Tumor necrosis factor- (TNF-) like weak inducer of apoptosis (TWEAK, also named TNFsf 12) is a member of the TNF-family of cytokines with multifunctional properties [
Recently, a role for sTWEAK in cardiac pathophysiology has been proposed [
Therefore, we conducted a follow-up study in a cohort of stable patients with dilated cardiomyopathy to evaluate the usefulness of serial sTWEAK determinations and their intraindividual variation over time in relation to their symptoms. Patients were seen on a yearly basis in our outpatient clinic with serial determination of clinical, functional, and laboratory parameters with follow-up assessment after four years.
A cohort of seventy-eight stable patients diagnosed with dilated cardiomyopathy were identified and followed on a yearly basis in the heart failure clinic of our university hospital that serves as a tertiary referral center in southern Germany. Eligible patients had to have the diagnosis of dilated cardiomyopathy, to be at least 18 years of age, and to have a reduced left ventricular systolic function with an ejection fraction of less than 50% on echocardiographic evaluation. All examinees enrolled in the present study had to have a stable clinical condition and medication for at least 1 month before inclusion. Patients with malignant or inflammatory diseases, acutely decompensated heart failure (NYHA class IV), a history of organ transplantation, or significant acute/chronic renal failure (serum creatinine > 2 mg/dL) were not included in our study. Coronary angiography had been performed in all patients. Patients with significant coronary artery disease, considered to be responsible for the reduced left ventricular function, were also excluded from our study. Blood samples of these patients were taken on 3 consecutive follow-up visits once a year in addition to clinical and functional evaluation of the patients including a 6-minute walk test (6MWT) and a peak oxygen consumption study (peak VO2max). Soluble TWEAK, sCD163, and NT-proBNP levels were determined in different aliquots of the same blood sample. All patients included in our study completed a four-year follow-up visit or a telephone call. All patients gave written consent.
Blood samples were drawn in all patients once a year during a follow-up visit on three consecutive occasions. Plasma samples were generated within 30 minutes of collection by centrifugation at 1000 ×g for 10 minutes at 4°C. To avoid repetitive freeze-and-thaw cycles, aliquots were generated, immediately frozen, and stored at −80°C until analysis. Plasma samples were taken as part of a multiple biomarker registry project in patients with dilated cardiomyopathy and heart failure, for which only serial plasma samples were available. Soluble TWEAK levels were determined with an ELISA by use of a commercially available kit tested for determination of human plasma samples (Bender Medsystems, Vienna, Austria) according to the manufacturer’s instructions. Briefly, a 1 : 2 diluted test sample was incubated for 3 hours at room temperature in wells precoated with an anti-human sTWEAK antibody together with a biotin-conjugated anti-human TWEAK antibody which binds to human TWEAK captured by the first antibody. Streptavidin-HRP binds to the biotin conjugated anti-human TWEAK. Following incubation unbound biotin conjugated anti-human TWEAK and streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells and wells are incubated for approximately 10 to 20 minutes. A colored product is formed in proportion to the amount of soluble human TWEAK present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A standard curve is prepared from 7 human TWEAK standard dilutions and human TWEAK sample concentration is determined. Absorbance was measured with an automatic ELISA reader (Tecan Spectra, Crailsheim, Germany). Human sTWEAK is detected with this kit at a threshold of 9.7 pg/mL. Intra-assay and interassay coefficients of variation were 7.9% and 9.2%, respectively, according to the manufacturer. All measurements were performed in duplicate by an investigator unaware of patients’ characteristics and outcome.
Soluble CD163 levels were determined with the MacroCD163 ELISA assay (IQ Products, Trillium Diagnostics). Soluble CD163 was only determined once at the time point of study initiation from the same sample as the first sTWEAK value was determined. Briefly, a polyclonal antibody recognizing CD163 is immobilized on the surface of a microtiter plate. After incubation with the sample or the standard containing recombinant CD163, a second biotinylated monoclonal antibody recognizing CD163 is added. Detection of the latter is done by adding streptavidin-HRP. Using TMB (3,3′,5,5′-tetramethylbenzidine) as a substrate for HRP, the amount of CD163 in the sample can be determined. Intra-assay variability of the assay is 3–6%; interassay variability is 5–8%, when measuring duplicates according to the manufacturer. As with sTWEAK determination, all samples were measured in duplicate.
NT-proBNP was measured from different aliquots of the same plasma sample. Measurements were performed at the clinical core laboratory of the University Hospital Heidelberg with an ELISA (Roche Diagnostics, Mannheim, Germany).
Data are presented as median (interquartile range) or count and percentages. For all continuous variables that were not normally distributed (tested by the Kolmogorov-Smirnov test), log transformation was performed. ANOVA was used for comparison of 3 or more different groups. The optimal plasma sTWEAK cutoff values to predict an adverse outcome in the present study population were calculated by a receiver operating characteristic (ROC) curve analysis. Estimates of the cumulative event rate were evaluated by the Kaplan-Meier method. Patients were compared according to the sTWEAK cutoff value with the use of log-rank test for the 4-year survival curves. Probability values < 0.05 were considered statistically significant. Statistical analyses were performed with Prism 5.0 (GraphPad Software, San Diego, California) and MedCalc 9.3.0.0 (MedCalc, Mariakerke, Belgium) software.
We serially analyzed a cohort of 78 patients diagnosed with dilated cardiomyopathy and evaluated sTWEAK levels, NT-proBNP levels, and functional parameters on a yearly outpatient basis in our heart failure clinic. The median ejection fraction (EF) of our patient cohort was 29% (interquartile range (IR) of 20 to 48%) and the median left ventricular end-diastolic diameter (LVEDD) was 60 mm with an interquartile range of 52 to 74 mm at the time point of study initiation. Median age of our patients was 55 years (IR: 43 to 64 years). Median of sTWEAK at study start was 603 pg/mL (IR: 423 to 839 pg/mL) and that of NT-proBNP was 482 ng/L (IR: 211 to 1505). Twenty-eight patients (35.9%) had atrial fibrillation, but only 4 (5.1%) developed thromboembolic events during follow-up. Overall NYHA class of the patient cohort was inversely related to peak oxygen consumption (VO2max) and walking distance during a 6MWT (peak oxygen consumption (VO2max): 22.0 (IR: 16.6 to 27.2) versus 17.7 (IR: 14.3 to 20.0) versus 13.7 (IR: 12.2 to 17.6); 6MWT: 587 (IR: 518 to 638) versus 492 (IR: 430 to 559) versus 395 (IR: 311 to 467); NYHA class I versus II versus III each;
Characteristics of the study cohort with dilated cardiomyopathy (
Age (years) | 55 (43–64) |
Gender (m/f) | 56/22 |
Ejection fraction in % | 29.5 (20–45) |
Left ventricular end-diastolic diameter (in mm) | 60 (52–66) |
6-minute walk test (distance in m) | 503 (417–596) |
Peak oxygen consumption (L/min/kg) | 17.6 (14.5–23) |
NYHA class | |
Class I | 36 (46.1%) |
Class II | 37 (47.4%) |
Class III | 5 (6.4%) |
Class IV | 0 (0%) |
Medication | |
|
74 (94.9%) |
ACEI/AT antagonists | 77 (98.7%) |
Aldosterone antagonists | 41 (52.6%) |
Diuretics | 53 (67.9%) |
Glycosides | 23 (29.5%) |
Diabetes | 6 (7.7%) |
Creatinine (mg/dL) | 0.93 (0.8–1.2) |
sTWEAK in pg/mL | 603 (423–839) |
sCD163 in ng/mL | 2554 (1990–3479) |
NT-proBNP in ng/L | 482 (211–1505) |
Death/heart transplantation on 4-year follow-up | 4/1 |
Functional deterioration (≥1 NYHA class) | 41 (52.6%) |
Functional deterioration (≥2 NYHA classes) | 11 (14.1%) |
Values given as median (IR), number, or number (%).
In our patient cohort of stable patients with dilated cardiomyopathy, reported functional NYHA class correlated well with functional testing. With increasing NYHA class, peak oxygen consumption (VO2max) and walking distance in the 6-minute walk test (6 MWT) progressively declined (a), whereas NT-proBNP levels increased (b). In contrast, sTWEAK levels significantly declined with increasing NYHA class (b). (Box-Whisker-Plots;
Among our 78 patients, 4 deaths and 1 heart transplantation occurred within the 4-year follow-up period. Soluble TWEAK levels were lower in patients who died or were transplanted (372 pg/mL (239 to 405) versus 617 pg/mL (434 to 841); median (interquartile range);
Soluble TWEAK predicts a progressive course of the disease in patients with dilated cardiomyopathy. (a) Receiver operating curve (ROC) analysis of baseline sTWEAK levels in patients with dilated cardiomyopathy revealed a cutoff value of 423 pg/mL for the prediction of death or heart transplantation on serial follow-up. Kaplan-Meier curves differed significantly between patients with a sTWEAK level below 423 pg/mL and those with an above level. (b) In those patients with significant deterioration during serial follow-up (
We further compared those patients who reported later a significant deterioration of their functional capacity (≥1 NYHA class) over time to those with a stable condition on follow-up. In patients with progressive symptoms, sTWEAK levels were already significantly reduced at the first visit with a median of 432 pg/mL (IR: 322 to 568 pg/mL) (
Serial evaluation of sTWEAK levels allowed us to analyze whether sTWEAK showed intraindividual variations dependent on the actual clinical condition similar to the use of NT-proBNP. However, soluble TWEAK levels showed little variation in most patients throughout the follow-up period, no matter whether they developed progressive worsening of their clinical and functional parameters or not. Soluble TWEAK levels remained significantly lower in patients with deterioration compared to those with stable symptoms, indicating that interindividual rather than intraindividual differences predicted clinical deterioration (Figure
Interindividual differences in sTWEAK represent a risk factor for outcome in patients with dilated cardiomyopathy. To analyze the usefulness of sTWEAK as a biomarker in patients with dilated cardiomyopathy, we performed at least three serial sTWEAK measurements within all patients of our cohort. Interestingly, sTWEAK not only differed significantly between those patients with a progressive course of the disease and those with a stable course, but surprisingly remained very stable within individual patients with progressive deterioration, indicating that sTWEAK might represent a risk factor for disease progression rather than a biomarker for disease monitoring (
Recently, the ratio between sCD163 and sTWEAK has been proposed to improve the predictive value of sTWEAK levels in patients with various clinical conditions [
Here, we report for the first time the results of a serial study of sTWEAK levels in a cohort of patients with dilated cardiomyopathy. Similar to previous reports, a single sTWEAK determination predicted long-term outcome in stable patients with dilated cardiomyopathy and reduced left ventricular function. In contrast to NT-proBNP, we found that sTWEAK showed little variation on serial determination within individual patients, independently of any clinical or functional deterioration. Thus, sTWEAK values differed significantly at baseline between individuals with subsequent progressive clinical deterioration and those with a stable clinical course. Moreover, sCD163 levels did not add additional information for outcome in our patient cohort. Finally, sCD163 did not correlate to the reduction of sTWEAK levels in patients with dilated cardiomyopathy.
The natural disease progression in patients with dilated cardiomyopathy can be quite variable despite similar echocardiographic and hemodynamic parameters. We and others have recently reported that reduced sTWEAK levels predicted long-term outcome in patients with dilated cardiomyopathy [
Our study adds additional evidence that sTWEAK levels may not be useful for disease monitoring but may rather be related to an increased risk for disease progression in a subset of patients. This is of potential interest in the setting of dilated cardiomyopathy, since sTWEAK has also been related to several autoimmune disorders and vice versa autoimmunity has been suggested to play a role in the pathogenesis of patients with dilated cardiomyopathy. For example, autoantibodies against several molecular targets in the heart and additional features of immune activation like cytokine activation and intramyocardial inflammation are detectable even in apparently healthy relatives of patients with dilated cardiomyopathy [
Whether sTWEAK plays a role in the pathogenesis of dilated cardiomyopathy or whether the association between reduced levels and outcome represents just an epiphenomenon of changes in the regulation of the immune system remains to be determined. However, recent experimental data suggest that sTWEAK directly influences cardiac pathology. Jain et al. have reported the occurrence of dilated cardiomyopathy in transgenic animals overexpressing TWEAK [
In contrast to sFn14, the ratio between sTWEAK and sCD163 has been described to be of value in other clinical settings, although a significant interaction between both molecules has been questioned by others [
Absolute values of sTWEAK levels in healthy control patients have been reported in several studies using different detection assays in the past reviewed in [
We cannot completely rule out all confounders; nevertheless, our relatively small study cohort consisted of a homogenous patient population: coronary angiography was used in all patients to exclude significant coronary artery disease, which might have affected sTWEAK measurements. All patients included had three consecutive visits with determination of sTWEAK levels and functional testing and all of them completed 4-year follow-up. Almost all patients were on
In summary, our study is the first to provide data on serial sTWEAK determination in combination with serial clinical and functional assessment to evaluate the potential of sTWEAK as a biomarker in the setting of dilated cardiomyopathy. As a result, we found that the reduction of sTWEAK levels in patients with dilated cardiomyopathy may not be of value for disease monitoring but may represent a risk factor for disease progression and death. Further research will be necessary to elucidate the exact role of sTWEAK as a potential modulator of immune response especially in the setting of dilated cardiomyopathy.
The authors declare that there is no conflict of interests regarding the publication of this paper.