CLINICAL RESEARCH: MYOCARDIAL ISCHEMIA/INFARCTION
Human hibernating myocardium is jeopardized by apoptotic and autophagic cell death
Albrecht Elsässer, MD*,*,
Achim M. Vogt, MD ,
Holger Nef, MD*,
Sawa Kostin, MD ,
Helge Möllmann, MD*,
Woitek Skwara, MD ,
Christoph Bode, MD||,
Christian Hamm, MD* and
Jutta Schaper, MD
* Department of Cardiology, Kerckhoff Clinic, Bad Nauheim, Germany
Department of Cardiology, Ruprecht-Carls-University, Heidelberg, Germany
Department of Experimental Cardiology, Max-Planck-Institute, Bad Nauheim, Germany
Department of Thoracic and Cardiovascular Surgery, Kerckhoff Clinic, Bad Nauheim, Germany
|| Department of Cardiology, Albert-Ludwigs-University, Freiburg, Germany
Manuscript received March 18, 2002;
revised manuscript received January 21, 2004,
accepted February 17, 2004.
* Reprint requests and correspondence: Dr. Albrecht Elsässer, Kerckhoff Clinic, Department of Cardiology, Benekestr 2-8, 61231 Bad Nauheim, Germany.
a.elsaesser{at}kerckhoff.mpg.de
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Abstract
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OBJECTIVES: The aim of the present study was to objectify the loss of myocytes and the mechanism by which myocytes die in human hibernating myocardium (HHM).
BACKGROUND: Intracellular degeneration, reduced cellular protein synthesis, and the replacement fibrosis contribute to structural disintegration of HHM.
METHODS: In 14 patients, HHM was diagnosed by dobutamine echocardiography, radionuclide ventriculography, and thallium-201 scintigraphy. Functional recovery was documented by repeating the preoperative clinical investigations three months after successful coronary artery bypass graft surgery (CABG). During CABG, transmural biopsies were taken from the center of HHM regions and studied by electron microscopy, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) method, reverse transcription-polymerase chain reaction, and Western blotting. Control samples were taken from nondiseased human myocardium.
RESULTS: All patients showed significant improvement or normalization of the regional function of HHM. Ubiquitin-related autophagic cell death was evident ultrastructurally by the occurrence of autophagic vacuoles, cellular degeneration, and nuclear disassembly. Ubiquitin-protein complexes were found in 0.03 ± 0.008% (control: 0%, p < 0.005) of all myocytes. The proteasome 20S subunit/total myocytes were reduced from 63.3 ± 9.6% in control myocardium to 36.9 ± 8.4% in HHM. Complement-9, indicating oncosis, was found in only one of 14 biopsies. TUNEL-positive myocytes were 0.002 ± 0.0003%. Electron microscopy showed apoptotic cells in 3 of 14 samples. However, the bcl-2/bax ratio was significantly reduced. Moreover, caspase-3 messenger ribonucleic acid was 8.5 times upregulated, and caspase-3 was activated. Cell death was absent in controls.
CONCLUSIONS: In HHM, ubiquitin-related autophagic cell death and apoptosis cause a loss of myocytes. This plays an important role in progressive tissue damage and causes a reduction of the extent of functional recovery of HHM.
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Abbreviations and Acronyms
| | ATP | = adenosine triphosphate | | CABG | = coronary artery bypass graft surgery | | DNA | = deoxyribonucleic acid | | HHM | = human hibernating myocardium | | LV | = left ventricle/ventricular | | RNA | = ribonucleic acid | | RT-PCR | = reverse transcription-polymerase chain reaction | | SPECT | = single-photon emission computed tomography | | TUNEL | = terminal deoxynucleotidyl transferase-mediated nick end-labeling |
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In his seminal studies, Rahimtoola (1) defined hibernating myocardium as a state of persistently impaired myocardial contractile function at rest due to reduced coronary blood flow. Despite this myocardial undersupply, however, adaptations of myocardial energy metabolism and function (e.g., an increase in glycolytic adenosine triphosphate [ATP] formation and a reduction in ATP consumption by attenuated contractile performance) aid in decreasing the ischemic myocardial energy deficit (2,3). In contrast to chronic human hibernation, short-term hibernation is defined as sustained perfusion-contraction matching for several hours, as demonstrated in experimental studies. Furthermore, short-term hibernation is characterized by a lack of necrosis, complete functional recovery during reperfusion, and recovery of metabolic parameters during persistent ischemia, whereas in human hibernating myocardium (HHM), a complete restoration of the energetic situation cannot be achieved (3).
As a consequence, although the biochemical, contractile, and cellular adaptations observed in HHM might delay and alleviate ischemic myocardial injury, there is increasing evidence that ongoing myocyte degeneration, reduced cellular protein synthesis, and an increased rate of synthesis of replacement fibrosis finally cause irreversible myocardial disintegration, limiting the structural and functional recovery after revascularization. Our group has shown that in HHM the number of myocytes is reduced, and replacement fibrosis develops with increasing duration and severity of ischemia (4). However, it has not yet been clarified by which mechanism myocytes die in HHM. Therefore, the aim of the present study was to identify the type of cell death occurring in HHM.
Three different types of cell death might be responsible for the disappearance of myocytes: apoptosis, ubiquitin-related autophagic cell death, and oncosis (necrosis). Oncosis is independent of energy supply and caspase activity. Apoptosis and autophagic cell death are actively programmed, energy-dependent phenomena, but apoptosis is caspase-dependent and autophagy is caspase-independent. Apoptosis has been described to occur at various rates in ischemic or failing myocardium, but its importance is still under discussion (5). Autophagic cell death has been described in hypertrophied and failing myocardium (6,7). It involves post-translational modification of proteins by linkage to numerous ubiquitin molecules in preparation for degradation by proteasomal enzymes. Excessive storage of polyubiquinated protein complexes leads to a loss of cell nuclei and cell death (8). From the present study, it is concluded that myocyte cell death is an unfavorable prognostic finding, indicating that HHM is at risk of irreversible tissue damage and a loss of functional capacity.
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Methods
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Patients and study protocol.
Fourteen patients with coronary heart disease and reduced left ventricular (LV) function were included in the study. As described previously, after angiographic diagnostics, which resulted in the indication for coronary artery bypass graft surgery (CABG), the reduced global and regional ejection fractions were verified by radionuclide ventriculography (4,9). Tissue ischemia and myocardial viability were assessed by thallium-201 scintigraphy using a stress-rest reinjection protocol. Echocardiography with low-dose dobutamine showed the extent of functional capacity of the afflicted regions.
All patients underwent revascularization of the hibernating area by CABG. The patients were restudied three months after revascularization by all methods employed preoperatively in order to determine the degree of functional recovery after restitution of adequate myocardial perfusion (Table 1).
Informed, written consent from all patients for each investigation, as well as approval by the hospital's Institutional Review Board of the University of Freiburg, had been obtained.
Clinical methods.
Dobutamine two-dimensional echocardiography
Transthoracic echocardiographic studies were performed at rest and during intravenous infusion of dobutamine at a rate of 5 and 10 µg/kg body weight/minute (each dosage given over 10 min). According to the recommendations of the American Society of Echocardiography, regional function was evaluated in images of standard views using a scoring system (1 = normal; 2 = slightly hypokinetic; 3 = severely hypokinetic; 4 = akinetic; 5 = dyskinetic) (10). Wall motion score indexes for global (LV) function and for the hibernating areas were calculated at rest and during dobutamine infusion.
Thallium-201 scintigraphy
A stress-rest reinjection protocol was used. At peak exercise testing, a dose of 111 MBq was injected intravenously. Four hours after exercise, redistribution images were acquired, followed by the administration of an additional thallium dose (37 MBq) at rest. Reinjection images were documented 30 min thereafter. The data obtained by myocardial single-photon emission computed tomography (SPECT) were reoriented into vertical long-axis, short-axis, and horizontal long-axis slices, using the Wiener filter for the entire LV. The SPECT images were analyzed qualitatively in 13 sectors, as well as quantitatively for four representative tomograms, using the circumferential profile analysis, and were compared with the database of a reference population (probability <5% for significant coronary artery disease; 46 women and 38 men investigated at our center).
In the qualitative analysis, regional tracer activity of a stress defect and the extent of its redistribution on reinjection images were interpreted visually as completely reversible, incompletely reversible, or fixed defects. Quantitatively, thallium uptake on stress images <2 SD of the mean value in the reference population was classified as abnormal, and an increase in segmental uptake by 15% or more, as measured on reinjection images, was considered "reversible" in the baseline study.
Preoperatively, myocardial perfusion was estimated by comparing the lowest thallium uptake value in the hibernating area after reinjection with those values in the database of our reference population.
The four tomograms were subdivided into three descriptor territories with respect to the perfusion areas of the three main coronary vessels.
According to the confidence interval (±2 SD) of our reference population, a peak myocardial thallium uptake of 55% or more indicated unimpaired perfusion.
Radionuclide ventriculography
Using a dose of 740 MBq of technetium-99m–labeled in vivo red blood cells, resting and exercise LV wall motion was assessed by multigated radionuclide ventriculography. Calculation of regional and global ejection fraction was performed by drawing nine radii to the LV border dividing the ventricle into an equal number of sectors.
Coronary angiography
Coronary narrowing was documented in multiple projections and was assessed as the percentage of diameter stenosis. Left ventriculography was performed in the biplane view.
Comparison of the different methods.
For comparison, all LV segments were grouped into three vascular territories: the anterior and anterolateral wall was seen as the perfusion area of the left anterior descending coronary artery; the lateral LV wall was related to the left circumflex artery; and the inferior and posterior walls were allocated to the right coronary artery. The final assignment was adapted according to the findings of coronary angiography, especially for the inferior wall and the apex.
For the unequivocal diagnosis of HHM, all of the following criteria had to be fulfilled: - Echocardiography: improvement of regional function in at least two adjacent abnormal segments by a factor
 1 of the scoring system during dobutamine infusion on the baseline study and postoperatively at rest.
- Thallium-201 scintigraphy: reversibility of a defect and/or uptake value in the range of the mean normal uptake ±2 SD on the baseline study, as well as absent signs of ischemia or infarction and a normalization of the uptake values after revascularization.
- Radionuclide ventriculography: postoperative improvement of the preoperatively impaired regional ejection fraction by at least 5%.
- Coronary angiography: documentation of adequate revascularization by bypass grafts.
The study population fulfilled all criteria listed. Concordance of the diagnostic results of the different clinical methods in the baseline study and postoperative normalization of regional function ensure that the myocardial biopsies were representative of HHM.
Tissue sampling
During open-heart surgery, two transmural biopsies (cylinders of about 1 mm diameter, weighing 8 to 15 mg) from each patient were taken from the center of the area clinically defined as HHM. The tissue was either immediately fixed in buffered glutaraldehyde for electron microscopy or immersed in liquid nitrogen for immunohistochemistry, Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), and the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) method.
Electron microscopy
Small tissue samples were embedded in Epon after routine procedures. Ultrathin sections were viewed and photographed in a Philips CM 10 electron microscope.
Immunohistochemistry
Cryosections were fixed with 4% paraformaldehyde. The sections were incubated with the first antibody (rabbit-ubiquitin [Dako, Cambridgeshire, United Kingdom], anti-mouse 20S proteasome subunit  5 [Biotrend, Köln, Germany], and mouse complement-9 [C9; Novo Castra, New Castle, United Kingdom]), followed by treatment with a secondary biotinylated detection system using either anti-mouse or anti-rabbit immunoglobulins. The last incubation was carried out with fluoroisothiocyanate-linked streptavidin (Amersham, Braunschweig, Germany). The sections were viewed in a confocal laser microscope (Leica, Solms, Germany).
TUNEL
TUNEL was performed on cryosections with an in situ cell detection kit (Roche, Basel, Switzerland) using fluorescein labeling.
Morphometry
Myocytes labeled for either TUNEL, ubiquitin, or C9 were quantified by counting the entire area of five tissue sections (diameter  1 mm) obtained at different levels from each biopsy. In ubiquitin-labeled sections, only myocytes with large ubiquitin-protein complexes lacking a nucleus were counted. All positive cells were expressed as the percentage of the total number of myocytes evaluated (280 to 350 per tissue section, total about 1,500).
Western blotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting were performed in six control and 14 HHM samples. Antibodies included rabbit bcl-2 (Santa Cruz, California), rabbit bcl-xL (Santa Cruz), rabbit bax (Santa Cruz), rabbit-cleaved casp-3 (Pharmingen, San Diego, California), mouse p53 (Calbiochem, San Diego, California), and rabbit c-myc (Santa Cruz). Quantitation was done by scanning of the immunoblots on a STORM 860 (Amersham-Pharmacia-Biotech).
RT-PCR
Total ribonucleic acid (RNA) of HHM was isolated using the guanidinium acid phenol-chloroform extraction method. First, strand synthesis of total RNA was performed using ingredients corresponding to standardized protocols. Reverse transcription was carried out at 42°C, followed by incubation at 95°C, to stop the reaction.
The PCR reactions were performed in duplicate with 60 ng of complementary deoxyribonucleic acid (cDNA), using a Perkin Elmar Gene Amp 9700. Primers were as follows: bcl-2 human: base pair (bp) 395-cn 32-ant 65; bcl-xL: bp 363-cn 32-ant 60; bax human: bp 358-cn 32-ant 60; casp-3 human: bp 833-cn 37-ant 55; p53 human: bp 330-cn 31-ant 60; c-myc human: bp 402-cn 31-ant 64; and GAPDH human: bp 496-cn 24-ant 60.
For quantitative analysis, the amplification products were separated on 2% agarose gel, stained with ethidium bromide, visualized by ultraviolet irradiation, and photographed with Polaroid film 667, evaluating the band densities by volume integration (Image Quant, Molecular Dynamics, Freiburg, Germany). GAPDH served as an internal control for the calculation of densitometric results.
Control tissue
Biopsies from three patients undergoing operative correction of atrial septal defects and three donor hearts not used for transplantation served as controls.
Statistical analysis.
Data are presented as the mean value ± SE. To evaluate the clinical data, the Friedman and Dunn test, analysis of variance, Bonferroni test, unpaired t test, and Mann-Whitney rank-sum test were used for echocardiography and radionuclide ventriculography, and the Wilcoxon test, paired t test, and Mann-Whitney rank-sum test were used for thallium-201 scintigraphy. Analysis of variance and Bonferroni testing were used to analyze the morphologic data, Western blotting, and RT-PCR results. A value p < 5% was considered to represent a significant difference.
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Results
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Clinical results.
Echocardiography
The preoperative mean value of the wall motion score index of the hibernating regions decreased from 3.9 ± 0.1 to 1.1 ± 0.3 at rest postoperatively (p < 0.05). The extent of functional recovery was predicted by dobutamine infusion in the baseline study (1.2 ± 0.4) (preoperative/postoperative: p < 0.05; preoperative dobutamine/postoperative: p = NS) (Table 1).
Radionuclide ventriculography
Postoperative normalization of the regional function of HHM, depressed preoperatively, occurred (preoperative/postoperative: 26.6 ± 5.1%/61.3 ± 3.9%; p < 0.05) (Table 1).
Thallium-201 scintigraphy
Preoperatively, 67 of total 182 segments were classified as HHM by qualitative and quantitative analysis of the reinjection images. Three months after revascularization, all of the segments showed regular tracer uptake and distribution, without signs of ischemia or infarction on the stress-redistribution reinjection images. The values of thallium-201 uptake increased from 38.5 ± 1.5% in HHM preoperatively to 65.7 ± 2.0% three months postoperatively, indicating normal perfusion.
Coronary angiography
The degree of coronary artery stenosis supplying the hibernating regions varied between 75% and 100%. Postoperatively, an adequate revascularization was evident (Table 1).
Tissue analysis.
Electron microscopy
Numerous myocytes in HHM showed signs of degeneration, such as a lack of contractile material, as well as nuclear and mitochondrial changes, as described earlier (4). Degenerative material (i.e., either totally empty or lipid-filled vacuoles, myelin figures, and lipofuscin) and numerous autophagic vacuoles and the absence of nuclei indicated development of autophagic cell death (Fig. 1A).
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Figure 1 Electron microscopic images of human hibernating myocardium (HHM). (A) Degenerative changes with numerous autophagic vacuoles (AV) and a lack of nucleus, indicating autophagic cell death. (B) Widening of the interstitial space that contains fibroblasts (F), macrophages (small arrows), and cellular debris (CB). Note the lack of contractile material in one myocyte (arrow). (C) Apoptotic nuclear disassembly in a myocyte (thick arrows). Note the formation of an apoptotic body (thin arrows). All bars = 5 µm.
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The expanded extracellular space contained large amounts of fibrotic material, including collagen fibrils, lipofuscin-containing macrophages, and electron dense "ground substance" representing fibronectin, laminin, and the proteoglycans (Fig. 1B). These degenerative changes were absent in nondiseased control myocardium.
Evidence of apoptosis in single myocytes was found in three biopsies. Typical morphologic signs of apoptosis included: intact mitochondria and intact sarcolemma, nuclei of varying size and shape (showing distinct chromatin clumping), and formation of apoptotic bodies, which indicate nuclear disassembly (Fig. 1C). Due to sequestration of cellular particles from myocytes, the size and shape of apoptotic cells varied greatly. This cellular debris was embedded in the extracellular matrix or was engulfed by macrophages. Evidence of acute cell death (oncosis) was not found. Mitotic figures were never observed.
Immunohistochemistry
Ubiquitin
Ubiquitin-positive cells showed either a small punctate-labeling pattern or exhibited large accumulations of ubiquitin-protein complexes (Fig. 2). In control myocardium, ubiquitin was absent; in HHM, 0.03 ± 0.008% of all myocytes were labeled.
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Figure 2 Large ubiquitin-protein complexes (green) in myocytes (red) lacking a nucleus in the confocal microscope. Nuclei are stained blue (TOTO). Bar = 10 µm.
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Complement-9
Complement-9 was found in single myocytes in HHM in only one of 14 biopsies (Fig. 3).
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Figure 3 One myocyte is clearly labeled for complement-9 (green) in the confocal microscope. All other myocytes are counterstained with phalloidin (red); nuclei are blue (TOTO). Bar = 20 µm.
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Proteasome 20S subunit
In control myocardium, 63.3 ± 9.6% of all nuclei were labeled. In HHM, this was reduced to 36.9 ± 8.4% (p < 0.01) (Fig. 4).
Ki-67
In HHM and controls, about one myocyte nucleus per 1,000 showed positive labeling.
TUNEL
Single TUNEL-positive myocytes were found in 5 of 14 patients, resulting in an apoptotic rate of 0.002 ± 0.0003% (Fig. 5). In control myocardium, apoptosis was absent.
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Figure 5 Apoptosis by the TUNEL method (green). Myocytes are red (phalloidin), and nuclei are blue (TOTO). Bar = 20 µm.
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Western blotting and RT-PCR
Caspase-3
Messenger ribonucleic acid (mRNA) for caspase-3 was 8.5-fold upregulated in HHM, and the protein content was 1.4-fold increased. Cleaved caspase-3 was detected by Western blot analysis, but it was absent in controls (Figs. 6 and 7).
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Figure 6 Polymerase chain reaction analysis. (A) Representative original recordings. (B) Quantitative evaluation of changes in gene expression. *p < 0.05 human hibernating myocardium (HHM) versus control. MRNA = messenger ribonucleic acid.
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Figure 7 Western blot analysis. (A) Representative examples of original blots. (B) Quantitative data. *p < 0.05 human hibernating myocardium (HHM) versus control.
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Bcl-2
Bcl-2 gene expression and protein content was three- to fourfold downregulated (Figs. 6 and 7).
Bax
A three- to fourfold increase in gene expression and protein content was found (Figs. 6 and 7).
Bcl-xL
The bcl-xL mRNA was 3.3-fold reduced in HHM, but the protein content remained unaltered compared with control myocardium (Figs. 6 and 7).
c-myc
The mRNA and protein levels for c-myc were unchanged in HHM and normal myocardium (Figs. 6 and 7).
p53
No significant difference in protein quantity was found between HHM and control myocardium. Gene expression, however, was downregulated (Figs. 6 and 7).
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Discussion
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Myocyte degeneration, including a loss of myofilaments and nuclear abnormalities, and replacement fibrosis are common phenomena in HHM in contrast to normal myocardium. Recently, in an animal study of hibernation, structural alterations such as myolysis and increased intracellular glycogen content were detected in hibernating areas and in normally perfused myocardium representing an intra-individual control. It was concluded that the process of global myolysis is caused by chronic elevation in preload or stretch (11). These results cannot be confirmed by the present study. Furthermore, intra-individual control biopsies from patients with HHM showed morphologic changes similar to those observed in myocardium of nontransplanted donor hearts; changes in HHM biopsies were always significantly more severe than in control region (E. R. Schwarz, unpublished data, 2001). Removal of intra-individual control biopsies was not permitted in the present study.
The histologic and molecular biologic data of the current study revealed the presence of two distinct types of cell death in HHM: ubiquitin-related autophagic cell death and apoptosis.
Autophagic cell death and the ubiquitin-proteasome system.
The ubiquitin-proteasome system represents quantitatively the most important process responsible for turnover and destruction of proteins and organelles in several tissues. In addition, autophagy plays a major role (12,13).
The process of autophagy is characterized by different steps: cellular material is enclosed in double membrane vacuoles (autophagosomes) that subsequently dock to and fuse with lysosomes and are digested by lysosomal proteases. In a final step, fragments of myocytes elicit an extracellular response resulting in phagocytosis of cellular debris by macrophages and generation of replacement fibrosis (12,14).
In addition to lysosomal enzymes, autophagic cell death may involve the ubiquitin/proteasome system as well. Whereas autophagocytosis is random protein degradation, the ubiquitin-mediated proteolytic pathway is responsible for a highly selective turnover of intracellular proteins (13,15). However, failure to eliminate ubiquinated proteins disrupts cellular homeostasis and causes degeneration, finally resulting in cell death (13,16). Autophagic processes can lead to cell death, but they are also able to ensure survival of cells by elimination of damaging substrates.
The ubiquitin/proteasome system is composed of activating (E1), conjugating (E2), and ligating (E3) enzymes, as well as of the 26S proteasome multi-catalytic proteinase complex. Polyubiquinated proteins are targets for ATP-dependent degradation by proteasomes in the nucleus and cytoplasm (8,17). In HHM, the percentage of myocytes exhibiting cellular degeneration, including the occurrence of autophagic vacuoles, was at a mean value of 18%. The number of ubiquitin-positive cells appears exceedingly low. This, however, has to be considered as a static value obtained at a certain point in time. When calculated for a longer time period, such as one year, it becomes apparent that a significant number of myocytes have been lost from HHM.
Myocytes with large cytoplasmic ubiquitin-protein complexes that showed an absence of nuclear material were considered to be dead cells where transcription and translation are arrested and the cell will be removed from the tissue (7).
The proteasome content of the myocytes containing autophagic vacuoles was reduced by 50%, resulting in an accumulation of ubiquitin-protein complexes.
Recently, our group described the occurrence of autophagic cell death in myocardium from two different groups of patients: one with end-stage heart failure due to dilated cardiomyopathy and another with hypertrophy in transition to heart failure (7,18). One of the major findings explaining the mechanism of accumulation of ubiquitin-protein complexes was a significant increase of the conjugating enzyme E2 and a significant reduction of the cleaving enzyme isopeptidase (7). Unfortunately, the small amount of tissue derived from the HHM biopsies did not allow for an extensive Western blot analysis in order to clarify the primary defect in the proteasomal system, which will be the subject of future studies.
The time needed for the completion of autophagic cell death is not known at the present time, and therefore, it is difficult to put into perspective the rate of ubiquitin-positive cells reported here. Because autophagic cell death is the result of chronic degeneration, it may be a slow process taking days, weeks, or even months to finally kill the cell (17). Experimental studies using cell culture models are needed to clarify this issue.
Apoptotic cell death.
The specificity of the TUNEL method has been questioned: labeled cells might be apoptotic or oncotic or undergoing DNA repair (19,20). Therefore, a detailed evaluation of the different steps of the apoptotic cascade is needed to identify the endangerment of tissue by apoptosis.
It was shown here that the apoptotic cascade is activated in HHM by bcl-2–related apoptosis regulators and by caspase-3.
In HHM, the gene expression of the anti-apoptotic factors bcl-2 and bcl-xL was significantly reduced compared with control myocardium. The same was true for the bcl-2 protein content. However, the mRNA and protein content of the pro-apoptotic molecule bax was increased, whereas the bax gene transcription factor p53 was unaltered. In HHM, apoptotic cell death seems to be independent of p53. Different models of cardiac myocyte apoptosis in culture showed that hypoxia acidosis, extracellular lactic acid, reoxygenation, and reperfusion are strong stimuli for programmed cell death that is substantially independent of p53. These results were reproduced in p53 knockout mice (21). In HHM, a normal protein content of p53 was documented, whereas mRNA was reduced. The half-life time of the p53 protein might be rather long, so that the protein level is still normal in the presence of a reduced mRNA level. As published recently, pH is reduced significantly in HHM and could be an apoptotic trigger (22).
The bax/bcl-2 ratio was elevated, indicating an activation of the death program, which may lead to bax-dependent mitochondrial membrane permeability changes with a loss of membrane potential and cytochrome c release. Subsequently, cytosolic cytochrome c activates a downstream caspase program (23,24). In HHM, the gene expression of caspase-3 was upregulated and the protein content twofold increased compared with control myocardium. Furthermore, cleavage of caspase-3 was evident.
Our observations are, at first sight, contradictory to those by the group of Borgers (25). However, the lack of apoptotic signals may be due to methodologic differences, inhomogeneity of the tissue, or selection of the study population. This group investigated functional hibernation (i.e., dysfunctional but recoverable myocardium at unimpaired resting blood flow); our results rather reflect structural hibernation, as also confirmed by an increased degree of fibrosis. Therefore, both concepts—dedifferentiation or degeneration with replacement fibrosis and cell death—complement each other with regard to the current model for the pathophysiology of HHM.
Cell death in HHM.
In HHM, both types of programmed cell death were found to occur concurrently. In all biopsies, the mitochondria-dependent apoptotic pathway, with predominance of pro-apoptotic factors, was activated, but apoptotic nuclear profiles were found only at a very low number. This is similar to findings reported by Narula et al. (23) and might have been caused by an extremely rapid removal of dead cells by macrophages (26).
Ubiquitin-related autophagic cell death, as documented in all biopsies, is a consequence of disturbed protein homeostasis (4). However, at the present time, it is difficult to estimate what percentage of myocytes will die in a given period, because the duration of both events—autophagic and apoptotic cell death—is still unknown. Therefore, it seems preferable to state that myocytes will be lost from the tissue due to both mechanisms of cell death.
On the basis of the present study and previous investigations, it is proposed that ubiquitin-related autophagic cell death is not only occurring in brain tissue but also in cardiac myocytes (6,8).
Various proteins have been described that are subject to regulation by proteasomal degradation and play a functional role in apoptosis. These include the tumor suppressor gene p53, proto-oncogene c-myc, transcription factor c-fos, and bax, one of the pro-apoptotic members of the bcl-2 family (15,27,28). Activated caspase-3 can inhibit proteasome function, and this is followed by an increase in intracellular ubiquitin conjugates (29,30). In HHM, the occurrence of myelin bodies and vacuolar structures represents damaged proteins, which are ubiquinated but not degraded and form intracellular aggregates, as described for neurodegenerative situations (8,27). Furthermore, the accumulation of ubiquinated proteins might inhibit the ubiquitin/proteasome system, thereby triggering the autophagic response and might act as a signal for activating the apoptotic cascade in HHM (17). Ultrastructural evidence for cellular regeneration as mitotic figures was not found. About 0.01% of myocytes from both hibernating areas and control tissue showed positive labeling with the cell cycle-associated nuclear nonhistone Ki-67, which is expressed in all active phases of the cell cycle, but not in quiescent G0 cells. It is more abundant in DNA synthesis and mitosis, but it might also be observed when DNA synthesis is inhibited (18). Here, it was interpreted as indicating DNA replication and repair, as described in a previous report (18).
The clinical consequence of the processes described here is the persistent danger of irreversible tissue deterioration in HHM, with incomplete functional recovery after revascularization. Intracellular degeneration and loss of myocytes by apoptosis and autophagy lead to progressive synthesis of replacement fibrosis, which determines the functional capacity of the myocardium. As described recently, a rate of fibrosis above 32% represents the limit for complete functional recovery after revascularization (4). Therefore, restoration of perfusion at an early time point of the disease is a decisive factor, which will determine the clinical outcome of patients with HHM.
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Footnotes
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Drs. Elsässer and Vogt contributed equally to this work.
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References
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