Proinflammatory Cytokine Activation Is Linked to Apoptotic Mediator, Soluble Fas Level in Patients With Chronic Heart Failure Toru Kinugawa,1 MD, Masahiko Kato,2 MD, Kazuhiro Yamamoto,2 MD, Ichiro Hisatome,3 MD, and Ryuji Nohara,4 MD Summary The Fas/Fas Ligand system is a major apoptosis signaling pathway that is up-regulated in patients with chronic heart failure (CHF). Serum soluble Fas (sFas) levels increase in proportion to the CHF severity and may have prognostic value, therefore, sFas is a promising biomarker of heart failure. In this study, we attempted to identify the determinants of sFas levels in patients with CHF. Serum levels of tumor necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 & sTNF-R2), interleukin (IL)-6, soluble IL-6 receptor (sIL-6R), glycoprotein (gp)130, and sFas were measured in 106 pa- tients with CHF and 39 controls. All subjects performed a symptom-limited cycle ergometer exercise test with expired gas analysis. CHF patients had higher levels of TNF-α, sTNF-R1, sTNF-R2, IL-6, and gp130. Serum levels of sFas (con- trols versus CHF; 2.60 ± 0.88 versus 3.38 ± 1.23 ng/mL, P = 0.0004) were higher in CHF. On univariate analysis, age (P = 0.0003), NYHA functional class (P = 0.0012), peak VO2 (P < 0.0001), plasma norepinephrine (P = 0.0013), log IL-6 (P < 0.0001), log TNF-α (P = 0.0002), log sTNF-R1 (P < 0.0001), and log TNF-R2 (P < 0.0001) were significantly re- lated to log sFas levels. Multivariate analysis showed that age and log IL-6 and log sTNF-R1 levels were independently associated with log sFas levels (overall R = 0.603, P < 0.0001). Serum levels of sFas were increased in patients with CHF, and age and serum IL-6 and sTNF-R1 levels were independent determinants of sFas levels. These data suggest that proinflammatory cytokine activation is linked to the Fas/Fas Ligand system in patients with CHF. (Int Heart J 2012; 53: 182-186) Key words: Heart failure, Soluble Fas, Proinflammatory cytokine, Cardiopulmonary exercise test hronic heart failure (CHF) is a complex clinical syn- Methods drome characterized by exercise intolerance, hemo- C dynamic alterations, and neurohormonal and cytokine Subjects: We studied 106 patients with CHF (84 men and 22 activation. Studies have shown that proinflammatory cytokines women, 59.2 ± 11.9 years). Every patient had a left ventricular such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, ejection fraction < 45%. We also studied 39 age-matched as well as soluble TNF receptors (sTNF-R1 and sTNF-R2) are healthy individuals as controls (31 men and 8 women). The re- increased in circulating blood in patients with CHF.1-5) Apop- sults for the medical history, physical examination, electrocar- totic mediators have also been investigated and Fas/Fas Ligand diograms, chest X-rays, and echocardiograms were negative system activity was reported to be up-regulated in patients with for cardiovascular disease in these 39 control subjects. Control CHF.6) subjects did not take medications. We excluded patients with Fas is a type I cell-surface protein belonging to the TNF inducible ischemia, significant pulmonary disease, intermittent family.7) Fas can regulate the apoptotic process by acting as a claudication, or other disorders limiting exercise performance receptor for Fas Ligand.8,9) Fas is considered to be an inhibitor other than cardiac disease. Patients with chronic inflammatory of apoptosis, while Fas Ligand is an inducer of apoptosis. Al- disease, collagen disease, acute infection, and/or neoplasm though Fas acts as an inhibitory regulator, serum levels of the were also excluded. The protocol was approved by the Ethics soluble form of Fas (sFas) reflect the Fas/Fas Ligand system Committee of Tottori University and all subjects gave their activity.10) Previous studies have confirmed that serum sFas written informed consent to participate in the study. levels are increased in proportion to the severity of CHF,11-13) Measurement of blood samples: Blood specimens were drawn and may have prognostic value.14,15) Therefore, serum sFas can through a plastic cannula placed in the forearm vein after 30 be a promising biomarker of heart failure. The aim of this minutes of supine rest. Serum IL-6 levels were measured using study was to investigate the significant determinants of sFas chemiluminescent enzyme immunoassay (CLEIA, Fujirebio, levels among cardiopulmonary exercise variables, neurohor- Tokyo). Concentrations of serum TNF-α were determined by monal parameters, and proinflammatory cytokines and their the high-sensitivity human TNF-α test (Quantikine HS, R&D receptors in patients with CHF. Systems, Minneapolis, MN, USA). Serum soluble TNF recep- From 1 Kinugawa Cardiology Clinic, Osaka, Departments of 2 Cardiovascular Medicine, Faculty of Medicine and 3 Genetic Medicine and Regenerative Therapeutics, Graduate School of Medical Science, Tottori University, Tottori, and 4 Heart Center, Kitano Hospital, The Tazuke Kofukai Medical Research Institute, Osaka, Japan. Address for correspondence: Toru Kinugawa, MD, Kinugawa Cardiology Clinic, 2-13-28 Tsukamoto, Yodogawa-ku, Osaka, Osaka 532-0026, Japan. Received for publication January 17, 2012. Revised and accepted March 16, 2012. 182 Vol 53 No 3 SFAS IN HEART FAILURE 183 tor type 1 (sTNF-R1), type 2 (sTNF-R2), glycoprotein 130 Table I. Clinical, Echocardiographic, Neurohormonal, and Cardiopulmo- (gp130), soluble IL-6 receptor (sIL-6R), and sFas levels were nary Exercise Parameters in Control Subjects and in Patients With Chronic assessed according to the manufacturer’s specifications using Heart Failure an ELISA kit (Quantikine). Plasma catecholamines were de- Controls CHF termined by high performance liquid chromatography using (n = 39) (n = 106) the diphenylethylene diamine method. ANP and BNP levels Age (years) 55.1 ± 10.4 59.2 ± 11.9 were determined using a Shionoria RIA Kit and an S-1215 Male/Female 31/8 84/22 RIA kit, respectively. The intra-assay variability values in our Body height (cm) 166 ± 9 163 ± 8 laboratory for TNF-α, IL-6, sTNF-R1, sTNF-R2, gp130, sIL- Body weight (kg) 62.3 ± 10.6 61.7 ± 12.3 6R, sFas, norepinephrine, epinephrine, ANP, and BNP are Body mass index (kg/m2) 22.6 ± 2.8 23.0 ± 3.5 5.3%, 2.2%, 2.9%, 2.5%, 4.3%, 2.6%, 1.7%, 1.8%, 6.0%, New York Heart Association (I/II/III) - 43/46/17 6.1%, and 3.3%, respectively. Etiology Dilated cardiomyopathy - 55 (52%) Cardiopulmonary exercise testing: Cardiopulmonary exercise Old myocardial infarction - 21 (20%) testing was performed using an upright bicycle ergometer with Valvular heart disease - 22 (20%) 16) ramp protocol as described previously. Briefly, after 3 min- Hypertensive heart disease - 4 (4%) utes of unloaded cycling, the exercise load was increased in 10 Others - 4 (4%) or 20 watt/minute increments to symptom-limited maximal Echocardiographic data * work. Patients stopped exercise when they had severe leg fa- Left ventricular end-diastolic dimen- 49 ± 5 63 ± 11 tigue and/or dyspnea. Oxygen uptake (VO ), carbon dioxide sion (mm) 2 Left ventricular end-systolic dimension 32 ± 4 52 ± 11* output (VCO2), and minute ventilation (VE) were measured at (mm) rest and throughout the exercise period using a 280E Aero- Left ventricular ejection fraction (%) 68.3 ± 6.6 34.8 ± 4.8* monitor (Minato Medical Science, Osaka, Japan). Anaerobic Cardiothoracic ratio (%) 46 ± 4 57 ± 7* threshold was determined by the V-slope method. Peak VO2 Neurohormones Plasma norepinephrine (pg/mL) 206 ± 67 311 ± 224* was defined as the maximal VO2 attained during exercise. The slope of the VE-VCO relationship was calculated by linear re- Plasma epinephrine (pg/mL) 35 ± 25 34 ± 27 2 71.3 ± 64.2* gression analysis using the values of VE and VCO . Plasma atrial natriuretic peptide 21.2 ± 18.2 2 (pg/mL) Statistical analysis: Comparisons of the continuous variables Plasma brain natriuretic peptide 14.9 ± 14.1 187.9 ± 217.0* between the two groups were performed using the Mann- (pg/mL) Whitney U-test. Linear regression analysis was used to com- Cardiopulmonary exercise variables pare the relationship between the two variables. Univariate and Peak work rate (watts) 183 ± 85 106 ± 48* Anaerobic threshold (mL/minute/kg) 16.5 ± 3.8 11.1 ± 2.5* stepwise multivariate linear regression analyses were used to * Peak VO2 (mL/minute/kg) 29.2 ± 8.7 18.3 ± 4.9 detect independent factors associated with serum sFas levels * VE/VCO2 slope 26.7 ± 3.1 33.6 ± 7.4 among 14 variables. All analyses were performed with a Peak RER 1.20 ± 0.10 1.17 ± 0.11 StatView statistical program (Version 5.0, SAS Institute Inc. Cary, NC, USA). The differences were considered significant Values are mean ± SD. *P < 0.05 versus Controls. CHF indicates chronic when P values were less than 0.05. Data are expressed as the heart failure; VO2, oxygen uptake; VE, minute ventilation; VCO2, carbon mean ± SD. dioxide output; and RER, respiratory exchange ratio. Results Table II. Cytokines and Their Receptors in Control Subjects and in Pa- tients With Chronic Heart Failure Baseline characteristics in controls and in patients with CHF: Controls CHF The baseline characteristics of the study subjects are summa- (n = 39) (n = 106) rized in Table I. Two groups were matched for age, male to fe- male ratio, and body mass index (BMI). There were 43 pa- TNF-α (pg/mL) 2.66 ± 0.94 4.26 ± 4.36* * tients with New York Heart Association (NYHA) functional Soluble TNF-R1 (pg/mL) 626 ± 161 916 ± 438 Soluble TNF-R2 (pg/mL) 1231 ± 342 1841 ± 816* class I, 46 patients with class II, and 17 patients with class III. IL-6 (pg/mL) 1.17 ± 0.54 2.38 ± 2.38* The etiology of CHF was idiopathic dilated cardiomyopathy in Soluble IL-6R (pg/mL) 31.0 ± 13.5 33.0 ± 15.2 55 patients, old myocardial infarction in 21 patients, valvular gp130 (pg/mL) 252 ± 56 355 ± 174* heart disease in 22 patients, hypertensive heart disease in 4 pa- * tients, and others in 4 patients.