Methods
Study Population
We prospectively enrolled 200 ambulatory patients with clinically stable chronic HF of <3 months' duration who took part in the SICA-HF project. Recruitment for this project was commenced in March 2010, and we included all subjects from the Department of Cardiology, Charité Medical School, Campus Virchow-Klinikum, Berlin, Germany who were recruited until April 2012. All subjects provided written informed consent at enrolment, and the local ethics committee approved the protocol. The study is funded by the European Commission's 7th Framework programme (FP7/2007–2013) under grant agreement number 241558 and fulfils all principles of the Declaration of Helsinki.
The study protocol has been published previously. In brief, patients were eligible if they fulfilled the following criteria: age >18 years, clinical signs, and symptoms of chronic HF with a left ventricular ejection fraction (LVEF) ≤40% (HF with reduced ejection fraction, HFrEF) or with an LVEF >40% and a left atrial dimension ≥4.0 cm [HF with preserved ejection fraction (HFpEF)]. Patients with previous heart transplantation, cardiac, or embolic events within 6 weeks prior to the baseline examination, and patients on haemodialysis or with known pregnancy were excluded. Before inclusion, patients were screened by transthoracic two-dimensional echocardiography to assess the LVEF and other cardiac parameters. Blood was drawn early in the morning after an overnight fast and at least 15 min of supine rest to analyse the full blood count and routine clinical biochemistry parameters.
Assessment of Muscle Strength, Muscle Function, and Functional Capacity
All patients underwent a standardized protocol of different examinations of the muscle mass, muscle strength, and muscle performance. Dual energy X-ray absorptiometry (DEXA) was used to evaluate body composition. The DEXA scan measures total mass, lean, and fat mass. The lean mass has been used to estimate the muscle mass in our patients as previously described. The appendicular lean mass was defined as the lean mass of both arms and legs combined. A scanner model 'lunar prodigy' and 'lunar en Core 2002' software were used to analyse the data (both from GE Medical Systems, Madison, WI, USA). The knee extension strength was assessed using an isokinetic dynamometer (Multitrace 2, Lectromed, Jersey, Channel Islands). The maximal strength was measured in both legs in a sitting position with the patient's legs hanging freely, the ankle fixed by a pressure transducer. The best of three measurements was used. Arm strength was analysed using the handgrip dynamometer (Saehan Coporation Korea Hydraulic Hand Dynamometer, model SH5001). Likewise, the best of three measurements was used.
All patients underwent treadmill performance testing according to the modified Bruce protocol. The modified Naughton protocol was used in selected patients. Ventilation gases, such as the oxygen uptake (VO2 in mL), carbon dioxide production (VCO2 in mL), absolute peak oxygen consumption (absolute peakVO2 in mL/min), and anaerobic threshold, were recorded. A 6-min corridor walk test as well as a 4-m walking (4-m walk) test was performed according to standard protocols. Therefore, time was measured for the 4-m distance and gait speed was calculated.
Muscle wasting was defined according to previously published criteria suggested to diagnose sarcopenia. Thus, using data from the previously published Rossetta study, we defined muscle wasting as a muscle mass 2 SD below the mean of a healthy young reference group aged 18–40 years. The lean mass data from the DEXA scan were evaluated and the muscle mass index of patients was calculated. This index assesses the appendicular skeletal muscle mass (ASM in kg), calculated as the lean muscle mass of both arms and legs divided by the height (in metre) squared.
Assessment of Serum Cytokines
Venous blood was collected from an antecubital vein. Serum samples were immediately centrifuged and stored at –80°C until the final analysis. Serum levels of interleukin (IL) 1β, IL-6, and the tumour necrosis factor-α (TNF-α) were analysed using magnetic cytokine assays purchased from Bio-Rad Laboratories GmbH (Munich, Germany), the lower limits of detection being 0.1, 0.1, and 0.4 pg/mL, respectively.
Statistical Analysis
Data are presented as mean ± standard deviation (SD) or median with percentiles. For statistical analysis, StatView 5.0 (SAS Institute, Inc., Cary, USA) was used. Serum levels of IL-1β, IL-6, and TNF-α were non-normally distributed and, therefore, log-transformed to achieve a normal distribution. Analysis of variance (ANOVA), Student's unpaired t-test, Fisher's exact test, Pearson's simple regression, and logistic regression were used as appropriate. A two-tailed P-value ≤0.05 indicates statistical significance.