Cardiovascular Research

Cardiovascular Research 1999 43(4):947-957; doi:10.1016/S0008-6363(99)00096-6
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Cardiomyocyte remodelling during myocardial hibernation and atrial fibrillation: prelude to apoptosis

Gerrit D. Dispersyn^a, Jannie Ausma^a, Fred Thoné^b, Willem Flameng^c, Jean-Louis J. Vanoverschelde^d, Maurits A. Allessie^e, Frans C.S. Ramaekers^a and Marcel Borgers^a,b,*

^aDepartment of Molecular Cell Biology & Genetics, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
^bDepartment of Morphology, Life Sciences, Janssen Research Foundation, B-2340 Beerse, Belgium
^cDepartment of Cardiovascular Surgery, Catholic University of Leuven, Leuven, Belgium
^dDivision of Cardiology, University of Louvain Medical School, Brussels, Belgium
^eDepartment of Physiology, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands

^* Corresponding author. Tel.: +32-14-602-458; fax: +32-14-605-788

Received 23 July 1998; accepted 28 January 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Similar structural changes in the myocardium can be observed in chronic hibernating myocardium and in myocardium taken from hearts suffering chronic atrial fibrillation. We investigated whether or not these changes are indicative of apoptosis. Methods: Myocardial biopsies from 28 strictly selected patients with chronic hibernating myocardium and heart samples from 13 goats with pacing-induced chronic atrial fibrillation were used. Special attention was paid to processing the tissues immediately (fixation/freezing) in order to prevent artificial degenerative changes, thereby excluding false positive identification of apoptosis. Infarcted areas or infarcted border zones were excluded from our study. Apoptosis was detected with light and electron microscopy and terminal deoxynucleotidyl transferase nick end-labelling. Immunohistochemistry was used for detecting Bcl-2, P53 and PCNA-proteins associated with apoptosis/DNA damage. Results: The results obtained for chronic hibernating left ventricular myocardium were similar to those for chronic fibrillating atrial myocardium. No apoptotic nuclei, as characterised by extensive chromatin clumping, could be observed in normal or dedifferentiated cardiomyocytes under the electron microscope. The end-labelling assay did not reveal any cardiomyocytes with damaged DNA. Nor could we find any evidence of substantial expression of Bcl-2, P53 or PCNA, a result indicative of the absence of apoptotic threat or DNA damage. Conclusion: Cardiomyocyte dedifferentiation, but not extensive degeneration through apoptosis, can be observed in chronic hibernating myocardium and chronic fibrillating atrium. Dedifferentiation may be the best way to survive prolonged exposure to the unfavourable conditions imposed by increased wall stress, a relative lowered oxygen environment, or both.

KEYWORDS Apoptosis; Atrial function; Hibernation; Histopathology; Remodelling

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Myocardial hibernation is a condition whereby the heart downgrades its contractile function in response to a reduced blood supply. Consequently the heart tries to restore a normal- or near-to-normal-oxygen supply/demand ratio and succeeds in preserving myocardial viability [1]. Typical for myocardial hibernation is the recovery of function after restoration of the blood flow, which can occur rapidly (within hours; acute hibernation) or slowly to very slowly (weeks to months; chronic hibernation) [2].

A remodelling of the myocardial structure takes place in chronic hibernating myocardium (CHM). The structural changes that can be observed consist of depletion of sarcomeres (myolysis), sarcoplasmic reticulum and T-tubules, together with the appearance of large masses of glycogen, the reappearance of strands of rough endoplasmic reticulum, the transformation of normal mitochondria into numerous small abnormally shaped mitochondria and nuclei with altered chromatin distribution [3,4]. That these changes are not degenerative, but rather present a state of dedifferentiation, is suggested by the reoccurrence of ‘early development markers’ of heart muscle during chronic hibernation. For example, -smooth muscle actin is expressed in CHM and also in embryonic/foetal heart muscle cells, but not in adult cardiomyocytes [4].

To our knowledge, no validated animal model for CHM has been reported to date. However, there is a model involving experimentally induced chronic atrial fibrillation (CAF) in goats [5,6] that shows structural alterations resembling those seen in CHM. As in CHM, the main characteristic subcellular change concerns myolysis and glycogen accumulation. The affected cardiomyocytes also adopt a dedifferentiated phenotype, as indicated by the re-expression of some proteins normally expressed only in embryonic/foetal cardiomyocytes and the disappearance of proteins typical of adult heart muscle cells, such as cardiotin [7].

Although there is evidence to show that dedifferentiating cardiomyocytes in CHM and chronic fibrillating atrium (CFA) are not suffering from ischemia [8,9], some doubts remain as to whether dedifferentiation is a long-lasting viable state that has the potential to reverse to normal [10]. Cell death through apoptosis is indeed suggested by some very recent reports [11,12]. Apoptosis is a highly regulated and energy-consuming form of intentional suicide for the cell, executed via a genetically regulated programme [13]. Apoptotic regulation seems to involve multiple pathways, resulting in either apoptosis induction or apoptosis inhibition. The upregulation of Bcl-2 expression is known to be correlated with protection against apoptosis [14], and the overexpression of P53 results in apoptosis induction [15]. During apoptosis, the nuclear DNA is degraded in a specific way, resulting in double-stranded DNA fragments with sizes in multiples of 180 to 200 bp [16]. Cardiomyocyte death via apoptosis has already been reported in postnatal cardiogenesis [17,18], and in myocardial infarction [19–23], but it also plays a role in idiopathic dilated cardiomyopathy [24], arrhythmogenic ventricular dysplasia [25] and in the progression to heart failure [20,23,26].

The main objective of this work is to assess whether CHM or CAF eventually results in cardiomyocyte death through apoptosis. The search for apoptotic phenomena involved the use of electron microscopy and immunohistochemical detection of apoptosis-related proteins and DNA fragmentation.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Patients/chronic hibernating myocardium
The human cardiac tissue material used in this study consisted of transmural biopsies from 28 patients, the biopsies being obtained during coronary bypass surgery. Strict patient selection criteria were employed. The biopsies were taken from hypo/akinetic left ventricular segments at the time of coronary bypass surgery, as detailed in previous studies [27–29]. In brief, all the patients had severe LAD stenosis and a decreased wall motion and regional ejection fraction as evaluated by angiography and 2D echocardiography. None of the patients showed signs of infarction on ECG and viability of the tissue was verified by positron emission tomography (PET) and postoperative (3 to 6 months) recovery of regional contractile function and ejection fraction. The detailed individual patient characteristics regarding degree of stenosis, anterior wall abnormalities, flow-metabolic match or mismatch, and functional recovery after coronary bypass surgery, have been described in previous papers [28,29].

The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997; 35:2–3) and was approved by the ethical committees for research of the Universities of Leuven and Louvain. Importantly to this study, we restricted our observations to myocardial segments entirely devoid of infarctions, and we stress that all the tissue samples were processed immediately, in order to avoid post-operative degenerative changes.

2.2 Animals/chronic atrial fibrillation model
In this study, 20 female goats were used. CAF was induced in 13 of these animals, and 7 with sinus rhythm were used as controls. Animal handling was carried out according to the Dutch Law on Animal Experimentation (WOD) and the European Directive for Protection of Vertebrate Animals used for Experimental and other Scientific Purposes. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23 revised 1996).

CAF was induced by electrical pacing with a fibrillation pacemaker. The animals were kept in sustained atrial fibrillation for 9–23 weeks. The procedure has been described by Wijffels et al. [5]. At the end of the experimental period, the goats were anaesthetised and the hearts were quickly removed. Parts of the left and right atrial free walls were cut into small blocks and prepared for morphologic and cytochemical examinations [6].

2.3 Morphologic evaluation
Light and electron microscopy were used to search for morphologic apoptotic phenomena and to exclude infarcted areas. Immediately after they had been taken, samples were fixed in 3% glutaraldehyde buffered to pH 7.4 with 90 mM KH₂PO₄ for at least 2 h, washed in the buffer supplemented with 220 mM sucrose and postfixed with 2% OsO₄ in 50 mM veronal acetate buffer for 1 h. Dehydration in graded series of alcohol and embedding in epoxy resin were done by standard procedures. Ultrathin sections were cut from each sample, counterstained with uranium acetate and lead citrate, and examined in a Philips CM100 electron microscope.

For light microscopic evaluation, 2 µm thick sections were stained with periodic acid–Schiff (PAS) and 0.1% toluidine blue. This staining procedure is effective in visualising contractile elements and glycogen, enabling the degree of sarcomere loss (myolysis) and the content of glycogen to be determined. These changes were scored visually by methods detailed elsewhere [3]. Cells were classified as affected if sarcomere depletion accounted for more than 10%. All samples were examined for the possible appearance of changes characteristic of apoptosis, such as shrinking of the cell cytoplasm accompanied by a rise in cell density, condensation of the nuclear chromatin and its segregation into sharply delineated masses against the nuclear membrane, karyorhexis or the ‘budding’ phenomena and the occurrence of apoptotic bodies. At least two sections of both left and right atrial free wall from each goat were evaluated using light microscopy. The number of evaluated nucleated cardiomyocytes in each section varied between 262 and 368. Light microscopic evaluation of human biopsies comprised at least two sections per patient (one of the endocardial and one of the epicardial region). The mean number of evaluated nucleated cardiomyocytes in each section was 208, ranging from 82 to 465.

2.4 Immunohistochemical staining procedures
The following primary antibodies were used for immunohistochemistry.

(1) A mouse monoclonal antibody, clone 124 (DAKO A/S, Glostrup, Denmark), against the human Bcl-2 oncoprotein (molecular mass 25 kDA), specific for an epitope residing between amino acids 41 and 54.

(2) A mouse monoclonal antibody, clone DO-7 (DAKO), against the human tumour suppressor gene product P53, both the wild type and mutant type. The antibody reacts with an epitope between amino acids 19 and 26.

(3) The monoclonal antibody PC10, against proliferating nuclear antigen (PCNA), which was kindly provided by D. Lane, Dundee, UK [48].

(4) A polyclonal rabbit anti digoxigenin antibody (Sigma Immunochemicals, St. Louis, USA) used for the TUNEL assay.

Tissue samples were frozen in isopentane precooled with liquid nitrogen, immediately after having been taken, and stored at –70°C. Five micron frozen sections were cut and stored at –20°C until use.

For the detection of apoptosis, Bcl-2 and P53, at least six sections per patient and at least eight sections per goat (four of both left as right atrial free wall) were evaluated. The mean number of nucleated cardiomyocytes in each section from patient biopsies was 152 ranging from 27 to 442. The mean number of nucleated cardiomyocytes in the frozen sections from goat atrial free wall was 835 ranging from 440 to 1320.

2.5 Immunohistochemical detection of apoptosis
The terminal deoxynucleotidyl transferase (TdT) mediated dUTP Nick End Labelling method (TUNEL) was used for detecting cells with nuclear DNA fragmentation, suggestive of apoptosis [30]. Frozen sections 5 µm thick were air-dried before fixation in 4% paraformaldehyde (Merck, Darmstadt, Germany) in PBS brought to pH 7.4 (10 min). After the sections had been washed in PBS (3x10 min), endogeneous peroxidase activity was blocked by incubation in 0.3% H₂O₂ in PBS, followed again by three washing steps (5 min each) in PBS. The sections were then treated with a solution of 0.1 M citric acid and 0.5% Triton X-100 (t-octylphenoxypolythoxyethanol; Sigma) for 10 min at 0°C and rinsed (2x5 min) in PBS and in 25 mM Tris–HCl (pH 7.4) (3x5 min). For the TUNEL reaction, the sections were incubated for 1 h at 37°C in a TdT reaction buffer (10 µl 5xTdT reaction buffer (pH 6.6) supplemented with: 5 µl 25 mM CoCl₂, 0.5 µl TdT (25x10³ units/ml; Boehringer Mannheim, Mannheim, Germany) and 0.5 µl digoxigenin-11-2'-deoxy-uridine-5'-triphosphate (1mM; Boehringer Mannheim)) diluted to 50 µl with distilled water. The 5xTdT reaction buffer consists of 1M potassium cacodylate (Merck), 125 mM Tris–HCl (Sigma) and 1.25 mg/ml bovine serum albumin (BSA; Sigma). To finish the reaction, the sections were washed for 5 min in 4x saline–citrate solution (SSC) and then in 0.1% BSA in PBS (2x5 min). For the detection of the incorporated digoxigenin-linked nucleotides, the sections were incubated in a 1:100 dilution of the primary antibody (rabbit anti-digoxigenin) in PBS for 1 h at room temperature (RT), followed by three washing steps in PBS and incubation (45 min; RT) in a peroxidase-conjugated swine anti-rabbit antibody solution (diluted 1:100 in PBS). After the sections had been washed with PBS (3x10 min) and distilled water (5 min), peroxidase activity was detected, with 3-amino-9-ethylcarbazole (AEC, Sigma) used as chromogen. For this, 40 mg AEC was dissolved in 10 ml N,N dimethylformamide (Merck) and added to 190 ml 0.05 M sodium acetate buffer (pH 4.95). Hydrogen peroxide was added to a final concentration of 0.01% (v/v). After incubation for 10 min, the sections were rinsed with tap water, counterstained with haematoxylin (Sigma), and mounted with Kaiser’s glycerol gelatine (Merck).

2.6 Immunohistochemical detection of Bcl-2 and P53
Five-micron thick frozen sections were air-dried, fixed in methanol (–20°C for 30 s) followed by acetone (–20°C for 15 s), and air-dried again. To block endogenous peroxidase activity, the sections were incubated for 15 min in 0.3% H₂O₂ in phosphate buffered saline (PBS) and then rinsed in PBS (3x10 min). The sections were incubated in the primary antibody solution (diluted 1:25 in PBS) at RT for 1 h, washed in PBS (3x10 min), and incubated in a solution containing the secondary, biotinylated horse anti-mouse antibody (Vector Laboratories, Burlingame, USA), at a dilution of 1:100 in PBS, for 45 min at RT. An avidin-peroxidase complex (diluted 1:100 in PBS) (Vectastain ABC kit, Vector Laboratories) was applied for 30 min. After rinsing the sections with PBS (3x10 min) and distilled water (5 min), staining (with AEC as chromogen), counterstaining, and mounting was performed as described above.

This procedure was performed on cardiac tissue from patients with CHM, normal human cardiac tissue (from an explant donor), and cardiac tissue from both control goats and goats with CFA.

2.7 Immunohistochemical detection of PCNA
With exception of the fixation step and the applied antibodies, the procedure for PCNA detection was the same as for the detection of Bcl-2 and P53. After having been air-dried, the 5 µm cryostat sections were fixed in 3.7% formaldehyde (Acros Organics, Geel, Belgium) in PBS for 2 min at RT. The primary antibody, PC10, was applied diluted 1:25 in PBS, for 1 h at 37°C. The sections were incubated in a biotinylated goat anti-mouse antibody (diluted 1:100 in PBS) (Vector Laboratories) solution at RT for 30 min.

2.8 Control reactions
For the Bcl-2, P53 and PCNA detection assays, negative controls consisted of incubations in which the primary antibody was omitted from the solutions.

The negative controls for the TUNEL reaction consisted of incubations in a medium from which TdT was omitted.

As positive controls for the Bcl-2 detection, we used cryostat sections of human lymphoma tissue, human spleen, goat lymph node and goat spleen [31].

For the P53 and PCNA detection, the positive controls used were frozen sections of respectively UV-irradiated skin and reactive lymph node from human volunteers [32].

The positive control for the TUNEL reaction comprised frozen sections of guinea-pig thymus and DNAse-treated myocardium from mouse, control goat and goat with CAF. Frozen sections of mouse and goat myocardium were fixed in a 4% paraformaldehyde solution and subsequently rinsed in PBS, as described above. They were then incubated in a solution of 1 µg DNAse I (Sigma) in 100 µl reaction buffer (50 mM sodium acetate, 10 mM magnesium chloride and 2 mM calcium chloride) for 10 min at 37°C. The sections were rinsed three times in PBS at 4°C before the TUNEL assay was performed.

2.9 Gel electrophoresis and Western blot analysis
To control the immunohistochemical results concerning the Bcl-2 expression, ten frozen sections of 10 µm thickness were cut from each of the following tissue samples: human normal heart, human spleen, atrial tissue from goat in sinus rhythm, atrial tissue from goat with CFA and goat spleen. The human tissues were both derived from an explant donor. The sections were boiled for 5 min in sample buffer containing 62.5 mM Tris–HCl (pH 6.8), 8% glycerol, 2% SDS and 0.00125 bromophenol blue. After centrifugation, the protein content of the supernatant was assessed by a BCA protein assay (Pierce, Rockford, IL, USA), and all samples were diluted with sample buffer to the same protein concentration. After addition of 10% 2-mercaptoethanol (Bio-Rad, Hercules, CA, USA), the samples were boiled again for 5 min. One-dimensional SDS-gel electrophoresis was done using 12% Ready Gels® (Bio-Rad) in a Ready Gel® cell system (Bio-Rad). Equal amounts of protein were loaded on the gels, and those were run for 15 min at 60 V and 1 h at 120 V. Thereafter, proteins were blotted on a nitrocellulose membrane (Hybond®-C pure, Amersham Life Science, Buckinghamshire, UK) using a Transblot® SD semi dry transfer cell (Bio-Rad).

After rinsing the membranes with AD, they were blocked for 1 h at RT using PBS containing 5% non-fat dry milk (Bio-Rad) and 0.05% Tween-20 (Polyoxyethylene-20–sorbitan monolaurate, Acros). The same buffer was used to dilute the antibodies. After overnight incubation with the primary antibody (clone 124, DAKO, 1:25) at 4°C, the blots were rinsed with PBS containing 0.05% Tween-20, and subsequently incubated for 1 h at RT with the secondary antibody (Donkey anti-mouse Ig, horseradish peroxidase conjugated, Amersham, 1:2000). Then the blots were extensively washed and peroxidase activity was detected by chemoluminescence (Lumi-Light plus, Boehringer Mannheim). High performance chemiluminescence film (Hyperfilm® ECL, Amersham) was used for visualisation of the luminescent signals.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Morphologic evaluation
In the biopsies from the group of 28 patients with CHM, an average of 27.1% of the cardiomyocytes were affected by sarcomere depletion to more than 10% and by glycogen accumulation. The atria of the 13 goats showed similar changes in approximately 50% of the cells. In addition to these typical changes, specific alterations were noted in the nuclear morphology of cardiomyocytes derived from CHM patients. Two types of nuclei with altered morphology could be distinguished. In one type, the chromatin was evenly dispersed, closely resembling nuclei of embryonic/foetal cardiomyocytes (Fig. 1b). The other type showed an irregular chromatin distribution, with clumps at the nuclear membrane and throughout the nucleoplasm (Fig. 1c). The detailed quantification and evaluation of these nuclear changes has been described elsewhere [9]. In brief, redistribution of heterochromatin in a pattern similar to that found in foetal cardiomyocytes was seen in 67% of the affected cells (Fig. 1b). Clumping of chromatin was found in 13% of the affected cardiomyocytes (Fig. 1c), while 20% of the cells showed a normal margination of chromatin (Fig. 1a).

View larger version (80K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Electron microscopic details of a nucleus with normal heterochromatin distribution (a) (28500x) and nuclei of chronic hibernating cardiomyocytes. Two types of altered nuclear morphology could be distinguished: nuclei with homogeneously dispersed heterochromatin (b) (10570x), thereby resembling nuclei of foetal cardiomyocytes, and nuclei with clumping of heterochromatin throughout the nucleoplasm (c) (9765x).

 
We found none of the features characteristic of apoptosis, such as shrinking of the cell cytoplasm accompanied by a rise in cell density, condensation of the nuclear chromatin and its segregation into sharply delineated masses against the nuclear membrane, karyorhexis or the ‘budding’ phenomena, or the occurrence of apoptotic bodies.

3.2 Immunohistochemical evaluation
The detection of cells with DNA nicks, thus potentially apoptotic, with the TUNEL method revealed no positive cardiomyocytes from CHM (Fig. 2a), CFA (Fig. 2b) or control goats. In one section of CHM there was one cell, probably an interstitial cell of mesenchymal origin, that showed intense staining (Fig. 2a, insert). In the sections of atria from goats with CAF or sinus rhythm, a minority of the interstitial cells – possibly fibroblasts or endothelial cells – stained positive. Many thymocytes, spread throughout the guinea pig thymus, were TUNEL positive, as expected (Fig. 2c). In the DNAse-treated myocardial sections from mouse, control goat and goat with CAF, most of the nuclei – both from cardiomyocytes and interstitial cells – also stained positive (data not shown).

View larger version (124K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Apoptosis detection with the TUNEL procedure: Light microscopic images of CHM (a) (309x), CFA (b) (309x) and guinea pig thymus (c) (309x). None of the cardiomyocytes of CHM and CFA stained. Only one interstitial cell in CHM was found to be positive (open arrow) (insert of Fig. 2a; 251x). The green/brown perinuclear granules consist of lipofuscin (asterisks). Note the myolytic perinuclear areas (arrows). In the guinea pig thymus, used as positive control, scattered apoptotic thymocytes were found to be stained (arrowheads) (c). Light microscopic images of immunohistochemical Bcl-2 detection in CHM (d) (309x) and CFA (e) (309x). All the cardiomyocytes and interstitial cells are negative in myocardial tissue from CHM and CFA. Myolytic perinuclear areas (arrows). Scattered Bcl-2 positive cells (arrowheads) can be observed in goat spleen tissue, used as positive control (f) (373x). Immunohistochemical detection of P53: Light microscopic images of CHM (g) (309x), CFA (h) (309x) and UV-irradiated human skin (i) (125x). None of the cells present in CHM was found to be stained. Only two cells in CFA, of which one is shown in the insert of Fig. 2h (300x), were P53-positive (open arrow). However P53 did not show the normal nuclear distribution in this cell. Myolytic perinuclear areas (arrow). Many keratinocytes are P53-positive in UV-irradiated human skin used as positive control (arrowheads) (i).

 
As far as the expression of the oncoprotein Bcl-2 in cardiac tissue of human CHM (Fig. 2d), goats in CAF (Fig. 2e) and goats in sinus rhythm is concerned, no positively stained cardiomyocytes – myolytic or normally structured – could be observed. Normal human cardiac tissue was also free of positively stained cardiomyocytes (data not shown). Occasionally, interstitial cells (probably fibroblasts or endothelial cells) were found to be slightly stained positive for Bcl-2 (data not shown). In the control reactions with human lymphoma tissue and human spleen, many cells stained intensely for Bcl-2 (data not shown). A similar Bcl-2 reactivity could be observed in goat spleen (Fig. 2f) and lymph node (data not shown).

The tumour suppressor gen product P53 was equally not detectable in cardiomyocytes or interstitial cells derived from CHM patients (Fig. 2g), from normal human cardiac tissue (not shown) or from goats in sinus rhythm. Only two cells in cardiac tissue from goats in CAF displayed reaction product in the perinuclear cytoplasm, a localisation not conforming with the normal nuclear residence of P53 (Fig. 2h, insert). On the basis of their topographical localisation and their size, these two cells were probably cardiomyocytes. Several P53-positive nucleated keratinocytes were found in the epidermis of the UV-irradiated human skin (Fig. 2i).

Proliferating Cell Nuclear Antigen (PCNA) expression could not be detected in cardiac tissue of CHM, CFA or control goats. All cells, including the affected and unaffected cardiomyocytes, were PCNA negative, in contrast to many lymphocytes in human reactive lymph node tissue (data not shown).

3.3 Western blot analysis
The expression of Bcl-2 in cardiac tissue was also assessed using Western blot analysis. Human and goat spleen were used as positive controls. The results show that there is a large constitutive expression of Bcl-2 in human spleen. We also could detect some Bcl-2 expression in normal human heart, however the level of expression is lower, since the signal was much less, although equal amounts of protein were loaded (Fig. 3). Many attempts to demonstrate Bcl-2 expression in goat tissue did not come up with clear results. No signals or only faint signals could be detected in goat spleen, whereas goat heart (both from control goats and goats with CFA) was never positive (Fig. 3).

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Western blot analysis of Bcl-2 expression in human control heart (HH), human spleen (HS), cardiac muscle from goats in sinus rhythm (HCG) and goats with CFA (HFG), and goat spleen (GS). Significant expression of Bcl-2 in human spleen, lower expression in normal human heart. Goat spleen was Bcl-2 negative, or only slightly positive, whereas cardiac muscle from the goat (both normal and CFA goats) was always negative (panel a). The protein content of al the samples was the same as determined by Ponceau Red staining (panel b).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In chronic hibernating myocardium (CHM), a number of typical structural changes are seen in cardiomyocytes, suggestive of dedifferentiation, i.e. a phenotype switch to an earlier developmental stage [3,4], although it is uncertain whether this dedifferentiated state is stable. Some researchers, however, interpret some of the changes observed as degenerative, and believe that the process ends in cell death [10–12]. In this study, we tried to assess whether cell death via apoptosis occurs in CHM in humans and in chronic fibrillating atrium (CFA) in the goat, the only animal model available with structural changes resembling those of CHM [6,7].

4.1 Nuclear morphology
When we limited our observations strictly to viable areas, thus excluding areas of infarctions or infarction border zones, morphologic evaluation of the tissue samples revealed no apoptotic phenomena. We were unable to detect any ‘endstage’ apoptotic cells or apoptotic bodies. Although nuclear changes were seen in some cells in the affected areas, only the minority of cardiomyocytes from CHM had nuclei in which the heterochromatin distribution was irregular and patchy, showing some resemblance with nuclei of degenerative cells. The origin of this altered chromatin distribution is, however, not clear. It remains doubtful whether such changes are related to an early degenerative phenomenon, because the cells display no other subcellular degenerative alterations. The ultimate condensation of chromatin, so characteristic of apoptotic nuclei was not found, nor were any nuclear fragments detected. On the contrary, many dedifferentiated (myolytic) cells showed quite the opposite of condensed chromatin, i.e. virtual disappearance of clustered heterochromatin, giving the nuclei a pale look. The latter chromatin distribution pattern strongly resembles that seen in embryonic/foetal cardiomyocytes.

4.2 DNA damage and apoptosis-related proteins
Apoptosis in the heart has been reported to play a role in postnatal morphogenesis [17], but is also likely to be present in a variety of cardiomyopathies. With the TUNEL method, we were unable to identify any cardiomyocytes with TdT reactivity in cardiac tissue from CHM and CFA. DNA damage was thus probably absent in these cells. This conclusion is corroborated by that fact that TUNEL positivity was obtained in properly treated positive control tissue, such as cardiac samples after DNAse exposure and guinea-pig thymus. It is therefore unlikely that apoptosis is a frequently occurring phenomenon in CHM and CAF.

Although the exact mechanism is unknown, Bcl-2 is considered to be a protein that protects the cell against apoptosis [14]. No Bcl-2 expression could be detected immunohistochemically in the cardiomyocytes from CHM, CFA and normal heart (both from human and goat). Whether Bcl-2 expression is present in normal myocardium has not yet been unambiguously proven [33–37]. Also in this study, some data are not completely in concert: we found low expression levels of Bcl-2 in the normal human heart using Western blot, but we could not detect Bcl-2 immunohistochemically in the same tissue. We argue that the expression levels of Bcl-2 in normal human heart are too low to be detected with the immunohistochemical method used, and/or that cardiomyocytes are not the major source of this protein. Because Bcl-2 could also not be picked up immunohistochemically in CHM, we suggest that the expression level of Bcl-2 does not increase markedly in this setting. Unfortunately we were unable to confirm this hypothesis by Western blot analysis, because of the limited size of the biopsies, and their limited availability at present. Western blot analysis of goat cardiac tissue did not come up with clear results. Goat spleen, which was shown to have a high expression level of Bcl-2 using immunohistochemistry, was not positive or only slightly positive on Western blot, and cardiac tissue from goats was always negative. Therefore we hypothesise that during the process of tissue sampling (in order to prepare it for Western blot), most of the goat Bcl-2 – if not all – undergoes profound changes, so that it no longer can be detected by the primary antibody. It has been described before that an increase in Bcl-2 in cells may represent a reaction to oppose programmed cell death [37]. From the results presented here, a significant increase in Bcl-2 expression in cardiomyocytes from CHM and CFA is not likely, so one may assume that these cardiomyocytes are more prone to undergo apoptosis, if a trigger for this kind of cell death were present. However, we could not find evidence for increased apoptosis, nor increased Bcl-2 levels in CHM and CFA, so it is possible that these combined results indicate that the cardiomyocytes are not subjected to an apoptotic threat.

The P53 protein is known to be an apoptosis-inducing agent. When the DNA is damaged, an upregulation of P53 expression will lead to an arrest of the cell cycle, in order to provide time for DNA repair, or to induce apoptosis if DNA repair is inadequate, impossible or insufficient [15]. P53 is not expressed in adult myocardium, or only at very low levels, in contrast to embryonic/foetal cardiac tissue, where high levels of P53 expression have been observed [38]. However, to our knowledge no study has yet been published in which P53 expression in cardiomyocytes was assessed by use of immunohistochemistry. We could not find any evidence for either a substantial expression of P53 in normal cardiac tissue (from human and goats in sinus rhythm), or a substantial upregulation of P53 expression in normal (unaffected) or abnormal (affected) cardiomyocytes in CFA or CHM. Thus, DNA damage and/or apoptosis induction seems very unlikely in these tissues. The absence of P53 expression, although not actually supporting the dedifferentiation hypothesis [39], might be an indication of the incomplete dedifferentiation state of cardiomyocytes in CHM and CFA. Previous observations have shown that proteins present in very early stages of myocardial development, such as vimentin and the cytokeratins 8 and 18, are absent in CHM and CFA, thereby supporting the hypothesis of incomplete dedifferentiation [4,7].

Proliferating Cell Nuclear Antigen (PCNA) is a protein that is a requisite for DNA replication and DNA repair, and its detection is frequently used as a marker of cell proliferation. Inducing DNA damage leads to the upregulation of PCNA, so the absence of such an upregulation probably means that DNA damage is absent [40]. Some authors report that PCNA is also upregulated during apoptosis, even in the physiological forms of apoptosis, because the DNA strand breaks induce a DNA repair process, though a futile one [41]. Because P53 and PCNA work co-operatively in DNA repair mechanisms, their actions in the apoptotic process are probably also related. PCNA is rarely if ever immunohistochemically detectable in normal adult cardiac tissue [42,43]. Western and Northern blots can demonstrate very low levels of PCNA mRNA and protein, although the source of the PCNA expression – cardiomyocytes or other cell types – remains inconclusive [44]. Marino et al. [44] also noticed high expression levels in foetal and neonatal cardiac tissue. In our study, we could not detect any PCNA in cardiac tissue from CHM, CFA or the control goats. Again, this result, together with the finding that P53 expression is also absent, indicates that DNA damage is unlikely, and that there is no cellular, or DNA proliferation in CHM and CFA.

Because of the absence of any detectable upregulation of Bcl-2 expression and TUNEL reactivity in the cardiomyocytes under study, we have to assume that in CHM and CFA, the heart muscle cells, like normal cardiomyocytes, are not prone to undergo apoptosis.

This assumption is reinforced by the absence of detectable levels of P53 and PCNA expression in CHM and CFA, suggesting that there is no DNA damage in these cells.

4.3 Viability of hibernating myocardium
With respect to the viability of hibernating myocardium, some reported data show that the protective mechanisms in this tissue are insufficient to retain the viability of the cardiac tissue. We believe that at least part of the reason that the results from our study differ from those from other studies can be attributed to the patient selection criteria [10,12] and, perhaps of even greater importance, the tissue selection criteria [10–12]. Our studies included strict patient selection criteria, so that pathologies other than CHM were excluded. When evaluating cardiac tissue from CHM, we took great care to exclude areas of infarction and infarct border zones histologically. It is well established that apoptosis, albeit to a limited extent, does occur in these areas [19–22], and we have also been able occasionally to observe morphologically identified apoptotic bodies in infarction areas and their border zones (Borgers, unpublished data). We are therefore not sure that our present findings are truly different from the data published hitherto [12]. Although they reported finding apoptosis in human hibernating myocardium, the authors gave only a very vague description of the areas that were examined, i.e. whether or not they were in the vicinity of infarcted zones, and gave no data on the frequency of apoptosis. In addition, the ultrastructural image of an apoptotic cardiomyocyte as published in [12] is devoid of the typical morphological characteristics of dedifferentiated cardiomyocytes, such as sarcomere depletion and glycogen accumulation. Thus we propose that dedifferentiation and apoptosis may not be consecutive phenomena but may possibly occur in different cell populations. Another report [11] mentioned extensive apoptosis in a pig model of hibernating myocardium. Although concomitant infarctions might be responsible for some of the occurrence of apoptotic cells, as convincingly reported elsewhere [25], the percentages of apoptotic cells reported (9.8±4.6% in the subendocardial region) are improbably high. With such high frequencies noted between 24 h and 4 weeks after the induction of the coronary occlusion, no viable cells would remain after that period [45]. The low specificity of the TUNEL procedure [46,47] might be responsible for the detection of artificial DNA damage in the pig model [11].

An important issue to be addressed is related to the time period these cells can survive in their dedifferentiated state. It is expected that indeed, below a certain threshold of oxygen supply, even these cells get insufficient fuel to survive. It may well be that apoptosis is the degeneration pathway that would then be involved. Nor can it be excluded that at some stage in the adaptation to ischemia, perhaps at the onset, cell loss through apoptosis occurs, but might not be noticed at later stages because of the fast resolution of the apoptotic phenomena. Furthermore it is still possible that only very few cells are undergoing apoptosis, which can only be detected when much more tissue is evaluated. The main study limitation is therefore the small size of the human cardiac biopsies. The very few apoptotic cells which can be missed could be clinically relevant after prolonged periods of time if revascularisation is delayed. However the current data suggest that dedifferentiation of cardiomyocytes normally does not end up in apoptosis, and that the possible sporadic apoptotic cells are of no short term clinical importance.

Since all of the apoptosis-related processes and characteristic morphologic changes currently under study are absent in our tissue samples, we suggest that the hibernating state might be of prolonged duration, both in chronic hibernating myocardium and chronic fibrillating atrium. The adaptation process of cardiomyocytes in both pathologies seems to be stereotypic. It might be speculated that dedifferentiation is the best way to survive for a prolonged period of time in the altered environment, imposed by either increased passive wall stress, reduced oxygen supply or both. The ‘survival’ state that is created by activating a ‘foetal programme’, moreover, allows these cardiomyocytes to return to normal as soon as the blood supply is restored, indicated by the recovery of function after revascularisation.

In conclusion, dedifferentiation of cardiomyocytes as seen in CHM and CFA can be considered as an adaptive principle of protection allowing cardiomyocytes to survive for a prolonged period of time in which a relative oxygen shortage, an increased wall stress, or both, take place. The survival of dedifferentiated cardiomyocytes could be a prerequisite for recovery of contractile function after revascularisation in CHM or cardioversion in CFA.

Time for primary review 28 days.

	Acknowledgements

 
The author wishes to thank M. Jansen and G. Krekels for providing tissue samples needed for positive controls in this study, G. Schaart and M. Mercken for their practical suggestions in regard to the Western blotting and L. Leijssen for processing the images. Part of this study was supported by the Dutch Heart Foundation, NHS Grant 96-155.

	Notes

 
This work was presented in part at the 70th Scientific Sessions of the American Heart Association, Orlando, Florida, USA.

	References

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