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Technical Briefs |
1
Dept. of Pathol. and Lab. Med. and
2
Div. of Cardiol., Dept. of Med., Hartford Hosp., 80 Seymour St., Hartford, CT 06102;
a author for correspondence: fax 860-545-5206, e-mail gtsonga{at}harthosp.org
An estimated 400 000 or more new cases of cardiovascular disease are diagnosed annually in the US, accounting for the majority of morbidity and mortality from heart disease (1). Coronary artery stenosis accounts for many of these cases because of subsequent necrosis of myocardial tissues. Increasing evidence now suggests that the central dogma of myocardial infarction pathophysiology, cell death from necrosis, should now concomitantly include discussion of apoptosis, or programed cell death (1)(2)(3)(4)(5)(6)(7).
Necrosis of myocytes results from physiological imbalances caused by lack of oxygenated blood supply to these cells. This process of cell death is characterized by depletion of ATP concentrations, intracellular damage to organelles, cell swelling or hypertrophy, rupture of cell membranes, and induction of an acute inflammatory response. Quite distinctive from necrosis is a second method of cell death, apoptosis, which is a highly regulated and systematic form of programed cell death (8)(9). This energy-dependent process involves loss of cell-to-cell contact, cell shrinkage, condensation of nuclear chromatin, and eventual endonucleolytic fragmentation of genomic DNA.
Apoptosis functions as a regulator of biological homeostasis and is often associated with cells that are progressing through the cell cycle. Thus, until recently, investigators had believed that this mechanism of programed cell death was not associated with terminally differentiated adult cells such as myocytes, neural cells, or hepatocytes. Recent studies, however, have shown that myocardial cell apoptosis can be induced by a variety of insults, including hypoxia, acute ischemia–reperfusion, myocardial infarction, cardiomyopathy–end-stage heart failure, and myocardial pressure stretch (1)(2)(3)(4)(5)(6)(7). Subsequent to various pathological stresses, the myocardium responds to bodily demands for increased cardiac output through a variety of compensatory physiological mechanisms, e.g., myocyte hypertrophy, increased expression of contractile proteins, and myocardial hibernation (10). Myocardial hibernation is thought to be a mechanism by which myocyte viability is preserved in response to ischemic attacks by a metabolically down-regulated process.
Previously, we established a porcine model of myocardial hibernation that resulted from reduced coronary flow induced by severe stenosis in the left anterior descending coronary artery (LAD) and demonstrated the presence of apoptotic myocytes by an immunohistochemical method (7)(10). The objective of the present study was to evaluate whether internucleosomal DNA fragmentation, a biochemical characteristic of apoptosis, could be detected in chronically hypoperfused myocardium containing apoptotic myocytes.
Five animals weighing 28–48 kg were studied and treated as previously described (10). After a midline thoracotomy and suspension of the heart in a pericardial cradle, the LAD was dissected free over 1–2 cm to accept a probe (Transonic Inc.) to measure coronary flow. A fixed stenosis was created to reduce the resting LAD coronary flow by 40%, and the LAD flow reduction was maintained in the 5 pigs for 24 h (n = 3), 7 days (n = 1), or 4 weeks (n = 1). Subsequent to LAD stenosis, the animals were restudied and then injected with 10 mL of 100 g/L KCl solution to induce infarction and death (one animal infarcted before injection). Their hearts were harvested immediately after arrest, and methylene blue was injected distally into the stenotic LAD to determine the ischematized area in the left ventricle (LV). The LV was cross-sectioned at 0.5-cm intervals from apex to base so that ischemic (areas stained with methylene blue) and normal tissues could be excised and stored on ice, or frozen at -70 °C, before being processed for DNA extraction. The remaining LV sections were fixed with formalin, 100 mL/L, and prepared for immunohistochemical tissue staining.
DNA extraction was performed with a nonorganic extraction protocol on fresh or frozen tissue specimens from the ischemic or nonischemic portions of the myocardium. Electrophoresis of genomic DNA was performed on 2% agarose gels, and the DNA was visualized by ethidium bromide staining and exposure to ultraviolet light. In situ detection of apoptotic cells was performed with the ApopTag Plus In Situ Peroxidase Detection Kit (Oncor), in which terminal deoxynucleotidyl transferase (TdT) is used to catalytically add digoxigenin-labeled nucleotides to the new 3'-OH ends of double- or single-stranded DNA formed during internucleosomal DNA fragmentation. In all animals, ischemic tissue samples were randomly chosen from the cross-sectional segments of myocardium in the hibernating region of the LV.
Internucleosomal DNA fragmentation was observed in 2 of 5 ischemic
samples from pig no. 1 (stenotic LAD <24 h) and in 1 of 4 ischemic
samples from pig no. 2 (stenotic LAD for 24 h) (Table 1
). No internucleosomal DNA fragmentation was observed in
ischemic tissues from the 3 remaining animals (Table 1
) nor in normal,
nonischemic tissue samples from any animal. Similarly, tissue samples
subject to in situ end-labeling of apoptotic myocytes were chosen at
random from the formalin-preserved myocardium. In all 5 animals,
positively labeled myocyte nuclei, indicative of apoptosis, were
observed in the myocardial regions supplied by the stenotic LAD (Table 1
). Pigs 2 and 3 had the highest degrees of apoptosis, with 6.7% and
2.8%, respectively, of the total number of myocytes counted showing
evidence of apoptosis. Both of these animals were stenotic for 24
h. In pig 4, which was stenotic for 7 days, 2.5% of the total number
of myocytes counted showed evidence of apoptosis, whereas in pig 5,
which was stenotic for 4 weeks, 1.9% of the total number of myocytes
counted showed evidence of apoptosis. In pig 1, which infarcted after
5 h of stenosis, 0.3% of the total number of myocytes counted
showed evidence of apoptosis. No positively stained myocyte nuclei were
detected in normal, nonischemic tissue samples from any animal.
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The nonchromatin, nucleoskeletal structures found in mammalian nuclei
comprise ribonucleoproteins, the nucleolus, and an in situ nuclear
protein matrix associated with internal peripheral structures of the
nucleus (11). Although little is known about the structure
and precise function of the nuclear matrix, DNA structure and
organization are understood to be inexorably linked to it. The
structure of chromosomal DNA is organized at numerous levels by the
nuclear matrix. The first-order structure of DNA is simply the 2-nm
right-handed double helix, whereas the 10-nm nucleosomes constitute the
second order of DNA structure (Fig. 1
). Nucleosomes are made up of core nucleosomal DNA wrapped
around a histone octamer (two molecules of each of the core histones,
H2A, H2B, H3, and H4) (12)(13). A distinct
histone protein, H1, mediates the generation of the third level of DNA
organization. Six nucleosomes, packed by interactions with the globular
domains of the H1 proteins, form a solenoid and generate a 30-nm DNA
filament that creates the DNA loop domain. The DNA loops, which are
~60 kb in size and represent the fourth level of DNA organization,
attach at their base to a central nuclear matrix structure. These loops
are wound into 18 DNA radial loops, which wrap around nuclear
scaffolding and form the fifth-order structure of DNA, a miniband unit
(14). Each miniband unit represents 1 turn of a chromatid.
Wound and highly compacted minibands are stacked along a central
nuclear protein scaffold to generate each chromatid.
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Internucleosomal DNA fragmentation has long been considered the
biochemical hallmark of apoptosis (15). Early studies of
apoptosis suggested that chromatin condensation, a morphological
characteristic of programed cell death, represented a specific form of
DNA degradation. When subjected to agarose gel electrophoresis, this
degraded DNA associated with chromatin condensation formed a DNA
ladder, depicting multiples of 180–200 bp. The generation of the DNA
ladder during apoptosis represents degradation at the second level of
DNA organization (Fig. 1
). Core nucleosomal DNA has been measured in
eukaryotes to be 146 ± 2 bp long; these base pairs form the two
turns of DNA wrapped around the histone octamer. The H1 histone
proteins, which associate with the nucleosome to mediate the formation
of the DNA loops, protect an additional 20 bp of DNA. Each nucleosome
is joined to adjacent nucleosomes by regions of linker DNA. The linker
regions of DNA range from 20 to 40 bp and flank each side of a
nucleosome outside the region of interaction between H1 and nucleosomal
DNA. The formation of 180–200-bp oligonucleosomes results from
cleavage in this 20–40-bp linker DNA region between adjacent
nucleosomes by calcium-dependent endonucleases (Fig. 1
).
Results from our study indicate that the formation of the internucleosomal DNA ladder is not always detectable in apoptotic myocytes. Of the 21 DNA samples extracted from ischemic tissue and subsequently electrophoresed, only 3 showed the characteristic internucleosomal DNA fragmentation pattern—despite the immunohistochemical detection of apoptosis in all tissue samples from the same ischemic regions of hibernating myocardium. What accounts for the inconsistency between the immunohistochemical and biochemical detection methods? Perhaps the focal nature of apoptosis itself accounts for this discrepancy between the two methods of detection. Apoptosis occurs in an asynchronous fashion; at any given point during myocardial hibernation, only a limited number of myocytes will be progressing through programed cell death, as indicated by the small percentage of cells detected by immunohistochemical methods. Therefore, the process of apoptosis may be a reason for the lack of fragmentation detected by gel electrophoresis. Unlike cells found in the thymus, apoptosis is not as prominent in nonreplicating myocytes. In addition, cells undergoing apoptosis are being removed from the cell population in vivo by phagocytosis, another possible reason for the low numbers of apoptotic myocytes found. Our data showing smaller percentages of apoptotic cells in tissues with longer periods of stenosis support this. Finally, the degree of apoptosis in our samples may not have been severe enough to demonstrate internucleosomal laddering, as detected by gel electrophoresis and ethidium bromide staining. Thus, although present in a small percentage of cells, DNA laddering from apoptotic cells was well below the detection limit of our biochemical method.
Several studies have recently reported the marked absence of
internucleosomal DNA cleavage pattern in cell lines that have been
induced to undergo apoptosis, yet the classic morphological features
associated with apoptosis were displayed (16)(17)(18)(19)(20)(21).
However, endonucleolytic cleavage at levels of DNA organization higher
than the nucleosome has been described; large fragments, ranging in
size from 50 to 300 kb, have been observed in several apoptotic
phenotypes, including rat thymocytes induced with dexamethasone,
lymphocytes treated with teniposide, and epithelial cells induced to
undergo apoptosis by serum deprivation (17)(18)(19). DNA
degradation at the fourth level of chromatin organization would produce
DNA fragments in the 50- to 300-kb range (Fig. 1
) and could result from
endogenous endonuclease cleavage sites existing near the area of
interaction between the nuclear matrix scaffolding and DNA loops.
In conclusion, the present study shows that chronically hypoperfused myocytes do undergo apoptosis; however, internucleosomal DNA fragmentation, as detected, is not necessarily associated with this form of cell death. The number of myocytes progressing through apoptosis may not be sufficient for detection of internucleosomal laddering by gel electrophoresis and ethidium bromide staining. In addition, it is possible that efficient disposal of apoptotic myocytes through phagocytosis may account for lack of internucleosomal DNA fragmentation. If DNA fragmentation is prevalent in myocytes undergoing apoptosis, the type of DNA degradation may be occurring at a higher level of DNA organization. Further studies are needed to better understand the relationship between DNA fragmentation and apoptosis in chronically hypoperfused myocytes.
Acknowledgments
We thank Richard Cartun, Carl A. Pedersen, Anne Perkins, and Marylou Debear for their technical assistance with the immunohistochemical staining, and Richard Johnson for his technical assistance with preparation of the DNA organization schematic.
References
The following articles in journals at HighWire Press have cited this article:
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  A. M. A. Nasirudeen, Y. E. Hian, M. Singh, and K. S. W. Tan Metronidazole induces programmed cell death in the protozoan parasite Blastocystis hominis Microbiology, January 1, 2004; 150(1): 33 - 43. [Abstract] [Full Text] [PDF]
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