CLINICAL STUDY
Chronic therapy with metoprolol attenuates cardiomyocyte apoptosis in dogs with heart failure
Hani N. Sabbah, PhD, FACCa,
Victor G. Sharov, MD, PhDa,
Ramesh C. Gupta, PhDa,
Anastassia Todor, MDa,
Vinita Singh, PhDa and
Sidney Goldstein, MD, FACCa
a Department of Medicine, Division of Cardiovascular Medicine, Henry Ford Heart and Vascular Institute, Detroit, Michigan, USA
Manuscript received December 13, 1999;
revised manuscript received April 19, 2000,
accepted June 19, 2000.
Reprint requests and correspondence: Dr. Hani N. Sabbah, Cardiovascular Research, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, Michigan 48202
HSABBAH1{at}hfhs.org
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Abstract
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OBJECTIVES
The purpose of this study was to determine if therapy with beta-blockade is associated with reduced cardiomyocyte apoptosis.
BACKGROUND
Chronic treatment with beta-adrenergic blocking agents has been shown to improve left ventricular (LV) ejection fraction and attenuate progressive LV remodeling in heart failure (HF). Cardiomyocyte apoptosis has also been shown to occur in the failing heart.
METHODS
Moderate HF was produced in 14 dogs by intracoronary microembolizations. Dogs were randomized to three months therapy with metoprolol (MET, 25 mg twice daily, n = 7) or to no therapy at all (n = 7). At the end of three months, dogs were sacrificed, and nuclear DNA fragmentation (nDNAf), a marker of apoptosis, was assessed in LV tissue using the TUNEL assay. The number of cardiomyocytes with positive nDNAf labeling per 1,000 was quantified in LV regions bordering old infarcts and in regions remote from infarcts. Endonuclease activity and expression of the antiapoptotic protein Bcl-2 and the proapoptotic proteins Bax and caspase-3 were also evaluated in LV tissue.
RESULTS
The number of nDNAf events per 1,000 cardiomyocytes was lower in dogs treated with MET compared with untreated dogs with HF in the border regions (0.35 ± 0.07 vs. 5.32 ± 0.77, p < 0.001) as well as the remote regions (0.07 ± 0.05 vs. 0.39 ± 0.12, p < 0.05). Endonuclease activity was also significantly lower in MET-treated compared with untreated dogs (25 ± 3 vs. 37 ± 2 ng [3H]DNA rendered soluble/min/mg protein). Western blotting for Bcl-2, Bax and caspase-3 showed increased expression of Bcl-2, decreased expression of caspase-3 and no change in Bax in MET-treated compared with untreated dogs.
CONCLUSIONS
Chronic therapy with MET attenuates cardiomyocyte apoptosis in dogs with moderate HF. Attenuation of ongoing cardiomyocyte loss through apoptosis may be one mechanism through which beta-blockers elicit their benefits in HF.
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Abbreviations and Acronyms
| | CSQ | = calsequestrin | | HF | = heart failure | | LV | = left ventricular | | MET | = metoprolol | | nDNAf | = nuclear DNA fragmentation | | SDS | = sodium dodecyl sulfate | | TdT | = terminal deoxynucleotidal transferase | | TUNEL | = TdT-mediated dUTP nick-end labeling |
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Chronic therapy with beta-adrenergic blocking agents in heart failure (HF) is associated with improved global left ventricular (LV) function and chamber remodeling (1–3). In dogs with moderate HF produced by intracoronary microembolizations, we showed that chronic therapy with metoprolol (MET) improves LV ejection fraction and attenuates progressive LV chamber dilation (4). An understanding of the mechanisms that mediate these beneficial effects of beta-blockers in HF remains elusive. A possible hypothesis is that progressive deterioration of LV function in HF is mediated, in part, by ongoing loss of cardiomyocytes and that therapy with beta-blockers has a positive impact on this process. Studies in dogs with HF (5) and in explanted failed human hearts (6) have documented the occurrence of cardiomyocyte apoptosis, a process that may account, in part, for the loss of cardiomyocytes (5). Apoptosis is an active, energy requiring, genetically-controlled process that is characterized by nuclear DNA fragmentation (nDNAf) (7). Biochemically, apoptosis is characterized by internucleosomal cleavage of DNA by endonucleases whose activity increases during apoptosis (8). Recent studies have suggested that factors inherent to HF, including angiotensin-II (9), norepinephrine (10) and tissue hypoxia (11) can stimulate cardiomyocyte apoptosis. In this study, we tested the hypothesis that the benefits of chronic treatment with the beta-blocker MET in dogs with moderate HF may be mediated, in part, by attenuation of cardiomyocyte apoptosis. In addition to quantifying the number of nDNAf events, the effects of MET on endogenous endonuclease activity, a key surrogate of ongoing internucleosomal DNA cleavage, was also evaluated. The expression of Bcl-2, Bax and caspase-3 in the LV myocardium was also examined because of the recognized proapoptotic effects of Bax and caspase-3 and the antiapoptotic effect of Bcl-2.
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Methods
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Animal model and study protocol.
The canine model of chronic HF used in this study was previously described in detail (12). In this model, chronic LV dysfunction is produced by multiple sequential intracoronary embolizations with polystyrene latex microspheres (77 to 102 µm diameter). A unique feature of this model is the progressive deterioration of LV function that occurs long after cessation of coronary microembolizations (4,12). In this study, 14 dogs weighing between 21 and 34 kg underwent coronary microembolizations to produce HF (4). The cohort of dogs used represents a subset of a previously published larger study in which three months therapy with MET was shown to improve LV ejection fraction and attenuate progressive LV chamber dilation (4). The detailed methods of embolization, anesthesia and hemodynamic assessment were reported as part of the larger study (4). Three weeks after a target LV ejection fraction of 30% to 40% was reached, embolizations were discontinued, and dogs were randomized to three months oral monotherapy with MET (25 mg twice daily, n = 7) or no therapy at all (n = 7). Hearts were harvested at the end of three months of or active therapy. Hearts from 6 normal dogs were also harvested and used for comparisons. The study was approved by the Henry Ford Hospital Care of Experimental Animals Committee and conformed to the position of the American Heart Association on research animal use.
Identification of nuclear DNA fragmentation.
At the end of three months of therapy, the dog’s chest was opened and the heart rapidly removed and placed in ice-cold cardioplegia solution. From each heart, transverse slices (3 to 4 mm thick) were obtained from the LV midventricular level and cut into several blocks, each labeled for anatomical location. Transmural blocks were fixed in formalin and embedded in paraffin. Sections 5 µm thick were prepared, deparaffinized in xylene and rehydrated through graded ethanol washes. Sections were then fixed in freshly prepared 4% paraformaldehyde and treated with 10 mg/ml proteinase K and triple stained as shown in Figure 1, A to C. Sections were first stained using the Apoptosis Fluorescein Detection System (Promega Corp., Madison, Wisconsin) to identify cells showing nDNAf. In this process, fluorescein-12-deoxyuridine 5'-triphosphate (dUTP) is catalytically incorporated at the 3'-OH fragmented DNA ends of apoptotic cells using the terminal deoxynucleotidal transferase (TdT) enzyme, which forms a polymeric tail as in the TUNEL (TdT-mediated dUTP nick-end labeling) assay. Negative control studies, namely, tissue sections processed for TUNEL without TdT were performed for each dog. To identify cardiomyocytes, sections were stained overnight at 4°C with a monoclonal ventricular heavy chain antimyosin antibody (Chemicon, Temecula, California). For visualization, a rhodamine conjugated secondary antimouse antibody was used. Lastly, to identify nuclei, sections were stained using the specific nuclear stain, Hoechst 33342 (Molecular Probes, Inc., Eugene, Oregon). Using the above sequence of staining, nDNAf events were visualized microscopically as yellow-green under fluorescein light (Nikon DM510 filter; Tokyo, Japan); myosin was seen as red under rhodamine light (Nikon DM 580 filter), and cell nuclei were seen as blue (Nikon DM 455 filter). From each section, 80 light microscopic fields (magnification x40) were used to count the number cardiomyocyte nuclei positively labeled for nDNAf. Forty fields were selected at random from myocardial regions bordering scar tissue (old infarcts) and 40 fields from myocardial regions remote from scars. The selection of regions bordering scars was made due to the high incidence of cardiomyocyte apoptosis previously identified in these regions (5). The number of cardiomyocytes positively labeled for nDNAf was counted in each field. The number of cardiomyocytes per field was determined by counting an average of 40 fields in each ventricular sample. By combining these data with the estimated numbers of positively dUTP-labeled cardiomyocytes, the number of nDNAf events per 1,000 cardiomyocytes was determined (6). In tissue sections stained with antimyosin antibody, a scar or infarct was defined as a myocardial region devoid of myocytes and occupying a surface area equivalent to that of at least 1,000 myocytes, as visualized at a magnification of 10x and as previously described (13). Confirmation that these regions indeed represented scar tissue was made by immunostaining sections with antibodies for collagens I and III. The region bordering a scar or infarct was defined using antimyosin stained sections as a peri-infarct region consisting of 10 rows of myocytes adjacent to the scar counted radially starting at the scar to viable tissue interface. A remote myocardial region was defined as any area that was at least 100 myocytes away from any infarct in all directions. These definitions, while empirical, are consistent with previous work from our laboratory (13,14).
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Figure 1 Panels A, B and C: Section of left ventricular myocardium showing cardiomyocytes bordering scar tissue from an untreated dog with heart failure (magnification x 750). (A) Shows a cardiomyocyte positively stained (red) with antimyosin antibody, identifying the cell as a cardiomyocyte. The unstained center region is the nucleus. (B) Shows the same cardiomyocyte nucleus stained positively (blue) with the specific nuclear stain Hoechst 33342. (C) Shows the same left ventricular section as in (A) and (B). The nucleus is positively stained (yellow-green) for nuclear DNA fragmentation.
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Assessment of endonuclease activity.
Endonuclease activity was determined as previously described (8) with minor modifications using tissue obtained from six normal dogs, six untreated dogs with HF and six dogs with HF treated with MET. From each dog, approximately 100 mg of frozen LV powder was homogenized in 1 ml of freshly prepared and ice-chilled buffer A (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 0.3 mM sucrose, 50 mM sucrose, 2 mM benzamidine, 1 µg/ml aprotinin and 2 µg/ml leupeptin). The resulting homogenate was filtered, aliquoted, rapidly frozen in liquid nitrogen and immediately stored at –70°C. The enzyme activity assay was performed in a total assay volume of 100 µl reaction volume that consisted of 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 5 µg Escherichia coli DNA ([3H]DNA = 20,000 dpm) and 50 µg LV homogenate. The reaction was initiated by adding the homogenate and terminated after incubating at 37°C for 1 h by adding 20 µl of 14% perchloric acid followed by 20 µl of 25 mg/ml bovine serum albumin. The resulting mixture was vortexed, centrifuged at 12,000 rpm for 5 min, and an aliquot of 100 µl of the clear supernatant was counted for radioactivity in the presence of 5 ml of scintillation fluid using a scintillation counter. Endonuclease activity was expressed as ng [3H]DNA rendered soluble/min/mg protein.
Western blotting for Bcl-2, Bax and caspase-3.
Tissue levels of Bcl-2, Bax and caspase-3 in LV myocardium were examined using Western blotting. Protein levels were determined in tissue sodium dodecyl sulfate (SDS) extract. Expression of Bax and caspase-3 was assessed in six untreated dogs with HF and six dogs treated with MET. Bcl-2 expression was assessed in five dogs from each of the two study groups. To determine whether differences in tissue levels of Bax, Bcl-2 and caspase-3 exist between normal dogs and untreated dogs with HF, LV tissue from six normal dogs was used, and the results were compared with untreated dogs with HF. In addition, tissue levels of calsequestrin (CSQ), a sarcoplasmic reticulum protein, was also examined in all normal dogs, in untreated dogs with HF and in MET-treated dogs. Calsequestrin does not change in HF and, as such, was used as an internal control (15). The SDS-extract of approximately 25 mg of tissue powder was prepared from the LV free wall of each dog as previously described (16). Equal volumes of the SDS-extract and sample buffer (62.5 mM Tris-HCl, pH 6.8; 20% glycerol and 40 mM DTT with 0.001% bromophenol blue) were combined, and the resulting mixture was incubated in a boiling water bath for 10 min. An aliquot of the boiled mixture, corresponding to 30 µg for Bcl-2 and caspase-3, or 10 µg for Bax and CSQ, was separated on 12% SDS-polyacrylamide gel, and the separated proteins were then transferred to nitrocellulose as previously described (16). To determine Bcl-2 and caspase-3 tissue levels, samples from normal dogs, untreated dogs with HF and MET-treated dogs were incubated with a 500-fold diluted monoclonal mouse anti-Bcl-2 or anti-caspase-3 antibody (Transduction Laboratory, Lexington, Kentucky). For determination of Bax tissue levels, samples were incubated with a 200-fold diluted rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California). For determination of CSQ tissue levels, samples were incubated with 250-fold diluted polyclonal rabbit skeletal muscle antibody raised against CSQ purified from canine ventricular sarcoplasmic reticulum. Monoclonal antibody binding protein was visualized calorimetrically using either antimouse IgG raised in sheep and polyclonal antibody binding protein, using antirabbit IgG raised in donkey. The intensity of the bands was quantified using a Bio-Rad Model GS-670 imaging densitometer. The densitometric unit of measurement used was optical density x mm2.
Data analysis.
Repeated-measures analysis of variance was used to examine the nDNAf outcomes to take into account the two regions (border zone and remote zone) that were examined. For endonuclease activity and CSQ one-way analysis of variance was performed. For other outcomes, namely Bax, Bcl-2 and caspase-3, Western blots were performed at different times for MET-treated versus untreated dogs with HF and for untreated dogs with HF versus normal dogs. Since the band density measurements for separate runs are not directly comparable due to different exposure times, two separate t tests were performed. In all instances, the Hochberg-Holms (17) modification of the Bonferroni adjustment was used to account for multiple comparisons. In the Hochberg-Holms method, if the largest of two or three p values is less than 0.05, then all the tests with smaller p values are considered significant. Only p values meeting this criterion are reported as significant. All data are reported as the mean ± standard error of the mean.
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Results
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Effect of treatment with MET on nDNAf.
In dogs with HF, regardless of treatment, nDNAf was identified in cardiomyocytes remote from any scars as well as in constituent cardiomyocytes of regions bordering scars. On statistical grounds, there was a significant interaction between regions and MET effect for nDNAf (p < 0.001). Therefore, separate tests of the MET effect were made for each region, namely border zone and remote zone. Regardless of therapy, the number of nDNAf events of cardiomyocyte origin was always higher in LV regions bordering scars compared with LV regions remote from any scars (Table 1). A typical high-powered micrograph depicting nDNAf in a cardiomyocyte is shown in Figure 1. There was a significantly lower incidence of nDNAf events of cardiomyocyte origin in regions bordering old infarcts of MET-treated compared with untreated dogs with HF. Similarly, there was a significantly lower incidence of cardiomyocyte nDNAf events in LV regions remote from infarcts of dogs that were MET-treated compared with untreated dogs with HF (Table 1).
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Table 1 Number of nDNAf per 1,000 Cardiomyocytes in Untreated Dogs With HF and Dogs with HF Treated With Metoprolol
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Effect of treatment with MET on endonuclease activity.
Tissue endonuclease activity was significantly higher in untreated dogs with HF compared with normal dogs (36 ± 2 vs. 23 ± 2 ng[3H]DNA rendered soluble/min/mg protein, p < 0.001). Consistent with findings of a reduced number of nDNAf events with MET, endonuclease activity was significantly lower in MET-treated compared with untreated dogs with HF (25 ± 2 vs. 36 ± 2 ng[3H]DNA rendered soluble/min/mg protein, p < 0.01). There was no significant difference in endonuclease activity between normal and MET-treated dogs (Fig. 2).
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Figure 2 Bar graph (mean ± SEM) depicting endonuclease activity measured in left ventricular tissue homogenate prepared from six normal dogs (NL), six dogs with heart failure that were untreated (HF) and six dogs with HF treated with metoprolol (HF-MET).
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Expression of CSQ, Bax, Bcl-2 and caspase-3.
Figure 3 depicts expression of CSQ in three normal dogs, three untreated dogs with HF and three MET-treated dogs. There was no difference in the expression of CSQ among six normal dogs, six untreated dogs with HF and six MET-treated dogs (12.0 ± 1.1 vs. 11.1 ± 1.0 vs. 11.4 ± 1.0 densitometric U). Figure 4 shows a Western blot depicting the expression of Bax in three normal dogs and three untreated dogs with HF. Densitometric analysis of Western blots showed no differences in the expression of Bax between six normal dogs and six untreated dogs with HF (2.4 ± 0.2 vs. 2.3 ± 0.1 densitometric U). Figure 4 depicts expression of Bax in three untreated dogs with HF and three MET-treated dogs. Densitometric analysis of Western blots showed no differences in the expression of Bax between six untreated dogs with HF and six MET-treated dogs (6.2 ± 0.6 vs. 5.6 ± 0.7 densitometric U).
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Figure 3 Western blot depicting the expression of calsequestrin (CSQ) in three normal (NL) dogs, three dogs with heart failure (HF) that were untreated and three dogs with HF treated with metoprolol (HF-MET).
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Figure 4 Top: Western blot depicting the expression of Bax in three normal (NL) dogs and three untreated dogs with heart failure (HF). Bottom: Western blot depicting the expression of Bax in three dogs with HF that were untreated and three dogs with HF treated with metoprolol (HF-MET). Western blots were performed at different times for MET-treated versus untreated dogs with HF and for untreated dogs with HF versus NL dogs and, therefore, while directionally consistent, the band density measurements are not directly comparable due to different exposure times.
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Figure 5 depicts expression of Bcl-2 in three normal and three untreated dogs with HF. Expression of Bcl-2 was increased in untreated dogs with HF compared with normal dogs. Densitometric analysis of Western blots showed an increase in the expression of Bcl-2 in six untreated dogs with HF compared with six normal dogs (8.2 ± 0.1 vs. 2.5 ± 0.2 densitometric U, p < 0.001). Figure 5 also shows expression of Bcl-2 in five untreated dogs with HF and five MET-treated dogs. Upregulation of Bcl-2 is evident in MET-treated compared with untreated dogs with HF. Densitometric analysis of Western blot showed a significant increase in the expression of Bcl-2 in MET-treated compared with untreated dogs with HF (17.5 ± 1.2 vs. 9.9 ± 1.3 densitometric U).
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Figure 5 Top: Western blot depicting the expression of Bcl-2 in three normal (NL) dogs and three untreated dogs with heart failure (HF). Bottom: Western blot depicting the expression of Bcl-2 in five dogs with heart failure that were untreated (HF) and five dogs with HF that were treated with metoprolol (HF-MET). Western blots were performed at different times for MET-treated versus untreated dogs with HF and for untreated dogs with HF versus NL dogs and, therefore, while directionally consistent, the band density measurements are not directly comparable due to different exposure times.
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Figure 6 shows expression of caspase-3 in three normal and three untreated dogs with HF. Caspase-3 was clearly over-expressed in untreated dogs with HF compared with normal dogs. Densitometric analysis of Western blots showed an increase in the expression of caspase-3 in six untreated dogs with HF compared with normal dogs (2.5 ± 0.2 vs. 0.3 ± 0.1 densitometric U, p < 0.001). Figure 6 also shows a Western blot of the expression of caspase-3 in three untreated dogs with HF and three MET-treated dogs. Downregulation of caspase-3 is evident in MET-treated dogs compared with untreated dogs with HF. Densitometric analysis of Western blots from all six untreated dogs with HF and six MET-treated dogs showed a significant decrease in the expression of caspase-3 in dogs treated with MET (7.5 ± 0.8 vs. 24.9 ± 2.8 densitometric U) (p < 0.001).
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Figure 6 Top: Western blot depicting the expression of caspase-3 in three normal (NL) dogs and three untreated dogs with heart failure (HF). Bottom: Western blot depicting the expression of caspase-3 in three dogs with HF that were untreated (HF) and three dogs with HF that were treated with metoprolol (HF-MET). Western blots were performed at different times for MET-treated versus untreated dogs with HF and for untreated dogs with HF versus NL dogs and, therefore, while directionally consistent, the band density measurements are not directly comparable due to different exposure times.
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Discussion
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The results indicate that chronic therapy with the beta1-adrenergic receptor blocker, MET, attenuates cardiomyocyte apoptosis in LV myocardium of dogs with HF. In MET-treated dogs the incidence of cardiomyocyte apoptosis was lower in both LV regions bordering old infarcts and in regions remote from any infarcts. The higher incidence of cardiomyocyte apoptosis in peri-infarct regions is consistent with previous reports in both animals and patients with HF and may be due to the susceptibility of these regions to hypoxia or ischemia, both of which are potential triggers for apoptosis (18). The overall incidence of cardiomyocyte apoptosis was approximately 2.9 cardiomyocytes per 1,000, which is consistent with findings in failed human hearts, whereby an incidence of 2.4 cardiomyocytes per 1,000 was reported (6). The observations are also consistent with findings by Yue et al. (19) who demonstrated a reduction in cardiomyocyte apoptosis in a rabbit model of myocardial ischemia/reperfusion after treatment with the nonselective beta-blocker, carvedilol. The observation of a reduced incidence of TUNEL-positive cardiomyocytes in MET-treated dogs is in line with the finding of reduced LV tissue levels of endonuclease activity. The latter is a family of enzymes responsible for internucleosomal cleavage of DNA and whose activity increases during apoptosis (8). Our findings of increased endonuclease activity in the LV tissue of dogs with HF that were untreated is also consistent with findings of Yao et al. (20), who showed that deoxyribonuclease I, which is indistinguishable from endonuclease, is significantly increased in the myocardium of patients with end-stage HF compared with the myocardium of nondiseased human hearts.
Angiotensin-II, norepinephrine and cardiomyocyte apoptosis.
It has become abundantly clear in recent years that interference with the renin-angiotensin system in the form of angiotensin-converting enzyme inhibition as well as interference with the sympathetic nervous system in the form of beta-blockade in HF can positively impact LV function and remodeling. The mechanisms responsible for the beneficial effects of these agents in HF are not fully understood. Exposure of cultured adult rat cardiomyocytes to angiotensin-II induced apoptosis that was blocked by treatment with the AT1-receptor antagonist, losartan (9). Angiotensin-II can also stimulate the release of norepinephrine from nerve terminals. Enhanced and sustained activity of the sympathetic nervous system is a central feature of HF. Exposure to high levels of catecholamines has long been postulated to be toxic to cardiomyocytes. Mann and colleagues (21) showed that norepinephrine exerts a direct toxic effect on cardiac myocytes in vitro and showed that this effect was mediated by beta-adrenergic receptor stimulation and associated with increased cyclic adenosine monophosphate and calcium influx. This concept was supported by the results of a recent study that showed that exposure of adult rat cardiomyocytes to norepinephrine leads to apoptosis through activation of the beta-adrenergic receptor pathway (10). Observations made in this study are in agreement with recent reports indicating that norepinephrine-mediated apoptosis occurs via a beta1-adrenergic receptor pathway (22).
Effects of beta-blockade on Bcl-2 and caspases.
In this study, we observed an upregulation in the expression of Bcl-2 in untreated dogs with HF compared with normal dogs; in our observation there was no increase in Bax. We also observed further upregulation in the expression of the Bcl-2 in MET-treated dogs compared with untreated dogs with HF; we observed no associated increase in Bax. The increase in Bcl-2 seen in untreated dogs with HF and in MET-treated dogs may have limited the formation of Bax homodimers (23). These data suggest the possibility that MET induces the expression of Bcl-2 independent of HF and that this independently confers protection. The means by which beta-blockade elicits upregulation of Bcl-2 in the setting of HF is not known. Alterations in Bcl-2 have been shown to modulate apoptosis in at least three ways. First, Bcl-2 has been shown to prevent hypoxia-induced apoptosis (24). In this study the highest incidence of cardiomyocyte apoptosis occurred in regions bordering old infarcts that are susceptible to hypoxia/ischemia (25). Beta-blockers reduce myocardial oxygen consumption secondary to afterload and preload reduction as well as reductions of heart rate and wall stress (4), which, in turn, can increase the threshold for apoptosis. Second, Bcl-2 regulates an antioxidant pathway at sites of free radical generation (26) such that an increase in Bcl-2 may be crucial in preventing programmed cell death. Third, Bcl-2 may also be directly involved in the mechanism of calcium-induced cell death as alluded to earlier. It has recently been shown that the calcium-activated protein phosphatase, calcineurine, can bind to Bcl-2, preventing its translocation into the nucleus. Since calcineurine has been implicated in calcium-mediated apoptotic cell death, its association with Bcl-2 may be relevant to cell loss in the setting of elevated cytosolic calcium conditions such as in HF (27).
Another factor involved in the regulation of apoptosis is the interleukin-converting enzyme family of cysteine proteases, also known as caspases. Recent studies have suggested that caspases can mediate apoptosis in cultured cardiomyocytes based on the ability of certain caspase inhibitors, such as zVAD-fmk, to block the apoptotic process (28). In this study the development of HF was associated with increased expression of caspase-3 (CPP-32), while treatment with MET was associated with decreased expression of caspase-3. Again, the exact mechanism by which MET mediates reduced expression of caspase-3 is not known. In a recent study, Black and colleagues (29) showed colocalization of caspase-3 with apoptotic cardiomyocytes after myocardial ischemia/reperfusion in the rat. Other studies have also suggested a close association between Bcl-2 and activation of caspase cascade, with the latter prevented by upregulation of Bcl-2 (30). Results of our study are consistent with these suggestions in that the observed increase in the expression of Bcl-2 in MET-treated dogs was associated with reduced expression of caspase-3.
Effects of beta-blockade on other mediators of apoptosis.
The above rationale conferring an antiapoptotic function to beta-blockers through stimulation of Bcl-2 expression and caspase-3 inhibition is only one possible mechanism by which beta-blockade can act to preserve cardiomyocytes in the setting of HF. Another possible action, alluded to earlier, may be mediated by reduction of circulating levels of norepinephrine (10). Enhanced norepinephrine release can mediate apoptosis through activation of protein kinase A, leading to increased calcium entry via voltage-dependent calcium channels (10). While not examined in this study, it is also possible that newer, so-called third generation beta-blockers can attenuate cardiomyocyte apoptosis by downregulation of the stress-activated protein kinase signaling pathway or by inhibiting Fas receptor expression (19). Finally, it is by no means certain that the modulation of cardiomyocyte apoptosis seen in this study is unique to beta-blockers and to interference with the sympathoadrenergic activation. We previously reported attenuation of cardiomyocyte apoptosis in peri-infarct regions in dogs with HF treated with an angiotensin-converting enzyme inhibitor. The fact that both classes of drugs appear to limit cardiomyocyte apoptosis in HF argues in favor of multiple pathways to the prevention of programmed cell death in the failing heart.
Study limitations.
This study is not without limitations. Identification of nuclear DNA breaks using the TUNEL assay was used to quantify cardiomyocyte apoptosis. Some studies have suggested that internucleosomal DNA cleavage can occur in both apoptosis and necrosis (31). Other studies, however, have shown that the intensity of the dUTP labeling when a cell is undergoing apoptosis is as much as 20-fold higher than when the same type of cell undergoes necrosis (32). Thus, when both apoptosis and necrosis are present, the very low intensity labeling of necrotic cells makes identification very difficult, if not impossible. Furthermore, in this investigation, cardiomyocyte nDNAf was examined in dogs with HF studied three months after the last microembolization when all infarcts were healed. In several studies in which this canine model was used, we were not able to identify cardiomyocyte necrosis in the late chronic stage of HF, namely long after (3 to 4 months) completion of intracoronary microembolizations (4,5,12–14). Another limitation to the use of the TUNEL method to quantify the absolute number of cardiomyocytes undergoing apoptosis is that not all cardiomyocytes have their nuclei in the plain of the histologic section and, for this reason, cannot be judged as undergoing apoptosis or not. This limitation is minimized, however, when comparisons are performed among study groups, as in this work, using identical histologic techniques. Furthermore, the directional changes observed using the TUNEL method are in line with changes in endonuclease activity, a marker of apoptosis. In this study, the pro form of caspase-3, and not the active form, was measured. It is possible, therefore, that the decrease of pro-caspase-3 observed in dogs treated with MET is secondary to cleavage of pro-caspase-3 to generate active caspase-3; the latter was not measured in this study. This possibility is not a likely explanation for our observation. An increase in active caspase-3 in MET-treated dogs would not be consistent with the marked reduction in TUNEL-positive cardiomyocytes, a significant decrease in endonuclease activity and a significant increase in Bcl-2 in dogs with HF treated with MET compared with untreated dogs with HF. Finally, the study design, one time point at the end of three months of therapy, did not allow for determination of the rate at which apoptosis occurs during the progressive phase of the disease, and, therefore, no firm implications can be derived at this time with regard to the role of beta-blockade in modulating cardiomyocyte apoptosis during the course of progressive LV dysfunction.
Conclusions.
The observations made in this study indicate that chronic treatment with the beta-blocker, MET, in dogs with moderate HF attenuates cardiomyocyte apoptosis. The attenuation of cardiomyocyte apoptosis with MET therapy is associated with a reduction in endonuclease activity, an increase in Bcl-2 expression and a reduction of caspase-3 without changes in Bax—all of which favor cell survival. Preventing or attenuating ongoing loss of functional cardiac units in HF by apoptosis may be one mechanism by which beta-blockers preserve LV function and chamber remodeling in the failing heart.
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Footnotes
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Supported, in part, by a grant from the National Heart, Lung, and Blood Institute, HL-49090-06, and by a grant from Astra Pharmaceuticals.
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