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European Journal of Heart Failure 2007 9(4):377-383; doi:10.1016/j.ejheart.2006.09.012
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© 2007 European Society of Cardiology

Relevance of apoptosis in influencing recovery of hibernating myocardium

Annalisa Angelinia,*,1, Giuseppe Maiolinob,1, Giovanni La Cannac, Claudio Ceconie, Fiorella Calabresea, Elena Pettenazzoa, Marialuisa Valentea, Ottavio Alfierid, Gaetano Thienea and Roberto Ferrarie

a Department of Pathological Anatomy, University of Padua Via Gabelli, 61, 35121 Padova, Italy
b Division of Cardiology, University of Padua Italy
c Department of Non-invasive Diagnostic Cardiology, San Raffaele Foundation Institute and University Hospital Milan, Italy
d Department of Cardiac Surgery, San Raffaele Foundation Institute and University Hospital Milan, Italy
e Department of Cardiology, University of Ferrara and Fondazione S. Maugeri IRCSS Gussago, Brescia, Italy

* Corresponding author. Tel.: +39 0 49 8272260; fax: +39 0 49 8272285. E-mail address: annalisa.angelini{at}unipd.it


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Background: Hibernating myocardium (HM) is viable but dysfunctional myocardium which can recover following revascularization. Myocyte necrosis is virtually absent in HM; however, cellular loss may take place by apoptosis, although this is controversial.

Aim: To assess the presence of apoptosis and its relevance in HM.

Methods: During coronary artery by-pass surgery (CABG), 21 patients underwent transmural biopsy in the dysfunctional left anterior descending artery tributary area of the left ventricle (LV), with kinetic recovery at follow-up, thus fulfilling the HM criteria. All patients underwent echocardiographic follow-up at 12 months. All biopsies were evaluated by light microscopy, electron microscopy (EM), and molecular analysis.

Results: All biopsies were structurally altered, showing increased fibrosis and myocytes with variable size. Myocyte dedifferentiation was not detected by immunohistochemistry or EM. On stepwise linear regression, 1 year LVEF was predicted by the apoptotic index (β=–0.973, p=0.002), the normotrophic cell percentage (β=0.449, p=0.038), and mean fibrosis (β=–0.412, p=0.51).

Conclusions: Our biopsy study detected a wide range of morphological substrate heterogeneity in HM with degenerative features. We have demonstrated for the first time in humans that myocyte apoptosis is an important phenomenon in HM, negatively influencing LV functional recovery after CABG.

Key Words: Hibernating myocardium • Apoptosis • Pathology

Received March 6, 2006; Revised July 28, 2006; Accepted September 19, 2006


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
The term hibernating myocardium (HM), referring to persistently impaired left ventricular function at rest due to reduced coronary blood supply, that can partially or completely recover on restoration of adequate blood supply, was first used by Rahimtoola [1]. This concept was expanded further by Bolli [2] who postulated that HM could also result from repetitive myocardial stunning, caused by stress-induced ischaemic episodes. Pagano et al. [3,4] confirmed this hypothesis by demonstrating with positron emission tomography that HM blood flow is similar before and after coronary artery bypass surgery (CABG) and comparable to controls, whereas the coronary vasodilatory reserve is reduced.

The pathological effects of HM are: myofilament depletion, glycogen deposition, nuclear and mitochondrial abnormalities, and increased extracellular fibrosis [5-8]. Acute ischaemic death of myocytes is virtually non-existent in HM [9,10]; however, cellular loss could occur by programmed cell death or apoptosis [7,11], although this is controversial [12,13]. Thus, our aim was to assess the presence of apoptosis in HM and its relevance in left ventricular (LV) functional recovery.


   2. Materials and methods
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
We enrolled 21 patients with angiographically assessed coronary artery disease and LV dysfunction at rest (LV ejection fraction (LVEF) ≤50%), referred to the Department of Cardiology, University of Brescia, for CABG. One patient was excluded from the analysis because the tissue sample obtained during surgery was inadequate. The decision for surgery was based on clinical and angiographic assessment.

Inclusion criteria were: detection of HM only in the left descending coronary artery (LAD) territory (as assessed by dobutamine echocardiography and partial or complete thallium-201 redistribution); narrowing ≥70% of the LAD; persistence of segmental left ventricular akinesis detected by serial echocardiograms for at least 1 month before revascularization; absence of rest angina or clinical instability during the echocardiographic and thallium studies; no unstable angina or acute myocardial infarction in the last 6 months before the study; no other concomitant cardiopulmonary or severe valvular disease.

All the patients underwent successful revascularization, without the occurrence of peri-operative myocardial infarction, which was excluded by ECG, echocardiogram monitoring, and CPK-MB or troponin determination. The study was approved by the Hospital Joint Ethical Committee on Clinical Investigation, and each patient provided written informed consent.

Patients underwent all diagnostic tests within 1 month prior to CABG, without any intervening cardiac event. The stability of the wall motion abnormalities was confirmed by echocardiographic examinations, which were repeated for each patient the day before surgery. All patients underwent CABG using the left internal mammary artery and saphenous vein grafts.

2.1. Low-dose dobutamine echocardiography
Complete two-dimensional and M-mode echocardiography and Doppler ultrasound was performed using an HP Sonos 1500 with 2.5, 3.5, and 5 MHz transducers. Dobutamine was infused intravenously using an infusion pump for 5 min at 5 and at 10 µg*kg–1 body weight per minute, without complications. Beta-adrenergic blocking or inotropic agents were withdrawn 48 h before the test.

To evaluate regional wall motion the left ventricle was divided into 16 segments [14]; regional systolic wall motion and thickening were evaluated in each segment by two experienced observers who were not aware of the identity of patients, the clinical findings, the order of echocardiograms, and the thallium-201 scintigraphy and angiography findings. The inter- and intra-observer variability for distinguishing akinetic from either normal or hypokinetic segments was 2% and 1%, respectively. Any inter-observer discrepancies in the grading of segments were resolved by consensus with two other well trained observers. A previously described scoring system was used [15]. Wall motion score was calculated for each patient as the sum of the scores of all 16 segments divided by the number of segments evaluated. Evaluation of wall motion and thickening in each segment was always performed >1 cm from the adjacent segment to avoid misinterpretation due to the tethering phenomenon.

2.2. Rest-redistribution thallium-201 scintigraphy
All patients underwent rest-redistribution thallium-201 scintigraphy. After an overnight fast, tomographic images were acquired 10 min (rest) and 4 h (redistribution) after injection of 74 MBq of thallium with a single-headed gamma camera (SP4 HR, Elscint) equipped with a general purpose, low energy, collimator (FWHM=11 mm). Both rest and redistribution thallium-201 images were acquired in 60 steps of 20-30 s each over a circular 180° orbit from the right anterior to the left posterior oblique projection. The data were collected on a 64x64 matrix with a 1.3 zoom. The set of transaxial images were reconstructed using a standard filtered back projection (Wiener filter). From the three-dimensional voxel matrix, three sets of 18 mm thick slices realigned along the main cardiac axes were then derived (short axis, horizontal and vertical long axis). From short axis slices a polar map was obtained. To compare with the echocardiographic images, analysis of regional thallium-201 myocardial uptake was performed using a 16-segment model. A considerable effort was made to match the scintigrams to the 16 left ventricular segments used in the echocardiographic studies. The uptake of tracer was graded subjectively in each segment using a scoring system based on reduction in peak thallium-201 uptake compared with a normally contracting segment: 1 (<25%); 2 (25-50%); 3 (>50 to 85%); 4 (>85%). Delayed images (redistribution) were classified for presence or absence of increase in thallium-201 uptake (one or higher grade compared with baseline). Interpretation of the tomographic studies was performed by consensus of two experienced investigators who were not aware of the clinical, echocardiographic and angiographic findings. A myocardial segment was graded as viable when the resting score was 1 or 2 and there was complete or partial redistribution, and was graded as non-viable when the resting score was 1-2 and there was no evidence of redistribution at delayed images.

2.3. Intra-operative echocardiography
After induction of anaesthesia, a 5-MHz phased array transoesophageal transducer was introduced into the oesophagus and connected to an HP 1500 echocardiogram for routine intra-operative monitoring of left ventricular function. In each patient a pulmonary artery Swan-Ganz® thermodilution catheter and a radial artery catheter were inserted. All vessels with significant (≥70%) stenosis were bypassed by a single surgeon who was unaware of the pre-operative echocardiographic and scintigraphic findings. The epicardial echocardiogram was performed as previously reported [15].

2.4. Histological, immunohistochemical and ultrastructural analysis
During CABG, under echocardiographic guidance, two transmural biopsy specimens of the anterior left ventricular wall were obtained from the centre of the area where HM was found (dobutamine echocardiography and partial or complete thallium-201 redistribution). The biopsies' epicardial face was marked with India ink. One biopsy was immediately immersed in phosphate-buffered 3% glutaraldehyde, postfixed in veronal acetate-buffered 2% osmium tetroxide, dehydrated in graded series of ethanol and embedded in Epon. The other biopsy was stained with haematoxylin-eosin and Azan Mallory. The first stain was used to measure at optical microscopy, with a computerized morphometric system, the myocyte diameter of 85-100 cells at 40x magnification in the epicardium, mesocardium, and endocardium. This evaluation was done only on myocytes transversely sectioned (central nucleus and boundaries approximately circumferential). The Azan Mallory stained biopsy was utilized to measure the mean percentage of fibrosis in the epicardium, mesocardium and endocardium, using 60 fields for specimen at a magnification of 40x. With TUNEL technique (terminal transferase (TdT) mediated dUTP nick end-labelling) the apoptotic index was determined ((number of positive myocytes for field/total myocyte number for field)x100). The immunohistochemical analysis was done with anti-{alpha}-smooth muscle actin antibodies. Dedifferentiation, regeneration, degeneration signs and apoptosis were assessed by electron microscopy.

2.5. Follow-up
All patients were re-evaluated by baseline transthoracic echocardiography, a mean of 3 and 12 months (range 2-4 months and 11-13 months, respectively) after surgery.

2.6. Statistical analysis
All quantitative variables are presented as mean±standard deviation. Data were tested for Gaussian distribution with the Kolmogorov-Smirnov test and, if required, were transformed by logarithm, then a two tailed Student's t-test was used to identify the differences between groups. The Fisher exact test was used to compare differences among categorical variables. Multiple linear regression analysis was performed to predict the 1 year LV ejection fraction (EF) and the recovery of LV function at 1 year (LVEF at 1 year-baseline LVEF). Variables considered in the analysis were: time interval between symptom onset and CABG; the baseline LVEF; normotrophic cell percentage; the mean percentage fibrosis in the myocardium; the apoptotic index. Differences were considered significant at p<0.05.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
Twenty patients completed the study (18 males and 2 females), their clinical and echocardiographic characteristics are shown in Tables 1 and 2. Sixteen patients had a previous myocardial infarction, in 10 patients this was of the inferior LV wall and in 6 patients of the anterior LV wall. Mean left ventricular EF was 24.7±8.2% preoperatively, 32.1±13.9% after 3 months, and 40.2±9.3% after 1 year, with a statistically significant difference between pre-CABG and 1 year values (p<0.0001). Left ventricular sizes were significantly reduced from preoperative to 1 year values: end-diastolic left ventricular diameter was 65.7±5.7 mm vs. 60.4±6.1 mm (p=0.001), respectively; end-systolic left ventricular volume was 139.0±44.5 ml vs. 111.4±54.4 ml (p=0.029), respectively. Mean preoperative NYHA class was 2.9±0.9, which decreased to 1.5±0.7 (p<0.0001) at 1 year. At follow-up, all patients had a partial or complete recovery of the LAD tributary territory kinetic.


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  		Table 1 Demographic and clinical characteristics of the subjects

 


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  		Table 2 Echocardiographic and histological characteristics of the subjects

 
A large number of specimens showed structural alterations, with myocytes of variable size, many were atrophic or hypertrophic, with increased fibrosis, although in variable amounts (Fig. 1). Myocytes were characterized by cytoplasmic vacuolization (Fig. 2), predominantly in the perinuclear site, and lipofuscin storage. The nuclei appeared bizarre, displaying alterations in size and shape, and were increased in width. More than 60% of patients showed TUNEL-positive myocytes. Myocyte dedifferentiation was not detected with immunohistochemical analysis (anti-smooth muscular {alpha}-actin antibodies) or with electron microscopy. Ultrastructural analysis confirmed the presence of hypertrophic and degenerated myocytes, myofilament content reduction, glycogen storage, the presence of numerous small mitochondria and nuclear irregularity with apoptosis features (Fig. 3).


Figure 01
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  		Fig. 1 Representative examples of collagen deposition detected in hibernating myocardium specimens, illustrating the morphological substrate heterogeneity of fibrosis. a) Azan 12x; b) Azan 25x.

 


Figure 02
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  		Fig. 2 Histologic section demonstrating, at higher magnification, atrophic myocytes and cytoplasmic vacuolization embedded in fibrosis. Azan 80x.

 


Figure 03
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  		Fig. 3 a) Early stage apoptotic cell (arrow) with fragmentation and margination of chromatin and increase in lipid droplets. b) Apoptotic bodies inside myocyte (arrow). EM 3500x.

 
The mean fibrosis was 20.3±13.7%. Fibrosis was 27.3±17.2%, 20.1±13.5% and 13.1±10.1% in the endocardium, mesocardium and epicardium respectively, with a significant difference between the epicardium and endocardium (p<0.0001). Cellular diameters were 29.5±7.3 µm, 32.3±5.8 µm e 33.8±6.0 µm in the endocardium, mesocardium and epicardium respectively, with a significant difference between the epicardium and endocardium (p=0.018). The percentage of hypotrophic , normotrophic and hypertrophic cells was 2.6±3.6%, 7.8±6.6% and 89.6±9.6%, respectively. The mean apoptotic index was 0.093±0.098%.

At multiple linear regression analysis (backward) the variables that predicted the 1 year EF were apoptotic index (β=–0.973, p=0.002), percentage of normotrophic cells (β=0.449, p=0.038), and mean fibrosis (β=–0.412, p=0.51) (R2-corrected=0.944, p=0.008). Moreover, the predictors of recovery of LV function at 1 year (LVEF at 1 year-baseline LVEF) were baseline LVEF (β=–0.738, p=0.001) and apoptotic index (β=–0.358, p=0.017) (R2-corrected=0.898, p=0.001).


   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
This histological evaluation of biopsy specimens with HM, detected altered architecture, showing variability in myocyte size, myocytes were either atrophied (2.6±3.6%) or hypertrophied (89.6±9.6%), which is consistent with previous studies [16], and with increased fibrosis (20.3±13.7%), although the amounts were variable in each patient. Myocytes were characterized by cytoplasmic vacuolization, predominantly in the perinuclear site, and by lipofuscin storage. The nuclei appeared bizarre, displaying alterations in size and shape, and were increased in width. This is in agreement with previously published papers [5-8,10,17-20]. In the literature there are two antithetic points of view: some authors interpret the myocyte changes as a dedifferentiation pattern [5,12,13,17,21,22], others as myocyte degeneration [6,7,19,23,24]. Our study supports the latter hypothesis. In fact, we found cellular alterations typical of cellular degeneration: myocyte vacuolization, the storage of lipofuscin and myocyte apoptosis (0.093±0.098%). Moreover, we didn't detect any signs of myocyte dedifferentiation, either at immunohistochemical analysis (absence of smooth muscular {alpha}-actin) in contrast to results reported by Ausma et al. [21,22], or at the ultrastructural analysis with electron microscopy. The latter technique confirmed the presence of hypertrophic and degenerated myocytes, the reduction of myofibril content, the storage of glycogen, the presence of numerous small mitochondria, and nuclear irregularity with features in keeping with apoptosis.

The presence of apoptosis and its relevance in human HM is controversial. There are only a few studies of myocyte apoptosis, and the techniques used are often different: TUNEL [7,11,13], evaluation of apoptosis related proteins (P-53 and Bcl-2) [11,13], ultrastructural analysis [7,11,13], and detection of 3-caspase mRNA content [11]. Some authors do not endorse the occurrence of apoptosis [12,13], whereas others support its relevance in HM pathogenesis [7,11]; suggesting that it could be a continuous process that stimulates the development of fibrosis triggering a vicious circle between myocyte damage and reparative mechanisms [25]. Moreover, recently Abbate et al. [26] raised questions about possible underestimation of the apoptotic phenomenon.

In a recent review, Dispersyn et al. [27] disputed the role of myocyte apoptosis in HM. Based on their personal experience, they suggested that apoptosis is a negligible phenomenon in HM; they reported finding only 3 myocytes with electron microscopy evidence of alterations suggestive of apoptosis in biopsies of 530 patients with coronary artery disease. The authors argued that even a tiny percentage of cellular loss, if protracted during time, could produce tissue damage which precludes any possibility of recovery after CABG. Finally, they stressed the point that the TUNEL technique for the detection of myocyte apoptosis causes false positives and false negatives [28].

The discrepancy between our results and those of others could be linked to differences in methodology and/or patient selection criteria, as already hypothesized by Dispersyn et al. [27]. Methodologically, it should be emphasized that use of the TUNEL technique could be criticized [27], but we supported it with apoptosis detection by electron microscopy. The suggestion of Dispersyn et al. [27] that a tiny amount of cell loss, if prolonged, could preclude the recovery of LV function is based on the assumption that myocyte apoptosis is constant in time; however, to the best of our knowledge this has not yet been proved. On the contrary, in an animal model of chronic myocardial dysfunction [29] it was demonstrated that, over time, the prevalence of apoptotic myocytes increases [30]. This led us to think that programmed cellular death may not be a constant phenomenon over time and, as well as increasing in the first phase of chronic myocardial dysfunction (transition from stunned myocardium to HM as stated by Fallavolita et al. [29]), it could decrease in the second phase.

The results of our study expand on previous work by Elsasser et al [11], demonstrating for the first time in humans that myocyte apoptosis is an important event, by negatively influencing recovery of left ventricular function after CABG. This finding is supported by the following evidence from our study. The multiple linear regression model to predict the 1 year EF showed the apoptotic index to be the major determining factor. Moreover, the recovery of LV function, i.e. the difference between the baseline and 1 year LVEF, was predicted by the apoptotic index. From these results we can deduce that the higher the degree of myocyte apoptosis in HM, the lower the 1 year EF and the recovery of LV function following CABG.

In an animal model, Lim et al. [30] demonstrated a decrease in myocyte nuclear density in HM compared to controls. Our results and data in the literature demonstrate an increase in fibrosis in human HM [6,7,10,17,21,22,24] that, at least in part, compensates for the myocyte loss. But what is the causal factor of myocyte loss? Our data and reports in the literature [9,10] demonstrate that myocyte necrosis is virtually absent in HM. Taken together this could support the hypothesis that in HM, apoptosis may cause the progressive myocyte loss.

What triggers apoptosis in HM, is controversial. In the myocardium it could be induced by multiple insults: ischaemia reperfusion phenomena [31], hypoxemia [32], myocardial infarction [33,34], severe heart failure [35], myocyte overstretching [36]. All these mechanisms could play a role, but the first two appear the most plausible in HM.

In HM, the percentage of normotrophic cells and the mean myocardial fibrosis can predict the left ventricular EF 1 year after CABG. Thus, with increasing structural alterations namely, less normotrophic cells and more fibrosis, left ventricular functional recovery and the EF 1 year after CABG will decrease.


   5. Conclusions
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
Our biopsy study detected a wide range of morphological substrate heterogeneity in HM, without signs of dedifferentiation or regeneration. The structural abnormalities which demonstrated degeneration were: the reduction of content of myofibrils, the storage of glycogen, and the presence of irregular nuclei. We have demonstrated for the first time in humans that in HM, myocyte apoptosis is a relevant phenomenon, negatively influencing left ventricular functional recovery after CABG. Structural alterations in HM can determine recovery of LV function.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
1 These authors have equally contributed to this manuscript. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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