahimtoola SH. Hibernating myocardium has reduced blood flow at rest that increases with low-dose dobutamine. Circulation 1996;94:3055–61. [4] Tillisch JR, Brunken R, Marshall R, Schwaiger M, Mandelkern M, Phelps ME, Schelbert H. Reversibility of cardiac wall motion abnormalities predicted by positron tomography. N Engl J Med 1986;314:884–8. [5] Vanoverschelde J-LJ, Wijns W, Depré C, Essamri B, Heyndrickx GR, Borgers M, Bol A, Melin JA. Mechanisms of chronic regional postischemic dysfunction in humans. Circulation 1993;87:1513–23. [6] Canty JM, Klocke FJ. Reductions in regional myocardial function at rest in conscious dogs with chronically reduced regional coronary artery pressure. Circ Res 1987;61(Suppl II):II107–16. [7] Shen Y-T, Vatner SF. Mechanisms of impaired myocardial function during progressive coronary stenosis in conscious pigs. Hiberation versus stunning? Circ Res 1995;76:479–88.

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