CLINICAL STUDIES
Upregulation of the Bcl-2 family of proteins in end stage heart failure
Najma Latif, PhDa,
Mahboob A. Khan, PhDa,
Emma Birks, MRCPa,
Aldo O’Farrell, MSca,
Jules Westbrook, BSca,
Michael J. Dunn, PhDa and
Magdi H. Yacoub, FRCS, FACCa
a Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, Heart Science Center, Harefield Hospital, Harefield, United Kingdom
Manuscript received September 8, 1999;
revised manuscript received December 30, 1999,
accepted February 21, 2000.
Reprint requests and correspondence: Dr. N. Latif, Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, Heart Science Center, Harefield Hospital, Harefield, England. UB9 6JH. UK
najma.latif{at}harefield.nthames.nhs.uk
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Abstract
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OBJECTIVES
To elucidate the pattern of expression of four members of the Bcl-2 family of proteins and to correlate this with terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation.
BACKGROUND
Apoptosis has been implicated as a possible mechanism in the development of heart failure. However, the mechanisms involved remain unclear.
METHODS
We have studied the expression of four members of the Bcl-2 family that are involved in the regulation of apoptosis and analyzed DNA fragmentation as a marker of apoptosis and as a biochemical criterion to distinguish between apoptosis and necrosis in dilated cardiomyopathy (DCM), ischemic heart disease (IHD) and normal donors.
RESULTS
Western blot analysis and immunocytochemistry of the proapoptotic and antiapoptotic Bcl-2 proteins demonstrated significantly higher levels of all these proteins in the diseased groups compared with normal donors. Additionally, Bax was significantly higher in the IHD group compared with DCM. Terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling analysis demonstrated a significantly higher percentage of TUNEL-positive cells in the diseased groups compared with the control. Genomic DNA extraction of ventricular myocardial tissue showed no demonstrable DNA laddering for any of the groups.
CONCLUSIONS
The significant increases in the levels of the proapoptotic proteins Bak and Bax and the higher percentage of TUNEL-positive cells in both diseased groups suggests the presence of ongoing apoptosis. However, increases in the antiapoptotic proteins, Bcl-2 and Bcl-xL, suggest a possible concommitant, compensatory antiapoptotic mechanism in patients with heart failure.
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Abbreviations and Acronyms
| | DCM | = dilated cardiomyopathy | | IHD | = ischemic heart disease | | TUNEL | = terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling |
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In heart failure myocardial cell loss had been traditionally attributed to tissue necrosis based on the morphological observation of dead myocytes and replacement fibrosis. However, it has now been accepted that apoptosis occurs in human hearts under various pathologic conditions including acute myocardial infarction (1–4), arrhythmogenic right ventricular dysplasia (5), complete heart block (6), long QT syndrome (7,8), Uhl’s anomaly (9), atherosclerosis (10), restenosis (11) and also in end-stage dilated and ischemic cardiomyopathy (12,13) and end-stage hypertrophic cardiomyopathy (14). However, these latter reports have relied heavily on the techniques of TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling) and DNA fragmentation.
Apoptosis is a complex, multistep, biochemical process that involves a decision step, which is mediated, in part, by the Bcl-2 family of proteins and an execution phase, which is mediated by the caspases. The Bcl-2 family of proteins consists of anti- and proapoptotic members, and the Bcl-2 protooncogene (15,16) suppresses apoptosis. Bcl-x encodes for two important protein isoforms by alternate splicing, of which the longer, Bcl-xL, inhibits apoptosis, whereas the shorter form, Bcl-xS, facilitates apoptosis by acting as a dominant inhibitor of Bcl-2 and Bcl-xL (17).
Bax (Bcl-2 associated X protein) and Bak (Bcl-2 homologous antagonist/killer) have powerful death-promoting abilities and primarily enhance apoptotic cell death after an appropriate stimulus (18–21). The ability of Bax to block apoptosis is critically dependent on the ratio of Bcl-2 to Bax. When Bcl-2 is in excess, Bax/Bcl-2 heterodimers are formed, and cells are protected. However, when Bax predominates, Bax homodimers are formed, and cells are susceptible to programmed cell death (22). In general, susceptibility of a cell to undergo apoptosis is regulated, in part, by the relative levels and competing dimerizations between the Bcl-2 family members. The Bcl-2 protein has been shown to bind to Bak, Bax, Bcl-xL, Bcl-xS, mcl-1, Bad and itself (18,23–25), and Bcl-xL similarly interacts with Bax, Bak, Bcl-2, Bcl-xS, mcl-1 and Bad (21).
To gain further insights into the possible biochemical/cellular pathways that lead to apoptosis, we have investigated the expression of four members of the Bcl-2 family of proteins: two antiapoptotic, Bcl-2 and Bcl-xL and two apoptotic, Bax and Bak. Additionally the TUNEL assay was used to assess the degree of apoptosis, and DNA fragmentation was used as a marker of apoptosis and as a biochemical criteria to distinguish between apoptosis and necrosis in heart failure.
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Materials and methods
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Patients.
The study group consisted of 20 patients with dilated cardiomyopathy (DCM) and 27 patients with ischemic heart disease (IHD). Details are given in Table 1. To confirm the diagnosis, all patients underwent an assessment that included a medical history, clinical investigations, two-dimensional M-mode and Doppler echocardiography, cardiac catheterization, evaluation of hemodynamic function and coronary arteriography for the DCM group. Ethical permission was obtained from Hillingdon Health Authority Ethics Committee.
Twenty-seven normal donors (23 men, 4 women, [mean age 38, age range 15–58 years]) without any cardiac complications were used as a control group for comparative purposes.
Tissue.
Myocardial tissue from the left ventricle was excised distal from the site of any myocardial infarcts from the diseased group of patients. Control myocardial specimens from the left atrium (n = 10) and left ventricle (n = 17) were obtained from 27 normal cardiac transplant donors without any heart disease or cardiac complications. No differences were detected in the Bcl-2 family of proteins between atrial and ventricular samples; hence, these control samples were grouped together.
SDS-PAGE and Western blotting.
Total protein homogenates (100 µg) were denatured and separated on 12% T SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels and transferred to nitrocellulose (Hybond C, Amersham, United Kingdom). Nitrocellulose membranes were probed using primary antibodies against the Bcl-2 family members (Santa Cruz, California). Visualization of the protein bands was accomplished using SuperSignal Ultra chemiluminescence substrate (Pierce).
Stripping membranes.
For probing, tubulin blots were incubated in stripping solution (100 mmol/L 2-mercaptoethanol, 2% w/v SDS, 62.5 mmol/L Tris Chloride, pH 6.7) for 30 min at 50°C and then probed using antitubulin (Sigma, Dorset, United Kingdom).
Densitometry.
The level of expression of the various Bcl-2 family of antibodies was standardized to tubulin reactivity in each respective lane and quantitated by laser densitometry, which was carried out using the QUANTITY ONE software (Bio-Rad laboratories, California) on a Sun Sparc station.
Immunocytochemistry.
Frozen sections (5–6 µm) were stained with PBS, a mouse irrelevant primary antibody and rabbit serum as negative controls and alpha-sarcomeric actin as a positive control. Antibodies of the Bcl-2 family were incubated on serial sections with a second layer of biotinylated rabbit antimouse or swine antirabbit immunoglobulins (Dako, Cambridgeshire, United Kingdom) followed by Extravidin peroxidase complex (Dako). Reactivity was detected using diaminobenzidine tetrahydrochloride (25 mg/ml) and hydrogen peroxide (0.01% w/v). All slides were counterstained in Mayer’s hemotoxylin. The reactivity of the different proteins was graded according to their intensity within the entire section as negative, weak, moderate or strong staining.
TUNEL assay.
The TUNEL assay was performed using the Apoptag kit (S7110-KIT, Oncor). For negative controls the TdT enzyme was omitted. DNAse 1 treated sections were used as positive controls. DNAse 1 (1.0 µg/ml) was dissolved in DN Buffer (30 mM Trizma base, pH 7.2, 4 mmol/L MgCl2, 0.1 mmol/L DTT) and applied for 10 min. Serial sections from each sample were used for a negative, a positive, TUNEL and also stained with mouse antihuman alpha-sarcomeric actin (1/50) (Sigma, Dorset, United Kingdom) to identify myocytes.
The number of TUNEL positive myocytes was counted in the entire section of each sample. In addition, the number of myocytes in the entire section was calculated using the Lucia quantitative package.
DNA extraction.
Genomic DNA was extracted from all samples using the QIAamp Tissue kit (Qiagen). The DNA was electrophoresed through a 0.8% agarose gel containing a 1:1000 dilution of SYBR Green I DNA stain (Flowgen, Staffordshire, United Kingdom). Genomic DNA from TNF-alpha treated U937 cells was used as an apoptotic DNA control. The DNA was visualized using a Storm Image 860 System (Molecular Dynamics, Amersham, United Kingdom).
Data analysis.
A Kruskal-Wallis test for one way analysis of variance on ranks followed by the Dunn’s test was used as a multiple-comparison procedure for all pairwise comparisons against the control group, and the Fisher’s Exact test was used to compare the numbers of patients after immunocytochemical analysis. A p value of <0.05 was considered statistically significant.
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Results
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Western analysis and protein quantitation for Bcl-2 family members.
Low levels of Bcl-2 (Fig. 1A), Bcl-xL (Fig. 1B), Bak (Fig. 1C) and Bax (Fig. 1D) were detected in the donor samples; however, higher levels were seen in the diseased groups. The Bcl-xS/L antibody detects both the short, proapoptotic Bcl-xS (24 kD), and the long, anti-apoptotic Bcl-xL (30 kD), form of Bcl-x and, consistently, bands could not be detected for Bcl-xS.
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Figure 1 (A) Top panel shows the expression of Bcl-2 in myocardial samples: donor (lanes 1–3), IHD (lanes 4–6) and DCM (lanes 6–10). The number at the left indicates MW in kD and lower band demonstrates tubulin reactivity as a marker of equal loading. Bottom panel shows the spread and the mean levels of Bcl-2 (indicated by the horizontal line) in donor, IHD and DCM. *p < 0.05. (B) Top panel shows the expression of Bcl-xL in myocardial samples: donor (lanes 1–3), IHD (lanes 4–6) and DCM (lanes 6–10). The number at the left indicates MW in kD, and lower band demonstrates tubulin reactivity as a marker of equal loading. Bottom panel shows the spread and the mean levels of Bcl-xL (indicated by the horizontal line) in donor, IHD and DCM. *p < 0.05. (C) Top panel shows the expression of Bax in myocardial samples: donor (lanes 1–3), IHD (lanes 4–6) and DCM (lanes 6–10). The number at the left indicates MW in kD, and lower band demonstrates tubulin reactivity as a marker of equal loading. Bottom panel shows the spread and the mean levels of Bax (indicated by the horizontal line) in donor, IHD and DCM. *p < 0.05. (D) Top panel shows the expression of Bak in myocardial samples: donor (lanes 1–3), IHD (lanes 4–6) and DCM (lanes 6–10). The number at the left indicates MW in kD, and lower band demonstrates tubulin reactivity as a marker of equal loading. Bottom panel shows the spread and the mean levels of Bak (indicated by the horizontal line) in donor, IHD and DCM. *p < 0.05. DCM = dilated cardiomyopathy; IHD = ischemic heart disease.
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Both IHD and DCM patients demonstrated significantly (p < 0.05) increased levels of Bcl-2, Bcl-xL, Bak and Bax compared with the donors, and, in addition, the IHD group demonstrated a significant increase in Bax above the DCM group (Table 2, Fig. 1D).
Immunocytochemistry.
Immunocytochemistry was carried out on 12, 16 and 18 donor, IHD and DCM samples, respectively. The pattern of expression of the various members of the Bcl-2 family mirrored that seen by Western blotting in that the donors showed low expression of all the Bcl-2 family of proteins, and their expression was increased in the diseased groups (Table 3, Fig. 2). Bcl-2 staining was generally very weak, uniform and localized within myocytes, not endothelial cells, in all the samples analyzed (Fig. 2, A and B). Bcl-xL staining was generally weak and uniform in all the samples although moderate staining was seen in isolated, focal regions. The staining, which was cytoplasmic, was localized to the myocytes, not endothelial cells, and some nuclear staining was observed (Fig. 2, C and D).
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Figure 2 Transverse sections of myocardial tissue showing Bcl-2 reactivity in donor (A) and in DCM (B), Bcl-xL reactivity in donor (C) and in DCM (D), Bak in donor (E) and in IHD (F) and Bax in donor (G) and in IHD (H). Original magnification x400. DCM = dilated cardiomyopathy; IHD = ischemic heart disease.
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Bak staining was generally moderate in the samples. Significantly more patients with IHD, but not with DCM, demonstrated moderate staining for Bak compared with the donors. The moderate to strong staining was localized to distinct, single myocytes surrounded by negative myocytes. In the diseased tissues, weak Bak staining was seen over the entire section with stronger expression in focal regions. Endothelial staining, especially of small vessels, was also seen (Fig. 2, E and F). Bax staining was not uniform over the sections, but varying intensities of Bax staining could be seen in focal patches, i.e., some focal staining was much darker than staining in other focal regions. Significantly more patients with IHD and DCM demonstrated strong Bax staining compared with the donors; however, there was no significant difference between the diseased groups. Some nuclei of myocytes and some endothelial cells lining small vessels were also positive for Bax staining. The Bax antibody provided the strongest staining patterns in the diseased tissues (Fig. 2, G and H).
TUNEL.
The TUNEL assay was performed on nine donor, seven IHD and nine DCM samples. In all the negative controls, where TdT was omitted, no positive reactivity was detected (Fig. 3A), and sections treated with DNAse 1 as a positive control showed positive staining of all the nuclei (Fig. 3B). Apoptotic cells were not seen uniformly across sections but were randomly scattered. In situ end labelling with TUNEL was performed in combination with staining for alpha-sarcomeric actin. This confirmed the identity of the TUNEL-positive cell as myocytic. Terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling positivity was predominantly confined to myocytes although occasional endothelial cells and leukocytes within vessels were seen as TUNEL positive within some sections from all groups.
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Figure 3 (A) Negative TUNEL reactivity, (B) positive TUNEL DNAse 1 treated control, (C) TUNEL positive reactivity (green) in a DCM sample, (D) propidium iodide (PI) staining (red) of section C, (E) double exposure of section C and D showing a myocyte that is positive for TUNEL and for PI (orange) and (F) alpha-sarcomeric actin staining of a serial section of C. DCM = dilated cardiomyopathy; TUNEL = terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling.
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In 2/9 donor samples no TUNEL positive myocytes were seen. In the remaining seven donor samples, very few myocytes stained positive, ranging from one to six TUNEL positive myocytes within the entire section. The percentage of TUNEL-positive cells in the donor samples was (7.1 x 10–3) (±6.8 x 10–3). The IHD and DCM samples demonstrated higher percentages of TUNEL-positive cells with values of 49 x 10–3 (±59 x 10–3) and 30 x 10–3 (±27 x 10–3), respectively (Table 4). Both diseased groups demonstrated significantly higher percentages of TUNEL-positive cells (p < 0.05) with a 7.0- and a 4.2-fold increase in TUNEL positivity respectively; however, there was no significant difference between the two diseased groups.
DNA fragmentation.
There was no detectable DNA fragmentation in any of the samples as assessed by laddering on agarose gels although in three diseased samples, very faint ladders could be discerned. Some of the diseased samples demonstrated a degree of smearing indicative of necrosis (Fig. 4).
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Figure 4 Visualization of genomic DNA extractions of myocardial tissue from donor (lanes 7 and 12), IHD (lanes 8–11) and DCM (lanes 1–6) patients using SYBR green I DNA staining. In lane 10 the beginnings of a ladder can be seen. L = 1kb DNA ladder markers; P = apoptosis positive control.
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Discussion
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This study demonstrates, for the first time, significantly elevated levels of four of the Bcl-2 family members in IHD and DCM patients compared with donor samples. Significant increases were seen in the proapoptotic Bax and Bak and also in the antiapoptotic Bcl-2 and Bcl-xL proteins in the IHD and the DCM patients compared with the donors. A significant increase in the expression of the apoptotic proteins Bak and Bax has not previously been reported in heart failure. However, we can confirm the findings of Olivetti et al. (12) who demonstrated a 2.4-fold increase in Bcl-2 in patients with heart failure. Our results demonstrated a 3.7- and 5.35-fold increase (mean 4.52) in the levels of Bcl-2 in patients with IHD and DCM, respectively.
Using immunocytochemistry we demonstrated that the pattern of expression of Bcl-2 proteins was lower in the donor samples and stronger in the diseased tissue. Positive reactivity was restricted to myocytes using Bcl-2 and Bcl-xL; however, endothelial cells as well as myocytes stained positive using antibodies reactive with the apoptotic proteins, Bak and Bax.
Terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling staining provides valuable information about the tissue distribution and, in combination with cell specific markers, the cell types undergoing cell death, but without electron microscopy, TUNEL positivity may be ascribed as necrosis. Recently Kanoh et al. (26) have demonstrated that TUNEL-positive myocytes are living cells with increasing activity of DNA repair, which puts greater emphasis on using a number of techniques to recognise apoptosis.
The percentage of TUNEL positive cells was significantly higher in the IHD and DCM patients compared with the donor samples, but there was no significant difference between the two diseased groups. The TUNEL data correlated well with the Western data where there was a significantly higher expression of the apoptotic proteins, Bak and Bax. Bearing in mind that the apoptotic cycle can be as rapid as 6 h to 24 h, such high percentages of TUNEL positive cells would imply that the loss of viable myocardial cells could potentially be considerable over a year. In our clinical samples of heart failure, no DNA laddering was evident, as apoptotic myocytes were detected in single, isolated cells, and these were extremely infrequent. This lack of DNA laddering is ascribed to the low level of TUNEL positive cells within a heterogeous piece of tissue. This result differs from that of others where DNA laddering was detected in all diseased samples (12) and only in patients with DCM and not in those with ICM (13).
Mechanisms of apoptosis in heart failure.
The mechanisms of apoptosis in IHD and DCM have yet to be clearly elucidated. Apoptosis and necrosis in heart failure may be induced by the same agents, with the type of cell death being dependent on the severity of the insult. Many factors, known or suspected to be present in the failing myocardium, have been shown to stimulate apoptosis in a variety of cells as well as cardiac myocytes. Such factors include inflammatory cytokines, reactive oxygen species, nitric oxide, hypoxia, reperfusion, growth factors and mechanical stretch (27). Of these, hypoxia has received considerable interest, especially with regard to IHD, as demonstrated by exposure of cultured neonatal rat cardiomyocytes to hypoxia, which induced apoptosis (28,29).
In addition, all patients were receiving several drugs for heart failure including angiotensin-converting enzyme inhibitors, digoxin, diuretics, inotropes and beta-adrenergic blocking agents. Their role in the induction process of apoptosis cannot be excluded, especially with regard to beta-adrenergic agonists, which have been associated with cardiac toxicity (30). Norepinephrine has also been shown to stimulate apoptosis in ventricular myocytes (31).
Although the etiology of DCM remains unknown, increasing evidence exists for this syndrome being a sequela of acute viral myocarditis, and an immunologic etiology has been substantiated by the presence of circulating autoantibodies. Several different viruses have been shown to act as triggers of apoptosis (32), and recent reports have demonstrated the ability of autoantibodies to induce apoptosis and, thereby, to have pathological significance (33,34).
The role of Bcl-2 family members.
Although the mechanism of action of the Bcl-2 family members has not been clearly defined, there is increasing evidence that these proteins can function as channels (35–37) for ions, proteins, or both, and as adaptor or docking proteins. As docking proteins they may pull other proteins, like apoptotic protease activating factor-1 (38), out of the cytosol, either sequestering them to the membrane alongside Bcl-2 (and probably inactivating them) (39) or targeting them for interactions with other membrane associated proteins. Bcl-xL indirectly binds pro-FLICE (fas associated death-domain-like interleukin-1 beta converting enzyme) (caspase-8) (40) raising the probability that Bcl-xL prevents the activation of caspases by sequestering procaspases.
Proapoptotic members (Bax) may interfere with channel formation, and it has been shown that Bcl-2 binds to Bax and blocks those channels (37), suggesting an association with the increase in mitochondrial permeability transition during apoptosis. This "megapore" opening results in the generation of oxygen free radicals, dumping of stored Ca2+ and the release of apoptogenic protease activators (cytochrome c and apoptosis-inducing factor [AIF]) into the cytosol in order to activate the caspases (41). Bcl-2 blocks the activation of caspases (42–44), probably by preventing the cell death-induced loss of mitochondrial membrane potential (45,46) and prevents the release of mitochondrial cytochrome c which, along with another cytoplasmic factor, activates caspase-3 (47,48).
This study demonstrates for the first time significant increases in two proapoptotic proteins, Bax and Bak, in patients with heart failure. Recently Narula et al. (49) have demonstrated the release of cytochrome c from mitochondria in patients with heart failure, and elevated levels of Bax and Bak may mediate this release, as it has been demonstrated that Bax and Bak accelerate the opening of voltage-dependent anion channel (50). The significant increases in the levels of these proapoptotic proteins, as demonstrated by Western blotting and immunocytochemistry, and the presence of increased numbers of TUNEL positive myocytes, suggests the presence of ongoing apoptosis. The absence of any demonstrable DNA laddering suggests that the level of apoptosis is very low but, considering the rate of the apoptotic cycle, over time this may prove to be significant enough to impair cardiac function. The increases of the antiapoptotic proteins may suggest the presence of a compensatory antiapoptotic mechanism in the diseased group of patients. Increasing evidence suggests that apoptosis is responsible, at least in part, for the progression of heart failure and the chronic loss of left ventricular function and mass. Whether increased apoptosis is the primary cause of heart failure or is secondary to an as yet unknown process remains to be elucidated. This loss of myocardial cells underlies the clinical manifestation of heart failure, and the success of any therapeutic intervention will depend heavily on a clear understanding of the mechanism of this cell loss.
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Footnotes
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This study was partly supported by the British Heart Foundation.
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Abstract
Materials and methods