Tumor Necrosis Factor-α–Converting Enzyme and Tumor Necrosis Factor-α in Human Dilated Cardiomyopathy
Background—Tumor necrosis factor-α (TNF-α) has been implicated in the pathogenesis of dilated cardiomyopathy (DCM). TNF-α–converting enzyme (TACE) has recently been purified and its complementary DNA cloned. The expression of TACE results in the production of a functional enzyme that has precursor TNF-α in the mature form. The aim of this study was to determine whether TACE is expressed with TNF-α in myocardium and whether levels of TACE and TNF-α are related to clinical severity of DCM.
Methods and Results—Endomyocardial tissues were obtained from 30 patients with DCM and 5 control subjects. TNF-α and TACE mRNA levels were measured by a novel real-time quantitative reverse transcriptase–polymerase chain reaction method. Expression of TNF-α and TACE proteins was determined by immunohistochemical analysis. TNF-α mRNA was expressed in DCM patients (TNF-α/GAPDH ratio 0.85±0.24) but not in control subjects. TACE mRNA expression was significantly greater in DCM patients than in control subjects (TACE/GAPDH ratio 2.52±0.59 vs 0.03±0.02, P<0.05). A positive correlation was found between TNF-α and TACE mRNA levels (r=0.779, P<0.001). TACE and TNF-α immunostaining was observed in myocytes in patients with DCM. When 2 subgroups of DCM were divided on the basis of left ventricular end-systolic diameter (LVESD) of 45 mm and left ventricular ejection fraction (LVEF) of 40%, the DCM subgroup with high LVESD (≥45 mm) showed significantly greater expression of TACE (P=0.02) and TNF-α (P=0.001) than did the low LVESD subgroup (<45 mm). In addition, the DCM subgroup with lower LVEF (<40%) showed higher expression of TACE (P=0.006) and TNF-α (P=0.01) than did the subgroup with high LVEF (≥40%).
Conclusions—This study has shown that increased myocardial TACE expression is associated with elevated myocardial TNF-α expression in both mRNA and protein levels in clinically advanced DCM.
Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine that contributes to cellular immunity and inflammatory reaction in a range of inflammatory diseases.1 Several studies suggest that the expression of TNF-α in myocardium plays an important role in the progression of dilated cardiomyopathy (DCM).2 3 4 Our previous study demonstrated that cardiomyocytes expressed excessive quantities of TNF-α and nitric oxide in human DCM.5 Kubota et al6 and Bryant et al7 reported that cardiac-specific overexpressing TNF-α transgenic mice showed decreased ventricular function and developed heart failure and increased mortality rates. TNF-α–converting enzyme (TACE) has recently been purified and cloned as a metalloproteinase disintegrin that specifically cleaves precursor TNF-α.8 9 The enzyme also releases and activates mature 17-kDa TNF-α into the extracellular space. However, it is unclear whether both TACE and TNF-α are expressed in myocardial tissue in DCM. In this study, we examined the expression of TACE and TNF-α in endomyocardial tissues of patients with DCM and control subjects by using a novel real-time 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-α.
Endomyocardial biopsies were obtained from 30 patients with DCM, and these tissues were examined. The DCM group included 23 men and 7 women with a mean age of 59±3 years (range 15 to 79 years). DCM was diagnosed according to the criteria of the World Health Organization and the International Society and Federation of Cardiology Task Force.10 Control myocardial tissue samples were obtained by endomyocardial biopsy from 5 subjects (4 men and 1 woman; mean age 46±13 years) suspected of cardiac disorder because of minor ECG and echocardiographic changes but showing no evidence of myocardial disease or other abnormality on morphological examination and cardiac catheterization. To confirm compatibility of expression of TACE and TNF-α between left and right ventricles, we compared expression in 3 pairs of samples obtained by biventricular endomyocardial biopsy. Consistency of expression from sample to sample was established by comparing 14 pairs of right ventricular endomyocardial biopsy samples from DCM patients. These study protocols were approved by our hospital ethics committee, and written informed consent was obtained from all subjects.
Positive Control Cells for TACE and TNF-α mRNA
Peripheral blood monocytes were prepared from normal donors. The monocytes were washed and resuspended in AIM-R medium (GIBCO BRL) supplemented with 2 mg of anti-human LeuTM-4 (CD3) (Becton Dickinson). They were then allowed to adhere to tissue culture flasks for 48 hours 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).11 The purity of extracted total RNA was confirmed by determining the ratio of absorbance at 260 nm to that at 280 nm. The extracted RNA was diluted to 100 ng/μL with double-distilled water. TaqMan glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control reagents were used for fluorogenic detection of human GAPDH transcript (Perkin-Elmer Applied Biosystems Division).
Oligonucleotides of Primers and Probes
Published cDNA sequences for human TNF-α,12 TACE,8 and GAPDH13 were used for primer and probe construction. The following primers and probes were used for relative quantification of targeted gene expression: for TNF-α: forward primer 5′-CTT CTC 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′-CCA TGA 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 GGT CGG AGT-3′, reverse primer 5′-GAA GAT GGT GAT GGG ATT TC-3′, and probe 5′-CAA GCT TCC CGT TCT CAG CC-3′. The PCR products of TNF-α, TACE, and GAPDH were amplified at 266, 190, and 226 bp, respectively.
Real-Time Quantitative RT-PCR
cDNA was synthesized from 100 ng of total RNA and 10-fold serial dilutions of human control RNA (Perkin-Elmer) by RT at 42°C for 30 minutes with the use of random hexamers and MuLV RT (Perkin-Elmer). PCR was performed with a TaqMan PCR core kit (Perkin-Elmer). The cDNA products were amplified with 40 cycles of PCR, with each cycle consisting of first denaturation at 95°C for 10 minutes, denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 30 seconds. A novel quantitative PCR method was developed with the use of real-time detection and 5′ nuclease assay by an ABI PRISM 7700 sequence detector (Perkin-Elmer).14 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.14 The probe was labeled with reporter fluorescent dye (6-carboxyfluorescein) at the 5′ end and a quencher fluorescent dye (6-carboxy-tetramethyl-rhodamine) at the 3′ end. When the probe is intact, reporter dye emission is quenched by the physical proximity of the reporter and quencher fluorescent dyes. However, during the extension phase of the PCR cycle, the nucleolytic activity of the DNA polymerase cleaves the hybridization probe and releases the reporter dye from the probe. Nuclease degradation of the hybridization probe quenched 6-carboxyfluorescein fluorescent emission, resulting in an increase in peak fluorescent emission at 518 nm.14 The ABI PRISM 7700 sequence detector measured fluorescent emission synchronized with the thermal cycler during each extension step. Relative quantification of GAPDH, TNF-α, and TACE mRNA was calculated by the comparative cycle threshold method outlined in user bulletin No. 2 provided by Perkin-Elmer.
Immunohistochemical analysis was performed on paraffin sections to investigate the source of TACE and TNF-α production. Monoclonal antibody against TACE, TACE-M220 (1:100, Immunex), and polyclonal rabbit anti-human TNF-α (1:1000, Genzyme) were used as primary antibodies. The tissue sections were deparaffinized with xylene for 20 minutes 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) was used as a secondary antibody. Peroxidase-labeled streptavidin (Histofine, SAB-PO kit, Nichiren Corp) was applied and visualized with the use of diaminobenzidine as a chromogen. The specificity of the immunohistochemistry was confirmed by substituting the primary antibodies with mouse IgG1 negative control (Dako) for TACE and rabbit immunoglobulin fraction (Dako) for TNF-α on control sections from patients with DCM.
All values are presented as mean±SE. Statistically, the difference in TNF-α and TACE expression levels between the DCM and control groups was analyzed by unpaired t test. Comparison between TNF-α and TACE mRNA expression levels was analyzed by Fisher’s test. Pearson’s correlation coefficients were used to examine the relation between mRNA expression level and clinical parameters. A value of P<0.05 was considered statistically significant.
PCR Detection in Real Time
An amplification plot is shown in Figure 1a⇓. Amplification was performed on a 10-fold serial diluted human control RNA, and the target was human GAPDH. The calculation of a value termed ΔRn used the following equation: ΔRn=Rn+−Rn−, where Rn+=emission intensity of reporter/emission intensity of quencher at any given time in the reaction tube, and Rn−=emission intensity of reporter/emission intensity of quencher measured before PCR amplification in the same reaction tube. During early cycles of PCR amplification, ΔRn values remained at baseline. When sufficient hybridization probe had been cleaved, intensity of reporter fluorescent emission increased. Most PCR amplifications reached a plateau phase of reporter fluorescent emission at high cycle numbers. The amplification plot was examined at a point representing the log phase of product accumulation. This was done by assigning an arbitrary threshold based on the variability of baseline data. When the threshold was chosen, the point at which the amplification plot crossed the threshold was defined as threshold cycle (CT). CT was shown as the cycle number at this point. Figure 1b⇓ shows linearity of dilution determined by making 10-fold serial dilutions of control RNA. The CT value was therefore predictive of the quantity of target sequence.
Expression of TACE and TNF-α mRNA
RT-PCR of positive controls revealed a single band corresponding to the size of TNF-α (266 bp), GAPDH (226 bp), and TACE (190 bp) (Figure 2⇓). There was a statistically significant rise in the level of TACE mRNA expression in right ventricular myocardium in DCM compared with control subjects (TACE/GAPDH ratio 2.52±0.59 vs 0.03±0.02, P<0.05). The expression of TNF-α mRNA was detected in right ventricular myocardium of DCM (TNF-α/GAPDH ratio 0.85±0.24) but not in control subjects. Figure 3⇓ shows the association between TACE/GAPDH ratio and TNF-α/GAPDH ratio in the DCM group. The plots show a strong and significant correlation (r=0.779, P<0.001). The mRNA expression of TACE and TNF-α showed no significant differences between right and left ventricular myocardium obtained from DCM (TACE/GAPDH ratio 2.87±1.75 vs 2.91±0.82, TNF-α/GAPDH ratio 1.80±1.55 vs 1.57±1.42, respectively). When paired biopsy samples taken from 14 patients with DCM were examined, no difference in TACE and TNF-α mRNA expression levels was found (TACE/GAPDH ratio 3.04±0.91 vs 3.30±1.10, TNF-α/GAPDH ratio 1.14±0.46 vs 1.21±0.47, respectively).
Immunohistochemical analysis was performed to identify the source of TACE and TNF-α protein in myocardial tissues. TACE immunostaining was found in the cytoplasm of cardiac myocytes in 16 patients with DCM (Figure 4A⇓). TNF-α immunostaining was localized not only in cardiac myocytes but also in endothelium (Figure 4C⇓). TNF-α immunostaining was observed in 14 biopsy tissues obtained from patients with DCM who showed positive myocardial TACE protein. Both TACE and TNF-α immunostaining were also found in cardiac myocytes of left ventricular tissues as well as in right ventricular samples. Figures 4B⇓ and 4D⇓ show an absence of nonspecific immunostaining in the myocardial tissues obtained from patients with DCM. The DCM subgroups with TACE and TNF-α proteins showed significantly higher expression of TACE and TNF-α mRNA than did subgroups without TACE and TNF-α proteins (TACE/GAPDH ratio 4.07±0.94 vs 0.75±0.19, P<0.05, TNF-α/GAPDH ratio 1.54±0.44 vs 0.24±0.11, P<0.05, respectively). Neither TACE nor TNF-α immunostaining was present in any of the specimens from the control group.
Comparison of Clinical Data
The differences in myocardial expression of TACE and TNF-α mRNA between the 2 subgroups of DCM divided by clinical parameters of left ventricular end-systolic diameter (LVESD) (45 mm) and left ventricular ejection fraction (LVEF) (40%) are shown in the Table⇓. The DCM subgroup with LVESD≥45 mm yielded a significantly higher expression of TACE and TNF-α mRNA (TACE/GAPDH ratio 4.20±0.91 vs 0.59±0.13, P=0.001; TNF-α/GAPDH ratio 1.31±0.41 vs 0.32±0.10, P=0.04) than did the subgroup with LVESD<45 mm. The DCM subgroup with LVEF<40% showed a significantly higher expression of TACE and TNF-α mRNA (TACE/GAPDH ratio 4.98±1.16 vs 1.10±0.37, P=0.006; TNF-α/GAPDH ratio 1.82±0.53 vs 0.28±0.10, P=0.01) than did the subgroup with LVEF≥40%. LVESD was correlated positively with mRNA expression of TACE (r=0.61, P<0.01; Figure 5a⇓) and TNF-α (r=0.41, P<0.05; Figure 5b⇓). Similarly, LVEF was significantly negatively correlated with mRNA expression of both TACE (r=−0.77, P<0.001; Figure 5c⇓) and TNF-α (r=−0.49, P<0.05; Figure 5d⇓).
This study has demonstrated that myocardial expression of TACE mRNA and protein was significantly increased in DCM patients, and these levels were associated with increased levels of TNF-α mRNA and protein in advanced DCM. It has been suggested that inflammatory cytokines such as interleukins and TNF-α induced by viral replication and autoimmune mechanisms may be pathogenetic factors underlying DCM.15 16 If inflammatory processes do contribute to the pathogenesis of DCM, then cytokine production may be an important factor in the pathogenesis of DCM. Our previous study showed that TNF-α is expressed in cardiomyocytes in patients with DCM, who usually have advanced left ventricular dysfunction and large ventricular volume.5 Yokoyama et al17 reported that a negative inotropic effect was directly mediated by TNF-α expression in the adult mammalian myocardium and that this might be caused by decreased levels of intercellular calcium. Recently, Kubota et al6 and Bryant et al7 reported that a transgenic mouse model with myocardial overexpression of TNF-α developed a primary phenotype consistent with DCM, displaying both cardiac dilation and left ventricular dysfunction. These transgenic mice also developed a failing heart with pulmonary and liver congestion and increased mortality rates.6 7 These facts strongly imply that TNF-α production by cardiac myocytes in vivo plays an important pathophysiological role not only in the pathogenesis of DCM but also in its progression. There is a further implication that an increase in cardiac expression of TNF-α may contribute to ventricular dysfunction and remodeling. However, the mechanism for release and activation of TNF-α in cardiomyocyte in DCM has remained uncertain.
Recent studies report the purification and cloning of the metalloproteinase disintegrin (called TACE), which specifically cleaves precursor TNF-α from membrane-bound precursor and releases mature TNF-α.8 9 Black et al8 also reported that TACE allele–deficient mouse cells were required to determine the extent to which TACE accounts for the production of soluble TNF-α in vivo. McGeehan et al18 and Mohler et al19 reported that metalloproteinase inhibitors suppress TACE processing of TNF-α precursor and lipopolysaccharide-induced TNF-α production. TACE may therefore regulate the processing of posttranslational myristoylation promoting membrane insertion and interaction of TNF-α. In this study, myocardial expression of TACE mRNA was greater in DCM than in control subjects. There was a positive correlation between TACE and TNF-α expression in myocardium in human DCM. Immunohistochemical analysis demonstrated that the protein biosynthesis of TACE and TNF-α was predominantly in cardiomyocytes. The DCM subgroup with TACE and TNF-α protein showed higher expression of TACE and TNF-α mRNA than the subgroup without TACE and TNF-α protein. These findings suggest a link between increased TACE and TNF-α expression in myocardium in this disorder.
DCM subgroups with higher left ventricular volume and lower LVEF showed significantly higher mRNA expression of TACE and TNF-α than did the lower ventricular volume and higher ejection fraction DCM subgroups. In addition, both TACE and TNF-α expression levels were positively correlated with left ventricular volume and negatively correlated with left ventricular systolic function. Although this study could not show whether TNF-α was a direct cause of myocytic injury, Bozkurt et al20 have recently reported that continuous infusion of TNF-α led to a time-dependent depression of ventricular function with ventricular dilatation in the intact ventricle, possibly caused by TNF-α–induced apoptotic cell death in myocytes. Although we were unable to confirm that expression of TACE came before that of TNF-α, the present observations suggest that increased TACE levels may be related to TNF-α expression in myocardium, making TACE a potentially important factor in the establishment of a new pathological concept and therapeutic challenge for human DCM.
In conclusion, the present study has shown that increased myocardial TACE expression is associated with elevated myocardial TNF-α expression as reflected in both mRNA and protein levels in clinically advanced DCM.
This study was supported by a grant-in-aid for general scientific research from the Japanese Ministry of Education, Science, Sports, and Culture (No. 09670742) and grants from the Keiryoukai Research Foundation (No. 57 and 65).
- Received December 8, 1998.
- Revision received March 17, 1999.
- Accepted April 9, 1999.
- Copyright © 1999 by American Heart Association
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