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(Circulation. 1996;94:1506-1512.)
© 1996 American Heart Association, Inc.

Articles

Expression of bcl-2 Protein, an Inhibitor of Apoptosis, and Bax, an Accelerator of Apoptosis, in Ventricular Myocytes of Human Hearts With Myocardial Infarction

Jun Misao, MD; Yukihiro Hayakawa, MD; Michiya Ohno, MD; Satoshi Kato, MD; Takako Fujiwara, MD, PhD; Hisayoshi Fujiwara, MD, PhD

the Second Department of Medicine (J.M., Y.H., M.O., S.K., H.F.), Gifu University School of Medicine, Gifu; and Kyoto Women's University (T.F.), Kyoto, Japan.

Correspondence to Hisayoshi Fujiwara, MD, PhD, Second Department of Medicine, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500, Japan.

	Abstract

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Background In general, myocyte death in myocardial infarctions (MIs) is attributed to necrosis, but recently the involvement of apoptosis has been suggested. The ratio of bcl-2 protein, an inhibitor of apoptosis, to Bax protein, an inducer of apoptosis, determines survival or death after an apoptotic stimulus. We speculated that bcl-2 or Bax expression is induced by ischemia and that it may be related to myocyte death in human hearts.

Methods and Results We studied immunohistochemically 37 autopsied human hearts (acute MI, n=15; old MI, n=12; normal hearts as a control, n=10) with the use of bcl-2 and Bax antibodies. There were no myocytes with positive bcl-2 immunoreactivity in the controls or hearts with old MI. However, myocytes with positive bcl-2 immunoreactivity were seen in 9 of 15 hearts (60%) with acute MI, in that it was localized only in salvaged areas surrounding the infarcted tissues. Myocytes with slightly positive Bax immunoreactivity were observed in the control hearts. In the salvaged myocytes surrounding the infarcted tissues, Bax was overexpressed in 2 of 15 hearts (13%) with acute MI but in 10 of 12 hearts (83%) with old MI.

Conclusions bcl-2 protein is induced in salvaged myocytes at the acute stage of infarction, but Bax protein is overexpressed at the old stage. The expression of bcl-2 and the overexpression of Bax may play an important pathophysiological role in the protection or acceleration of the apoptosis of human myocytes after ischemia and/or reperfusion.

Key Words: infarction • apoptosis • myocytes

	Introduction

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Apoptosis, which is programmed cell death, differs from necrosis in terms of ultrastructural and biochemical features, including cytoplasmic and nuclear condensation, subsequent formation of membrane-bound apoptotic bodies, and oligonucleosomal DNA degradation.¹ Apoptosis is characteristically not associated with inflammatory cell infiltration, unlike necrosis,² and it occurs in various cells from several organs.^{1 3 4 5 6} Apoptosis in myocytes is induced by postnatal morphogenesis, especially in the conduction system,⁷ and by hypoxia.⁸ According to studies of rabbits⁹ and human autopsy specimens,¹⁰ myocardial infarction might also include apoptosis, although in general it has been considered to consist of necrosis.

Inhibitors of apoptosis, such as bcl-2 protein,^{11 12 13 14} and inducers, such as Bax,^{14 15 16} have been identified. The bcl-2 protein (molecular weight of 25 kD) is encoded by a gene involved in the 14:18 chromosomal translocation.^{17 18} This cytogenetic abnormality, which is frequently found in human follicular lymphoma, brings the bcl-2 gene into juxtaposition with the immunoglobulin heavy-chain gene and causes the overexpression of bcl-2 protein.^{17 18} The bcl-2 protein is a cytosolic protein with a lipid anchoring domain that can target it to the mitochondria or the nucleus.¹⁹ It plays a role in the inhibition of apoptosis.^{12 13 20} Immunohistochemical analyses with the use of a monoclonal antibody against human bcl-2 protein have shown that in normal adult humans, bcl-2 protein is located in a variety of tissues that either have extended viability such as neurons or undergo self-renewal through mechanisms involving apoptosis, such as interfollicular lymphocytes in lymph nodes and colonic epithelium, but not in the heart.²¹ However, hearts with myocardial infarction were not examined.

Bax, a member of the bcl-2 family, is encoded by six exons.^{15 22} It homodimerizes and forms heterodimers with bcl-2 in vitro. Overexpressed Bax accelerates apoptotic death. The ratio of bcl-2 to Bax determines survival or death after an apoptotic stimulus.^{15 16 22} In the mouse, Bax protein is present in cells of various tissues, including hepatocytes, lymphocytes within the germinal center of lymph nodes, and myocytes.^{16 23} The expression is more widespread than that of bcl-2.²³ However, the expression of Bax protein in human myocytes is unknown.

In fatal myocardial infarction, the infarction size at risk is 76%, even in transmural infarction with abnormal Q waves in ECGs, indicating that considerable salvaged myocytes are at risk.²⁴ Thus, we speculated that bcl-2 and/or Bax protein expression is induced in the salvaged myocytes of the human hearts with myocardial infarction. However, in myocardial infarction, various inflammatory cells, including polymorphonuclear cells, macrophages, lymphocytes, and fibrocytes, infiltrate the infarcted tissues. These inflammatory cells, especially lymphocytes and/or macrophages, may have bcl-2 and/or Bax protein.^{21 23} Therefore, the expression of bcl-2 and/or Bax protein in the tissues as revealed by Western blotting does not mean that bcl-2 and Bax proteins are expressed in the myocytes. Expression of bcl-2 or Bax protein in the myocytes can be precisely determined immunohistochemically with the use of monoclonal or polyclonal antibodies against human bcl-2 or Bax protein.

Thus, in the present study, we examined immunohistochemically whether bcl-2 and Bax proteins are expressed in myocytes in human autopsied hearts with acute or old myocardial infarction.

	Methods

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Subjects
We studied 27 autopsied human hearts with infarction. Fifteen were from patients with acute myocardial infarction who died at 6 hours to 20 days from clinical onset, and 12 were from patients with old myocardial infarction who died >1 month after onset. None of the hearts had any other pathological conditions.

Ten hearts from age- and sex-matched individuals with no cardiac disease (no myocardial disease or significant stenosis of the three major coronary arteries) were used as control patients; they had died as the result of cancer, pneumonia, or renal failure and had no hypertrophy of the heart (heart weight, <350 g). Autopsy revealed that the percent stenosis of the three major coronary arteries was <50%, and routine histological examination of heart showed no abnormal findings, such as myocarditis. All of 37 autopsied hearts were fixed with 10% neutral buffered formalin within 6 hours after death and embedded with paraffin 4 days after fixation. A summary of the autopsy data is provided in the Table.

View this table: [in this window] [in a new window]   Table 1. Patient Characteristics

Among the 15 patients with acute infarction, the direct cause of death included ventricular fibrillation, congestive heart failure, rupture of dissecting aneurysm, and cardiac rupture (Table). They had no previous history of infarction or other organic heart disease. Among the 12 patients with old infarction, 8 had shown clinical and pathological evidence of congestive heart failure, and the other 4 had not. All of the 8 patients with old infarction and heart failure had shown severe symptoms of congestive heart failure (NYHA class IV) for 1 month before death and had died as a result of heart failure. The ventricular cavities were dilated, and congestion of the lungs, liver, and other organs was found at autopsy. On the other hand, patients with old infarction without heart failure were classified as NYHA class I or II and had died as the result of extracardiac causes (pneumonia in 3 patients and renal failure in 1). Abdominal lymph nodes obtained from autopsied patients were used as positive controls for bcl-2 and Bax stainings.

After fixation with 10% neutral buffered formalin, the hearts were cut transversely and serially from the base to the apex. A slice obtained from the midpoint between the base and apex, which contained the greatest focus of myocardial infarction, was then cut into five or six blocks, embedded in paraffin, cut into 4-µm sections, and stained with hematoxylin and eosin or Masson's trichrome. The histological features were then examined with light microscopy with special reference to myocardial infarction.²⁴

Immunohistochemical Procedure and Evaluations
A monoclonal mouse anti-human bcl-2 oncoprotein (M887, DAKO Inc) as the primary antibody was used for bcl-2 immunohistochemical staining. Indirect immunoperoxidase staining was performed as described with some modifications.²⁵ Deparaffinized tissue sections were placed in a glass jar filled with 10 mmol/L citrate buffer, pH 6.0, and placed in a 500-W microwave oven twice for 5 minutes. Intrinsic peroxidase activity was inhibited by 0.3% hydrogen peroxide in methanol for 30 minutes, and nonspecific binding was blocked with normal goat serum. The primary antibody was diluted 1:20 and incubated with the sections overnight at 4°C. The second antibody, peroxidase-conjugated F(ab')₂ fragment goat anti-mouse IgG (H+L) (Jackson Immunoresearch Laboratories, Inc), was incubated with the sections at a dilution of 1:500 for 40 minutes at room temperature. Sections were then stained with 0.4 mg/mL 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co) and 0.006% hydrogen peroxide in 50 mmol/L Tris-HCl, pH 7.4, buffer for 5 minutes at room temperature. Between each step, the sections were washed with distilled water or 10 mmol/L sodium PBS (pH 7.2). Finally, the sections were counterstained with hematoxylin.

A rabbit polyclonal antibody against human Bax protein (Bax: N-20, No. sc493, Santa Cruz Biotechnology, Inc) as the primary antibody was used for Bax immunohistochemical staining. The deparaffinized thin sections were treated by microwave irradiation twice at 500 W for 5 minutes with 10 mmol/L citrate buffer (pH 6.0). Intrinsic peroxidase activity was inhibited by 0.3% hydrogen peroxide in methanol for 30 minutes, and nonspecific binding was blocked with normal goat serum. Thereafter, the sections were soaked for 30 minutes in 5 µg/mL saponin in distilled water at room temperature. The primary antibody was diluted 1:500 and incubated with the sections overnight at 4°C. The sections were then incubated with peroxidase-conjugated F(ab')₂ fragment goat anti-rabbit IgG (H+L) for 40 minutes. After being soaked in 0.5% Triton X-100/PBS, sections were stained with DAB solution and counterstained with hematoxylin.

The specificity of the immunological reaction was controlled by replacing the primary antibody with mouse IgG₁ or rabbit immunoglobulin fraction of serum and by preabsorbing the primary antibody with the corresponding synthetic antigens. Positive controls were lymphocytes within the interfollicular regions of human autopsied lymph nodes for bcl-2 and cells within the germinal center for Bax.

Statistical Analysis
Quantitative data are expressed as mean±SD. Data from among the groups were compared with the use of one-way ANOVA with multiple comparisons and by ² analysis. Statistical significance was set at a level of P<.05.

	Results

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Microscopically, in the infarcted tissues at the acute stage, dead myocytes were deeply stained with eosin and had a granular, banded cytoplasm (coagulation necrosis) or supercontracted myofibrils (contraction band necrosis) (Fig 1). The nuclei of infarcted myocytes disappeared at the late acute stage. There was evidence of marked inflammatory cell infiltration and various degrees of hemorrhage. Then, granulation was seen, including the absorption of dead myocytes and an increase in the number of microvessels and fibrocytes. A specific feature of the old stage was scar formation. However, there was slight inflammatory cell infiltration.

View larger version (105K): [in this window] [in a new window]   Figure 1. Immunohistochemical findings of bcl-2 protein. A, bcl-2 immunohistochemical staining of human lymph nodes as a positive control. Most cells within the interfollicular regions of lymph node showed dark-brown positive immunoreactive staining against monoclonal anti-human bcl-2 protein antibody. In contrast, the majority of cells within the germinal center were negative (magnification, x40). Bottom right, High-power magnification of the interfollicular regions (x400). In this specimen, counterstaining with hematoxylin to identify the nucleus was not done because of clear observation of positive bcl-2 immunoreactivity in the lymphocytes. B1, bcl-2 immunohistochemical staining of salvaged tissues surrounding the infarcted areas in a heart with acute infarction. Dark-brown bcl-2 positive immunoreactivity is seen in the salvaged myocytes (x200). B2, A serial section of B1 incubated with anti-human bcl-2 antibody preabsorbed with excess bcl-2. The positive immunoreactivity of B1 disappeared completely (x200). C, bcl-2 immunohistochemical staining of inflammatory cells infiltrating the infarcted tissue. Dark-brown bcl-2 positive immunoreactivity is evident in some inflammatory mononuclear cells (x400). D, bcl-2 immunohistochemical staining of a normal control heart. There is no evidence of positive immunoreactivity (x400). E1, Hematoxylin and eosin staining in the early stage of acute infarction and salvaged cardiac tissue obtained from patient 4 (see Table). Bottom half, infarcted tissue. Top half surrounded by arrowheads, salvaged tissue (x200). E2, bcl-2 immunohistochemical staining of a serial section of E1. Dark-brown positive bcl-2 immunoreactivity is evident in the cytoplasm of salvaged myocytes. However, infarcted (dead) myocytes show negative immunoreactivity (x200). F1, Hematoxylin and eosin staining in the late stage (granulation) of acute infarction and salvaged cardiac tissue obtained from patient 14 (see Table). Bottom half, infarcted tissue. Top half surrounded by arrowheads, salvaged tissue (x200). F2, bcl-2 immunohistochemical staining of a serial section of F1. Dark-brown positive immunoreactivity is evident in the cytoplasm of salvaged myocytes (x200). G1, Hematoxylin and eosin staining of old infarction and salvaged cardiac tissue. Bottom half, infarcted tissue. Top half surrounded by arrowheads, salvaged tissue (x200). G2, bcl-2 immunohistochemical staining of a serial section of G1. Positive immunoreactivity is absent in the cytoplasm of salvaged myocytes (x200).

Immunohistochemistry of bcl-2 and Bax Proteins
In the positive control sections of lymph nodes, dark-brown immunoreactive bcl-2 products were found in the cytoplasm of lymphocytes within the interfollicular regions but not in the majority of cells within the germinal center (Fig 1A). Conversely, dark-brown immunoreactive Bax products were seen in the cytoplasm of cells within the germinal center but not in the cells within the interfollicular regions (Fig 2A1 and 2A2). There was positive bcl-2 immunoreactivity in the cytoplasm of salvaged myocytes of hearts with acute infarction and positive Bax immunoreactivity in the cytoplasm of myocytes of hearts with and without infarction (Figs 1B, 2B, 2C, and 2D). Some inflammatory mononuclear cells infiltrating into infarcted tissues showed positive bcl-2 and Bax immunoreactivity (Figs 1C and 2E). However, serial sections of these positive sections incubated with the primary antibody preabsorbed with synthetic antigens revealed no positive immunoreaction (Fig 1B1 and 1B2). Those incubated with mouse IgG₁ or rabbit immunoglobulin fraction of serum (negative controls) as the first antibody showed no positive immunoreactivity (Fig 2B1 and 2B2).

View larger version (174K): [in this window] [in a new window]   Figure 2. Immunohistochemical findings of Bax protein. A1, Bax immunohistochemical staining of human lymph nodes as a positive control. Most cells within the germinal center of lymph node showed dark-brown positive immunoreactivity against monoclonal anti-human Bax protein antibody. In contrast, the majority of cells within the interfollicular zone were negative (magnification, x50). A2, Bax immunohistochemical staining of a serial section of A1 in which counterstaining with hematoxylin was not done because of clear observation of positive Bax immunoreactivity in the lymph nodes. Note that only the central zone has positive immunoreactivity (x50). B1, Bax immunohistochemical staining of salvaged tissues surrounding the infarcted areas in a heart with old infarction. Strong dark-brown Bax positive immunoreactivity (overexpression of Bax) is seen in the salvaged myocytes. Note that the immunoreactivity shown in B1 is definitely stronger than in C and D (x200). B2, Serial section of B1 incubated with rabbit immunoglobulin fraction negative control as the first antibody. The positive immunoreactivity of B1 disappeared completely. B1 and B2, Arrows indicate the same small vessel in the serial sections (x200). C, Bax immunohistochemical staining of heart with acute myocardial infarction. Slight dark-brown Bax positive immunoreactivity is seen in salvaged tissues surrounding the infarcted areas in a heart with acute infarction. Left half surrounded by arrowheads, salvaged tissue. Right half, infarcted tissue (x200). D, Bax immunohistochemical staining of normal control heart tissue. Slight dark-brown Bax positive immunoreactivity is seen in most of myocytes (x200). E, Bax immunohistochemical staining of inflammatory cells infiltrating into the infarcted tissues. Dark-brown Bax positive immunoreactivity is evident in some of the inflammatory cells (x400).

There was no evidence of positive bcl-2 immunoreactivity in the left ventricular free walls or ventricular septa of 10 normal control hearts without myocardial infarction (Table and Fig 1D). However, myocytes with positive bcl-2 immunoreactivity were immunohistochemically demonstrated in 9 of 15 hearts (60%) with acute infarction (Table and Fig 1B1, 1E1, 1E2, 1F1, and 1F2). In these hearts, bcl-2 positive immunoreactivity was diffused in the salvaged myocytes surrounding the infarcted areas in the regions at risk (Fig 1B1, 1E2, and 1F2). However, dead myocytes within infarcted tissue (Fig 1E2 and 1F2) or myocytes of the left ventricular wall without infarction showed no positive immunoreactivity.

In all 12 hearts with old infarction, there were no myocytes with positive immunoreactivity (Table and Fig 1G1 and 1G2). Significant stenosis of >75% in one to three major coronary arteries was seen in all hearts of this group. Ten hearts showed significant cardiac hypertrophy of from 350 to 650 g, and eight patients had shown chronic (>1 month) and severe (NYHA class IV) congestive heart failure before death. Some inflammatory mononuclear cells showed positive immunoreactivity against bcl-2 protein in 9 of 15 hearts with acute infarction and 6 of 12 hearts with old infarction (Table and Fig 1C). None of the vessel walls showed positive immunoreactivity in the autopsied hearts.

Slight positive Bax immunoreactivity was seen in myocytes in the left ventricular wall of autopsied hearts without cardiac disease and the left ventricular wall within the nonrisk area of the hearts with infarction (Fig 2D). In salvaged myocytes within the risk area, definite Bax overexpression, compared with the degree of expression in myocytes of control hearts without infarction, was rare at the acute stage of myocardial infarction (2 of 15 hearts) (Fig 2C). However, Bax was frequently overexpressed at the old stage of myocardial infarction (10 of 12 hearts [83%]) (Fig 2B1 and Table). There were no findings of positive immunoreactivities in the infarction (dead) myocytes (Fig 2C). Inflammatory cells with positive Bax immunoreactivity were found in each of the acute (8 of 15 hearts) and old (7 of 12 hearts) stages of infarction (Fig 2E). However, there was no evidence of positive immunoreactivity in the vascular smooth muscle cells in all hearts (Fig 2B1).

	Discussion

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The results of the present study revealed that bcl-2 and Bax protein–like substances were expressed in the salvaged myocytes of hearts with acute and/or old infarctions. In positive control lymph nodes, cells within the germinal center showed negative bcl-2 and positive Bax immunoreactivity, but cells within the interfollicular regions were bcl-2 positive and Bax negative, as described previously.²³ Some inflammatory cells infiltrating the infarcted tissues were also positive for bcl-2 or Bax in the hearts with acute and old infarctions. When mouse IgG₁ or rabbit immunoglobulin fraction of serum was used as a first antibody, the positive bcl-2 or Bax immunoreactivity, respectively, disappeared. When the primary antibody was preabsorbed with corresponding antigens, the positive immunoreactivities disappeared. We used polyclonal antibody for detection of Bax protein, and slight Bax immunoreactivity was observed, even in the myocytes of control hearts. However, serial sections of these slight positive sections incubated with the polyclonal antibody preabsorbed with corresponding antigen and incubated with rabbit immunoglobulin fraction of serum as the first antibody showed no positive immunoreactivity. These results showed that each positive immunoreactivity reflects the presence of bcl-2 or Bax protein. However, we cannot deny the possibility that positive immunoreactivity may not be a specific reaction. In addition, we studied autopsied hearts fixed in 10% buffered formalin and embedded in paraffin. Although all hearts were fixed within 6 hours after death and embedded 4 days after fixation, the sensitivity of the immunoreactivity might have been decreased considerably. Therefore, negative findings in this study did not necessarily indicate the absence of bcl-2 or that Bax was not overexpressed.

Expression of bcl-2 and Bax Proteins in Myocytes
Hockenbery et al²¹ found through immunohistochemical means that bcl-2 protein is not expressed in cardiac tissues. However, they did not examine hearts with myocardial infarction. This study revealed bcl-2 protein expression in salvaged myocytes surrounding infarcted tissues in the areas at risk in hearts with acute infarction. bcl-2–positive myocytes were not seen in the infarcted (dead) myocytes or in those of the left ventricular wall in areas not at risk in the same heart with acute infarction. There was no evidence of positive immunoreactivity in hearts with old infarction or in normal control hearts. Among 12 hearts with old infarction, each had severe stenosis of major coronary arteries. The weight of six of them had increased by >400 g, and eight had signs of severe and chronic congestive heart failure. In acute infarction, bcl-2–positive myocytes were present in patients with and without acute congestive heart failure. Thus, the expression of bcl-2 protein in myocytes detectable in autopsied hearts does not occur in chronic ischemia, cardiac hypertrophy, or congestive heart failure but rather in the presence of acute ischemia and/or reperfusion. The shortest period from the onset of acute infarction to death in this study was 6 hours, and this heart showed positive immunoreactivity. This indicated that bcl-2 is expressed soon after the onset of infarction.

The Bax protein is found in various tissues.²³ This study also showed Bax expression, even in the myocytes of human hearts without cardiac disease. The overexpression of Bax was rare in salvaged myocytes at the acute stage of infarction, but it was frequent at the old stage. In the present study, the shortest period of overexpression from the onset of infarction was 14 days, and the longest period was 10 years (Table). In all hearts with old infarction, infarction-related arteries had significant stenosis of >75%, indicating that salvaged myocytes were chronically exposed to relative ischemia. These suggested that Bax overexpression is induced by chronic cellular response of the salvaged myocytes against various stress, such as chronic ischemia, mechanical stress of myocytes near scar formation, and so on.

Pathophysiological Role of bcl-2 and Bax Protein Expression in Myocytes
The bcl-2 proto-oncogene was originally identified as a transcriptional factor associated with the t(14:18) chromosomal translocation, the cytogenetic hallmark of follicular lymphoma.^{17 18} Several bcl-2 transgenic models have shown that bcl-2 significantly enhances the viability of B lymphocytes expressing the transgene without resulting in an increase in proliferation.²⁶ The extended viability impaired by bcl-2 is a consequence of the suppression of apoptosis. bcl-2 appears to be restricted to tissues in which apoptosis molds developing structures or accounts for cell turnover.²¹ bcl-2 as an antidote to programmed cell death may be required to save progenitor and long-lived cells.²¹ Recently, a protective effect of bcl-2 protein against apoptosis was identified in endothelial cells of the uterus²⁷ and cultured vascular smooth muscle cells,²⁸ in addition to lymphocytes.

However, it has been reported that the bcl-2 gene family consists of >15 members. They can be classified as antideath or prodeath. bcl-2 is a prototype for an antideath or survival factor. Bax is a member of the bcl-2 family and, when overexpressed, accelerates the apoptotic death induced by cytokine deprivation in an interleukin-3–dependent cell line.¹⁵ Overexpressed Bax also counters the death repressor activity of bcl-2.¹⁵ That is, the ratio of bcl-2 to Bax determines survival or death after an apoptotic stimulus.^{14 15}

This study revealed that in the salvaged myocytes at the early stage after the onset of infarction, bcl-2 protein is expressed and overexpression of Bax is rare. A study with the use of in situ nick-end labeling and DNA agarose gel electrophoresis has shown that apoptotic myocytes are among the dead myocytes of rabbit hearts with reperfusion after ischemia.⁹ In addition, infarcted myocytes have features of apoptosis in human hearts with infarction.¹⁰ These findings suggest that some myocytes are salvaged by the expression of bcl-2 in the early stage of infarction.

Recently, Sharov et al²⁹ reported that features of cardiocyte apoptosis were observed in regions bordering old infarcts in congestive heart failure. In the present study, most patients with old infarcts also showed severe congestive heart failure. In the salvaged myocytes of the regions bordering old infarcts, Bax was overexpressed and the bcl-2 expression seen in the acute stage of infarction had disappeared. The Bax to bcl-2 ratio is increased in cells induced to die by an apoptotic stimulus.^{14 15} Therefore, the overexpression of Bax in the old infarction may be related to the pathogenesis of apoptosis and congestive heart failure in the old infarction.

In myocardial tissues with infarction, numerous infiltrating inflammatory cells are observed at the acute stage, but only a few are seen at the old stage. In general, some of these inflammatory cells may have bcl-2 and Bax protein.^{21 23} The present study confirmed the fact. Recently, Savill et al³⁰ reported that apoptosis may be related to the disappearance of infiltrating cells at the chronic stage of inflammation. Therefore, the presence of inflammatory cells with positive bcl-2 and Bax immunoreactivity in the present study suggests that bcl-2 and Bax may play an important role on the apoptosis of these cells in myocardial infarction.

Finally, apoptosis is a very complex process, and methods are available for demonstrating the wide range of gene products involved at all its stages. We used antibodies to only two gene products, and thus this study must be considered limited in scope. Further examinations of Bad, Bak, Fas, and apoptosis itself are warranted.

We conclude that the expression of bcl-2 or the overexpression of Bax protein in the salvaged myocytes in human hearts with infarction may play an important role in the protection or the acceleration of cellular damage after infarction.

	Acknowledgments

 
This study was supported in part by research grants 05670641 (1993) and 07457598 (1995) from the Ministry of Education, Science and Culture of Japan. Thanks are given to Toshie Otsubo and Akiko Hara for assistance in preparing the manuscript; Masami Nariai, Kyoko Fuchino, and Youko Tei for providing technical assistance; and Daniel Mrozek for reading the manuscript.

Received January 9, 1996; revision received April 9, 1996; accepted April 14, 1996.

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