Journal of the American College of Cardiology
© 2000 by the American College of Cardiology
Published by Elsevier Science Inc.
Vol. 36, No. 4, 2000
ISSN 0735-1097/00/$20.00
PII S0735-1097(00)00827-5
Expression of Tumor Necrosis
Factor-alpha–Converting Enzyme and
Tumor Necrosis Factor-alpha in Human Myocarditis
Mamoru Satoh, MD, PHD, Motoyuki Nakamura, MD, Hidetoshi Satoh, MD,* Hidenori Saitoh, MD,
Ikuo Segawa, MD, Katsuhiko Hiramori, MD
Iwate, Japan
We determined whether tumor necrosis factor-alpha– converting enzyme (TACE) is expressed with tumor necrosis factor-alpha (TNF-alpha) in myocarditis.
BACKGROUND Tumor necrosis factor-alpha– converting enzyme, which has recently been identified as
belonging to the family of metalloproteinase disintegrin proteins, is responsible for the
conversion of TNF-alpha precursor to its mature form.
METHODS
We examined TACE and TNF-alpha expressions in endomyocardial biopsy tissues obtained
from 14 patients with myocarditis and five control subjects by using quantitative reverse
transcriptase polymerase chain reaction and immunohistochemistry.
RESULTS
Expression of TNF-alpha and TACE messenger ribonucleic acid (mRNA) was significantly
greater in the myocarditis group than in the control group. A positive correlation was found
between TNF-alpha and TACE mRNAs (r ⫽ 0.83, p ⬍ 0.05). Six patients with severe
myocarditis underwent repeat biopsies. Although TNF-alpha and TACE mRNAs were
expressed at high levels in the initial biopsies, a marked decrease was noted in the repeat
biopsies. The immunostainings for TNF-alpha and TACE were positive in the myocytes and
interstitial cells of myocardium obtained from patients with myocarditis. Expression of
TACE and TNF-alpha mRNAs was greater in the subgroup in New York Heart Association
functional class III or IV than in the subgroup in class I or II. Expression of TACE and
TNF-alpha mRNA was correlated positively with left ventricular volume (TNF-alpha: r ⫽
0.85; TACE: r ⫽ 0.80) and negatively with left ventricular systolic function (TNF-alpha: r ⫽
⫺0.85; TACE: r ⫽ ⫺0.85).
CONCLUSIONS These findings indicate that the expression of TNF-alpha and TACE may have important
implications in the pathogenesis of myocarditis and may influence advanced cardiac
dysfunction in myocarditis. (J Am Coll Cardiol 2000;36:1288 –94) © 2000 by the American
College of Cardiology
OBJECTIVES
Myocarditis has been considered to be a myocardial inflammatory reaction due to infection by a wide range of viruses
and autoimmune reactions (1). Several studies have used a
molecular approach to demonstrate the relation between
pathogenetic factors and viral agents in causation of human
myocarditis (2,3). In a murine acute viral myocarditis model,
tumor necrosis factor-alpha (TNF-alpha) messenger ribonucleic acid (mRNA) was strongly expressed in infiltrating
cells, indicating that these cells mediate the immune responses in viral myocarditis (4). A strong linear relation has
also been reported between mortality and TNF-alpha levels
in a murine model of congestive heart failure due to viral
myocarditis (5).
Other previous experimental and clinical studies have
suggested that there is an association between depressed
myocardial function and elevated TNF-alpha mRNA and
protein levels in the myocardium in human dilated cardiomyopathy (6,7). TNF-alpha is a multifunctional cytokine
From the Second Department of Internal Medicine and *Second Department of
Pathology, Iwate Medical University School of Medicine, Iwate, Japan. This study
was supported by a grant-aid for General Scientific Research from the Japanese
Ministry of Education, Science, Sports and Culture (nos. 09670742 and 122770348)
and a grant from the Keiryokai Research Foundation (no. 72), Morioka, Iwate, Japan.
Manuscript received December 3, 1999; revised manuscript received March 21,
2000, accepted May 1, 2000.
that mediates various pathologic processes, such as septic
shock, inflammation and cachexia (8). Although the major
biologic role of TNF-alpha is thought to be a host response
against systemic infections, this cytokine has been recently
implicated in the pathogenesis of a variety of human cardiac
diseases, including myocarditis, dilated cardiomyopathy and
congestive heart disease (9 –12).
Recently, the protease responsible for the shedding of
pro-TNF-alpha was identified and cloned (13,14). Tumor
necrosis factor-alpha– converting enzyme (TACE) belongs
to the family of metalloproteinase disintegrin proteins
(13,14). It has also been suggested that TACE might be an
important target enzyme for anti-inflammatory agents in
suppression of the final processing stage of TNF-alpha
production (13). However, it is uncertain whether TACE is
expressed with TNF-alpha in the myocardial tissues in
human myocarditis. In this study, we examined the expression of TACE and TNF-alpha in the endomyocardial
tissues of patients with myocarditis and control subjects by
using a quantitative reverse transcriptase polymerase chain
reaction (RT-PCR) method and immunohistochemical
analysis. We also explored the relation between clinical
characteristics and myocardial expression of TACE and
TNF-alpha.
Satoh et al.
Expression of TACE and TNF-alpha in Myocarditis
JACC Vol. 36, No. 4, 2000
October 2000:1288–94
Abbreviations
cDNA
CK
GAPDH
and
⫽
⫽
⫽
mRNA
NYHA
RT-PCR
⫽
⫽
⫽
TACE
⫽
TNF-alpha ⫽
Acronyms
complementary deoxyribonucleic acid
creatine kinase
glyceraldehyde-3-phosphate
dehydrogenase
messenger ribonucleic acid
New York Heart Association
reverse transcriptase polymerase chain
reaction
tumor necrosis factor-alpha– converting
enzyme
tumor necrosis factor-alpha
METHODS
Subjects. We examined 20 endomyocardial biopsy tissues
obtained from 14 patients with myocarditis by right ventricular endomyocardial biopsy. This group included 10
males and four females (mean age 37 ⫾ 5 years, range 13 to
73 years). Myocarditis was diagnosed on the basis of initial
symptoms (e.g., upper respiratory illness, fever and chest
pain), and clinical findings included tachycardia, arrhythmias, murmurs, rubs, cardiomegaly, elevated erythrocyte
sedimentation rate, leukocytes, cardiac enzyme, C-reactive
protein and electrocardiographic changes (e.g., conduction
disturbances, ST-T segment and Q wave abnormalities), in
conjunction with morphologic evidence of inflammatory
infiltrate in the biopsy materials. Myocarditis was histologically diagnosed according to the Dallas criteria (15). Each
biopsy sample was examined by three investigators who had
no knowledge of the clinical features and results of RTPCR and immunohistochemistry. Patients with a pathologic diagnosis of idiopathic myocarditis were carefully
selected for this study. Specific types of myocarditis, such as
rheumatic, septic, mycotic, eosinophilic types and myocarditis associated with collagen disease, sarcoidosis or another
known etiology, were excluded from this study. In addition,
all patients with myocarditis underwent coronary angiography at the time of each endomyocardial biopsy to exclude
ischemic heart disease and other secondary cardiac diseases.
The initial endomyocardial biopsies were performed two to
12 days (mean 8 ⫾ 1 days) after admission. Six of the 14
patients in New York Heart Association (NYHA) functional class IV at admission underwent repeat biopsies at 28
to 62 days (mean 35 ⫾ 5 days) after admission to confirm
the extent or completion of healing. Echocardiagraphy
determined left ventricular ejection fraction and diameter at
the time of each endomyocardial biopsy. Serial peripheral
blood samples were taken from patients with myocarditis
between one and 14 days after admission for measurement
of cardiac enzymes and inflammatory variables. Control
myocardial tissue samples were obtained by endomyocardial
biopsy from five subjects (three men and two women; mean
age 48 ⫾ 15 years) suspected of having a cardiac disorder on
the basis of premature beats and echocardiographic changes,
such as slight ventricular wall thickness. The resulting
1289
pathologic findings and close clinical examination showed
no evidence of myocardial disease and medical history of
infectious illness (e.g., myocarditis, sepsis and pneumonia),
and these subjects were designated as the control subjects
with normal cardiac function and morphology. These study
protocols were approved by our hospital’s Ethics Committee, and written, informed consent was obtained from all
subjects.
Positive control cells for TACE and TNF-alpha mRNA.
Peripheral human monocytes were prepared from normal
donors. The monocytes were washed and resuspended in
AIM-R medium (GIBCO BRL, Gaithersburg, Maryland)
supplemented with 2 mg of anti-human LeuTM-4 (CD3)
(Beckton Dickinson, San Jose, California). They were then
allowed to adhere to tissue culture flasks for 48 h at 37°C.
Extraction of total RNA. Total RNA was extracted by the
acid guanidinium thiocyanate-phenol-chloroform method
from endomyocardial tissues and cultured monocytes and
treated with RNase I (GIBCO BRL) (16). TaqMan
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
control reagents were used for fluorogenic detection of
human GAPDH transcript (Perkin Elmer Applied Biosystems Division, Foster City, California).
Oligonucleotides of primers and probes. Published complementary deoxyribonucleic acid (cDNA) sequences for
human TNF-alpha (17), TACE (13) and GAPDH (18)
were used for construction of primers and probes. The
sequence of primers and their associated fluorogenic probes
was designed using applications-based primer design software (Primer Express version 1.0, Perkin Elmer Applied
Biosystems Division). The following primers and probes
were used for relative quantification of targeted gene expression—for TNF-alpha: forward primer 5⬘-CTTCTC CTT CCT GAT CGT GG-3⬘, reverse primer 5⬘GCT GGT TAT CTC TCA GCT CCA-3⬘ and probe 5⬘CAG GCA GTC AGA TCA TCT TCT CGA AC-3⬘; for
TACE: forward primer 5⬘-ACC TGA AGA GCT TGT TCA TCG AG-3⬘, reverse primer 5⬘-CCATGA AGT GTT CCG ATA GAT GTC-3⬘ and probe 5⬘TTG GTG GTA GCA GAT CAT CGC TTC T-3⬘; for
GAPDH: forward primer 5⬘-GAA GGT GAA GGTCGG AGT-3⬘, reverse primer 5⬘-GAA GAT GGT GATGGG ATT TC-3⬘ and probe 5⬘-CAA GCT TCCCGT TCT CAG CC-3⬘. The PCR products of TNF-alpha,
TACE and GAPDH were amplified at the sizes of 266, 190 and
226 base pair, respectively.
Quantitative RT-PCR. We analyzed TACE and TNFalpha mRNA expression levels using a quantitative RTPCR method, as previously described (19). The cDNA was
synthesized and amplified from total RNA and tenfold
serial dilutions of human control RNA (Perkin Elmer
Applied Biosystems Division) by RT-PCR using the TaqMan EZ RT-PCR kit (Perkin Elmer Applied Biosystems
Division). The cDNA products were synthesized at 60°C
for 30 min and amplified with 40 cycles of PCR, with each
cycle consisting of denaturation at 94°C for 20 s and
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Satoh et al.
Expression of TACE and TNF-alpha in Myocarditis
JACC Vol. 36, No. 4, 2000
October 2000:1288–94
Table 1. Clinicopathologic Characteristics and mRNA Expression Levels in Patients With Myocarditis
Pt.
No.
1a
1b
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7
8
9
10
11
12
13
14
Age
(years)
Gender
50
M
48
M
28
F
16
M
32
M
40
M
73
16
36
65
56
26
13
16
F
M
M
F
F
M
M
M
Pathologic
Diagnosis
NYHA
class
Duration*
(days)
CK
(IU/liter)
LVESD
(mm)
LVEF
(%)
TACE/
GAPDH
Ratio
TNF-alpha/
GAPDH
Ratio
Active MC
Healing MC
Active MC
Healed MC
Active MC
Healing MC
Active MC
Healed MC
Active MC
Healing MC
Active MC
Healing MC
Healing MC
Healing MC
Active MC
Active MC
Healing MC
Healing MC
Active MC
Healing MC
IV
II
IV
I
IV
II
IV
I
IV
II
IV
II
III
II
II
II
II
III
II
II
7
62
10
29
5
28
14
30
2
34
8
29
10
9
8
5
8
12
9
10
766
48
29
53
29
52
38
49
38
59
32
51
32
45
31
28
26
33
49
35
30
42
64
44
65
39
68
43
60
39
66
32
68
48
63
67
64
63
46
66
60
4.34
1.92
3.47
1.06
4.83
1.63
3.51
1.99
5.20
2.25
4.75
1.58
1.73
0.73
0.95
0.75
0.49
0.74
1.84
0.83
4.08
4.42
5.78
4.91
5.52
3.26
3.28
1.34
7.48
3.46
7.10
2.59
3.34
0.99
0.77
3.42
2.57
3.78
2.52
1.23
658
826
513
519
838
308
244
495
366
336
983
230
363
*Duration from hospital admission.
a ⫽ initial biopsy; b ⫽ repeat biopsy; CK ⫽ creatine kinase (peak plasma concentration); F ⫽ female; GAPDH ⫽ glyceraldehyde-3-phosphate dehydrogenase; LVEF ⫽ left
ventricular ejection fraction; LVESD ⫽ left ventricular end-systolic diameter; M ⫽ male; MC ⫽ myocarditis; NYHA ⫽ New York Heart Association; Pt. ⫽ patient; TACE ⫽
tumor necrosis factor-alpha– converting enzyme; TNF-alpha ⫽ tumor necrosis factor-alpha.
annealing and extension at 62°C for 1 min. A quantitative
PCR method was developed using detection and 5⬘ nuclease
assay by an ABI PRISM 7700 sequence detector (Perkin
Elmer Applied Biosystems Division) (19 –21). This system
was based on the use of the 5⬘ nuclease activity of Taq
polymerase to cleave a nonextendable, dual-labeled fluorogenic hybridization probe during the extension phase of
PCR.
Immunohistochemal analysis. Immunohistochemical
analysis was performed on paraffin sections to determine the
cellular source of TACE and TNF-alpha production.
Monoclonal antibody against TACE, TACE-M220 (1:
100, Immunex, Seattle, Washington) and polyclonal rabbit
anti-human TNF-alpha (1:1000, Genzyme, Cambridge,
Massachusetts) were used as primary antibodies. The tissue
sections were deparaffined with xylene for 20 min and
thoroughly dehydrated with serial diluted ethanol. After
inhibition of endogenous peroxidase and blocking of nonspecific reactions, primary antibodies were applied. Biotinylated goat immunoglobulin (Histofine, SAB-PO kit,
Nichiren Corp., Tokyo, Japan) was used as a secondary
antibody. Peroxidase-labeled streptavidin (Histofine,
SAB-PO kit, Nichiren Corp.) was applied and visualized
using diaminobenzidine as a chromogen. The specificity of
the immunohistochemical analysis was confirmed by substituting the primary antibodies with mouse immunoglobulin G1 negative control (Dako, Glostrub, Denmark) for
TACE, and rabbit immunogloblin fraction (Dako) for
TNF-alpha on control sections from patients with myocarditis.
Statistical analysis. All data are presented as the mean
value ⫾ SEM. Statistically, the difference in TNF-alpha
and TACE expression levels between the myocarditis and
control groups was analyzed by using the unpaired t test.
Comparison of TNF-alpha and TACE mRNA expression
levels in serial biopsy samples was analyzed by using
two-way repeated measures analysis of variance. Peason’s
correlation coefficients were used to examine the relation
between levels of mRNA expression and clinical variables. A
value of p ⬍ 0.05 was considered statistically significant.
RESULTS
Quantitative RT-PCR. The clinical characteristics,
pathologic diagnosis and RT-PCR results of the myocarditis group are shown in Table 1. As shown in Figure 1, the
expression of TNF-alpha and TACE mRNA was significantly greater in the myocardial tissues of patients with
active myocarditis than in the tissues of control subjects
(TNF-alpha/GAPDH ratio 3.70 ⫾ 0.57 vs. 0.04 ⫾ 0.02,
p ⬍ 0.05; TACE/GAPDH ratio 2.44 ⫾ 0.48 vs. 0.03 ⫾
0.02, p ⬍ 0.05). There was a positive correlation between
the TACE/GAPDH ratio and TNF-alpha/GAPDH ratio
in the myocarditis group (r ⫽ 0.83, p ⬍ 0.05). In six
patients with severe myocarditis who underwent repeat
biopsy, although high levels of both TNF-alpha and TACE
mRNA were expressed in myocardial tissues obtained from
the initial biopsy when active myocarditis was present, both
TNF-alpha and TACE mRNA decreased markedly in the
repeat biopsies when cellular infiltration had become reduced and was intimately associated with necrotic myocytes
JACC Vol. 36, No. 4, 2000
October 2000:1288–94
Satoh et al.
Expression of TACE and TNF-alpha in Myocarditis
1291
Figure 1. Comparison of TACE/GAPDH (a) and TNF-alpha/GAPDH (b) levels in serial endomyocardial biopsy samples of patients and control subjects.
Both TNF-alpha/GAPDH and TACE/GAPDH mRNAs were expressed to a higher level in initial biopsy samples than in repeat biopsy samples
(TNF-alpha/GAPDH ratio 5.54 ⫾ 0.67 vs. 3.33 ⫾ 0.52, p ⫽ 0.03; TACE/GAPDH ratio 4.36 ⫾ 0.29 vs. 1.74 ⫾ 0.17, p ⬍ 0.01). MC ⫽ myocarditis.
(TNF-alpha/GAPDH ratio 5.54 ⫾ 0.67 vs. 3.33 ⫾ 0.52,
p ⫽ 0.03; TACE/GAPDH ratio 4.36 ⫾ 0.29 vs. 1.74 ⫾
0.17, p ⬍ 0.01) (Fig. 1). However, the repeat biopsy levels
were still higher in patients with myocarditis than in control
subjects (p ⬍ 0.05) (Fig. 1).
Immunohistochemical analysis. Immunohistochemical
analysis was performed to identify the source of TACE and
TNF-alpha protein within myocardial tissues. Immunostaining for both proteins was found in cardiac myocytes
and interstitial cells in myocardial tissues obtained from
patients with active myocarditis (Fig. 2a, c). Positive interstitial cells for TACE and TNF-alpha were predominantly
infiltrating mononuclear cells and macrophages. However,
both TACE and TNF-alpha immunostaining was markedly
decreased in cardiac myocytes and interstitial cells from
Figure 2. Immunohistochemical analysis for TNF-alpha and TACE in
serial endomyocardial biopsy tissues obtained from patients with myocarditis. Detection of TNF-alpha (a) and TACE (c) proteins with primary
and secondary antibodies in the initial biopsy. TNF-alpha and TACE
proteins were found in myocytes, infiltrating macrophages and mononuclear cells. Immunostaining of TNF-alpha (b) and TACE (d) was
markedly decreased in myocytes and interstitial cells in repeat biopsy.
Magnification for a and c, ⫻230; for b and d, ⫻240, reduced by XX%.
repeat biopsy tissues, coinciding with the healing stage (Fig.
2b, d). There was no evidence of nonspecific immunostaining in the myocardial tissues obtained from patients with
myocarditis. Neither TACE nor TNF-alpha immunostaining was present in any of the specimens from the control
group.
Comparison of clinical data. When patients with myocarditis were classified into two subgroups according to NYHA
functional class on hospital admission, both TACE and
TNF-alpha mRNA levels were expressed to a greater extent
in the subgroup in functional class III or IV (n ⫽ 8) than in
the subgroup in class I or II (n ⫽ 6) (TACE/GAPDH ratio
3.58 ⫾ 0.56 vs. 0.93 ⫾ 0.19, p ⫽ 0.02; TNF-alpha/
GAPDH ratio 5.04 ⫾ 0.59 vs. 1.92 ⫾ 0.44, p ⫽ 0.01) (Fig.
3). There was a positive correlation between left ventricular
end-systolic diameter and expression of both TNF-alpha
(r ⫽ 0.85, p ⬍ 0.05) and TACE mRNA (r ⫽ 0.80, p ⬍
0.05). The correlation with left ventricular ejection fraction
was negative for both proteins (TNF-alpha: r ⫽ ⫺0.85, p ⬍
0.05; TACE: r ⫽ ⫺0.85, p ⬍ 0.05). The differences in
TACE and TNF-alpha mRNA expression between the two
Figure 3. Comparison of TACE (a) and TNF-alpha (b) mRNA expression according to NYHA functional class. TNF-alpha and TACE mRNAs
were expressed to a higher level in the myocarditis subgroup in functional
class III or IV than in the subgroup in class I or II (TACE/GAPDH ratio
3.58 ⫾ 0.56 vs. 0.93 ⫾ 0.19, p ⫽ 0.02; TNF-alpha/GAPDH ratio 5.04 ⫾
0.59 vs. 1.92 ⫾ 0.44, p ⫽ 0.01).
1292
Satoh et al.
Expression of TACE and TNF-alpha in Myocarditis
Figure 4. Comparison of TACE (a) and TNF-alpha (b) mRNA expressions in two myocarditis subgroups classified according to maximal CK
concentration levels. TACE and TNF-alpha expression levels were significantly higher in the subgroup with CK ⬎500 IU/liter than in the subgroup
with CK ⬍500 IU/liter (TACE/GAPDH ratio 3.84 ⫾ 0.57 vs. 1.04 ⫾
0.20, p ⫽ 0.006; TNF-alpha/GAPDH ratio 5.29 ⫾ 0.62 vs. 2.12 ⫾ 0.42,
p ⫽ 0.001).
subgroups with myocarditis, classified by peak plasma concentration of creatine kinase (CK), are shown in Figure 4.
The subgroup with peak plasma CK ⬎500 IU/liter (n ⫽ 7)
had a significantly higher level of TACE and TNF-alpha
mRNA expression than the subgroup with CK ⬍500
IU/liter (n ⫽ 7) (TACE/GAPDH ratio 3.84 ⫾ 0.57 vs.
1.04 ⫾ 0.20, p ⫽ 0.006; TNF-alpha/GAPDH ratio 5.29 ⫾
0.62 vs. 2.12 ⫾ 0.42, p ⫽ 0.001) (Fig. 4). There was no
statistical relation between TACE and TNF-alpha mRNA
expression levels and other clinical variables representing an
inflammatory reaction (erythrocyte sedimentation rate, leukocyte and C-reactive protein) and cardiac enzymes (glutamic oxaloacetic transaminase and lactic dehydrogenase).
DISCUSSION
In this study, we have demonstrated that both TACE and
TNF-alpha mRNAs and proteins are expressed in the
myocardium of patients with myocarditis. A positive correlation was found between TACE and TNF-alpha mRNA
expression. Interestingly, TACE and TNF-alpha mRNAs
were expressed to a high level in advanced cardiac dysfunction in patients with myocarditis. Immunohistochemical
analysis revealed that the cellular source of TACE and
TNF-alpha proteins was not only infiltrating macrophages
and mononuclear cells but also cardiac myocytes. These
findings strongly suggest that myocardial expression of
TACE and TNF-alpha may be significantly implicated in
the myocardial damage that occurs during the inflammatory
process associated with myocarditis.
Myocardial expression of TACE and TNF-alpha. An
animal model of viral myocarditis has demonstrated that
proinflammatory cytokines, such as interleukin-1 and TNFalpha, are implicated in the pathogenesis of myocarditis in
the acute phase (4,5). A previous study has reported that the
JACC Vol. 36, No. 4, 2000
October 2000:1288–94
proinflammatory cytokines, especially TNF-alpha, are persistently expressed in myocarditis and dilated cardiomyopathy (11). Kubota et al. (22) and Bryant et al. (23) have
described a transgenic mouse model of myocarditis in which
TNF-alpha is expressed, particularly in cardiac myocytes. In
these models, the mice developed gross lymphocytic infiltration and myocytic necrosis that pathologically resembled
human active myocarditis. In addition, biventricular dilation
and cardiac contractile dysfunction were noted. Therefore,
these reports have strongly suggested that TNF-alpha
overexpression is sufficient to cause cardiac morphologic
changes and dysfunction, confirming the role of this protein
in human diseases, such as myocarditis and dilated cardiomyopathy. However, the mechanism of TNF-alpha production
in human myocarditis has not been ascertained. Recently,
the protease responsible for the shedding of pro-TNF-alpha
was identified and cloned (13,14). This protease—TACE—
belongs to the family of metalloproteinase disintegrin proteins (24,25). It has also been suggested that TACE is
expressed ubiquitously and that several TACE knock-out
cell types lose TNF-alpha processing activity, indicating
that TACE is responsible for pro-TNF-alpha shedding in
various cell types such as monocytes, T cells, neutrophils
and endothelial cells (13). In this study, TACE mRNA and
protein were expressed with TNF-alpha expression in myocardial tissues obtained from patients with myocarditis. The
cellular source of TACE and TNF-alpha proteins was
found to be not only infiltrating macrophages and lymphocytes but also cardiac myocytes. There was a positive
correlation between TACE and TNF-alpha mRNA expression. Although this study could not confirm whether increased expression of TACE has a direct causal relation to
the inflammatory process associated with myocarditis, several experimental studies have reported that inflammatory
conditions, such as endotoxin challenge and bacterial lipopolysaccharide stimulation on culture monocytes, mediated
TACE-like metalloproteinase overexpression, and that
metalloproteinase stimulated TNF-alpha production
(26,27). Therefore, these findings suggest that TACE may
induce TNF-alpha expression in cardiac myocytes and
infiltrating cells, such as macrophages and mononuclear
cells, during the paracrine or autocrine inflammatory process, or both.
Comparison between mRNA expressions and clinical
variables. One of the most important findings of this study
was a significantly elevated level of expression of TACE and
TNF-alpha mRNAs in patients with myocarditis with a
greater left ventricular volume and a lower left ventricular
ejection fraction. The myocarditis subgroup with high CK
levels revealed high expression of TACE and TNF-alpha
mRNAs. Although our previous study demonstrated that
TNF-alpha mRNA was expressed to a high level with
TACE expression in clinically advanced dilated cardiomyopathy (19), TNF-alpha mRNA in patients with severe
myocarditis was expressed at more than twice the level seen
in patients with advanced dilated cardiomyopathy. Both
JACC Vol. 36, No. 4, 2000
October 2000:1288–94
Satoh et al.
Expression of TACE and TNF-alpha in Myocarditis
1293
REFERENCES
Figure 5. The hypothetical role of TACE in myocarditis. First, viral
infection or autoimmune dysfunction, or both, occurred in myocardial
tissues. These immune responses in infiltrating inflammatory cells (infiltrating macrophages and lymphocytes) and cardiac myocytes mediated
expression of TACE and pro-TNF-alpha. Activated TACE cleaved from
pro-TNF-alpha to its mature form. The mature form of TNF-alpha
induced myocytic injury and left ventricular dysfunction through the
paracrine or autocrine process, or both.
TACE and TNF-alpha mRNA expression levels had decreased in repeat biopsy samples, but these levels were high
as compared with those in control subjects. These findings
suggest that the inflammatory process of TACE-mediated
TNF-alpha production may induce substantial myocytic
injury and impair cardiac systolic function. Studies by
Yokoyama et al. (28) and Bozkurt et al. (29) have reported
that TNF-alpha has the negative inotropic effects of myocytic injury and ventricular remodeling in cultured myocytes
in the experimental TNF-alpha infusion model. It has also
been reported that TNF-alpha–activated cytotoxic T cells
may cause direct myocytic injury (30,31). These observations imply that TACE expression may be related to
TNF-alpha expression during the inflammatory process of
myocarditis. Our speculation about the TACE-activated
pathway in myocarditis is shown in Figure 5. Myocardial
expression of TACE and TNF-alpha may play an important role in advanced cardiac dysfunction in human myocarditis. We speculate that TACE may be a potentially
important factor in the establishment of a new pathologic
concept of myocarditis and may also be a novel target for
therapeutic intervention in the inflammatory process of
myocarditis.
Conclusions. These findings indicate that the expression
of TNF-alpha and TACE may have important implications
in the pathogenesis of myocarditis and may induce advanced
cardiac dysfunction.
Reprint requests and correspondence: Dr. Mamoru Satoh, Second Department of Internal Medicine, Iwate Medical University
School of Medicine, 19-1 Uchimaru, Morioka 020-8505, Iwate,
Japan. E-mail: [email protected].
1. Woodruff JF. Viral myocarditis: a review. Am J Pathol 1980;101:
427–9.
2. Bowles NE, Richardson PJ, Olsen EGJ, Archard LC. Detection of
coxsackie-B-virus–specific RNA sequence in myocardial biopsy samples from patients with myocarditis and dilated cardiomyopathy.
Lancet 1986;1:1120 –2.
3. Satoh M, Tamura G, Segawa I. Enteroviral RNA in endomyocardial
biopsy tissues of myocarditis and dilated cardiomyopathy. Pathol Int
1994;44:345–51.
4. Seko Y, Takahashi N, Yagita H, Okumura K, Yazaki Y. Expression of
cytokine mRNAs in murine hearts with acute myocarditis caused by
coxsackievirus B3. J Pathol 1997;183:105– 8.
5. Shioi T, Matsumori A, Sasayama S. Persistent expression of cytokine
in the chronic stage of viral myocarditis in mice. Circulation 1996;94:
2930 –7.
6. Torre-Amione G, Kapadia S, Lee J, et al. Tumor necrosis factor-alpha
and tumor necrosis factor receptors in the failing human heart.
Circulation 1996;93:709 –11.
7. Habib FM, Springall DR, Davies GJ, Oakley CM, Yacoub MH,
Polak JM. Tumor necrosis factor and inducible nitric oxide synthase in
dilated cardiomyopathy. Lancet 1996;347:1151–5.
8. Vilcek J, Lee TH. Tumor necrosis factor: new insights into the
molecular mechanisms of its multiple actions. J Biol Chem 1991;266:
7313– 6.
9. Levine B, Kalman J, Mayer L, Fillit HM, Packer M. Elevated
circulating levels of tumor necrosis factor in severe chronic heart
failure. N Engl J Med 1990;323:236 – 41.
10. Bowles NE, Towbin JA. Molecular aspects of myocarditis. Curr Opin
Cardiol 1998;13:179 – 84.
11. Satoh M, Tamura G, Segawa I, Tashiro A, Hiramori K, Satodate R.
Expression of cytokine genes and presence of enteroviral genomic
RNA in endomyocardial biopsy tissues of myocarditis and dilated
cardiomyopathy. Virchow Arch 1996;427:503–9.
12. Satoh M, Nakamura M, Tamura G, et al. Inducible nitric oxide
synthase and tumor necrosis factor-alpha in myocardium in human
dilated cardiomyopathy. J Am Coll Cardiol 1997;29:716 –24.
13. Black RA, Rauch CT, Kozlosky CJ, et al. A metalloproteinase
disintegrin that releases tumour necrosis factor-␣ from cells. Nature
1997;385:729 –33.
14. Moss ML, Jin S-LC, Milla ME, et al. Cloning of a disintegrin
metalloproteinase that processes precursor tumour necrosis factor-␣.
Nature 1997;385:733– 6.
15. Aretz HT, Billingham ME, Edward WD, et al. Myocarditis: a
histopathologic definition and classification. Am J Cardiovasc Pathol
1986;1:3–14.
16. Chomcynski P, Sacchi N. Single-step method of RNA isolation by
acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156 –9.
17. Shirai T, Yamaguchi H, Ito H, Todd CW, Wallace RB. Cloning and
expression in Escherichia coli of the gene for human tumour necrosis
factor. Nature 1985;313:803– 6.
18. Ercolani L, Florence B, Denaro M, Alexander M. Isolation and
complete sequence of a functional glyceraldehyde-3-phosphate dehydrogenase gene. J Biol Chem 1988;263:15335– 41.
19. Satoh M, Nakamura M, Saitoh H, et al. Tumor necrosis factor-␣–
converting enzyme and tumor necrosis factor-␣ in human dilated
cardiomyopathy. Circulation 1999;99:3260 –5.
20. Gibson UEM, Heid CA, Williams PM. A novel method for real time
quantitative RT-PCR. Genome Res 1996;6:995–1001.
21. Fink L, Seeger W, Ermert L, et al. Real-time quantitative RT-PCR
after laser-assisted cell picking. Nature Med 1998;4:1329 –33.
22. Kubota T, McTiernan CF, Frye CS, et al. Dilated cardiomyopathy in
transgenic mice with cardiac-specific overexpression of tumor necrosis
factor-␣. Circ Res 1997;81:627–35.
23. Bryant D, Becker L, Richardson J, et al. Cardiac failure in transgenic
mice with myocardial expression of tumor necrosis factor-␣. Circulation 1998;97:1375– 81.
24. Maskos K, Fernandez-Catalan C, Huber R, et al. Crystal structure of
the catalytic domain of human tumor necrosis factor-␣– converting
enzyme. Proc Natl Acad Sci USA 1998;95:3408 –12.
25. Merlos-Suarez A, Fernandez-Larrea J, Reddy P, Baselga J, Arribas J.
Pro–tumor necrosis factor-␣ processing activity is tightly controlled by
1294
Satoh et al.
Expression of TACE and TNF-alpha in Myocarditis
a component that does not affect notch processing. J Biol Chem
1998;273:24955– 62.
26. Gearing AJ, Beckett P, Christodoulou M, et al. Processing of tumour
necrosis factor-alpha precursor by metalloproteinases. Nature 1994;
370:555–7.
27. Mohler KM, Sleath PR, Fitzner JN, Cerretti DP, et al. Protection
against a lethal dose of endotoxin by an inhibitor of tumour necrosis
factor processing. Nature 1994;370:218 –20.
28. Yokoyama T, Vaca L, Rossen RD, Durante W, Hazarika P, Mann DL.
Cellular basis for the negative inotropic effects of tumor necrosis factor-␣
in the adult mammalian heart. J Clin Invest 1993;92:2303–12.
JACC Vol. 36, No. 4, 2000
October 2000:1288–94
29. Bozkurt B, Kribbs SB, Clubb FJ Jr., et al. Pathophysiologically
relevant concentrations of tumor necrosis factor-␣ promote progressive
left ventricular dysfunction and remodeling in rats. Circulation 1998;
97:1382–91.
30. Woodley SL, McMillan M, Shelby J, et al. Myocyte injury and
contraction abnormalities produced by cytotoxic T lymphocytes. Circulation 1991;83:1410 – 8.
31. Seko Y, Ishiyama S, Nishikawa T, et al. Restricted usage of T cell
receptor V alpha–V beta genes in infiltrating cells in the hearts of
patients with acute myocarditis and dilated cardiomyopathy. J Clin
Invest 1995;96:1035– 41.