MS ID: CIRCULATIONAHA/2004/495895/R1
Online Data Supplement - Expanded Methods
Methods
Patient Selection
The Ethics Committee of the University of Leipzig approved the protocol of this study, and written informed consent was obtained from all patients and healthy subjects.
This study group consisted of 23 male patients £ 70 years with chronic heart failure (CHF, NYHA functional class II and III) as a result of and ischemic heart disease or dilative cardiomyopathy as assessed by cardiac catheterization. All patients had clinical and radiological signs of CHF and a left ventricular ejection fraction <40% as determined by ventriculography. They were required to be clinically stable for at least three months before enrollment into the study and had to have a physical work capacity at baseline greater than 25 W. Patients with CHF received their individually tailored medication consisting of angiotensin-converting enzyme inhibitors, digitalis, diuretics, and beta-blockers when tolerated. Cardiac medications were not changed during the study period. Exclusion criteria were significant valvular heart disease, uncontrolled hypertension, peripheral vascular disease, pulmonary disease, and musculoskeletal abnormalities precluding exercise training. A total of 12 age-matched men, who were admitted for non-specific chest pain to rule out coronary artery disease, served as healthy subjects (HS). They were normal by physical examination, ECG, chest x-ray, two-dimensional echocardiography, coronary angiography, left ventriculogram, and were not taking any medication during the study period.
Patients with CHF were randomized to either 6 months of exercise training (Training group, T) or sedentary lifestyle (Control group, C).
Training Protocol
The initial phase of the exercise program was performed on an in-hospital basis. During the first two weeks, patients exercised 4 to 6 times daily for 10 minutes on a bicycle ergometer adjusted to a work load at which 70% of peak oxygen uptake was reached. Before discharge from hospital, symptom-limited ergospirometry was performed to determine training target heart rate for home training that was defined as the heart rate reached at 70% of maximum oxygen uptake during symptom-limited exercise. Upon discharge, patients were provided with bicycle ergometers for home exercise training. They were encouraged to exercise close to their target heart rate daily for 20 minutes for a period of 6 months and were expected to participate in one group training session for 60 minutes each week consisting of walking, calisthenics and non-competitive ball games.
Patients assigned to the control group continued their sedentary lifestyle and remained on their individually tailored cardiac medication.
Exercise Testing and Respiratory Variables
Exercise testing was performed on a calibrated, electronically braked bicycle in an upright position with work load increasing progressively every 3 min in steps of 25 W beginning at 25 W. Respiratory gas exchange data were determined continuously throughout the exercise test.1
Skeletal Muscle Biopsies
Percutaneous needle biopsies were obtained from the middle part of the vastus lateralis muscle under local anesthesia two days before maximal ergospirometry at baseline and again after six months of training. The biopsies, which have a size of approximately 50mg were either snap frozen in liquid nitrogen and stored at –80°C or fixed with 4% buffered formaldehyde before paraffin embedding.2 All investigators involved in the analysis of the skeletal muscle samples were blinded to patient identity, patient assignment (i.e., training, control or healthy), and intervention status (i.e., samples before or after the 6 months of the study period).
Measurement of mRNA
MnSOD (U: 5´-GAG ATG TTA CAG CCC AGA TAG C-3´, L: 5´- AAT CCC CAG CAG TGG AAT AAG G-3´), CuZnSOD (U: 5`-CAA AGA TGG TGT GGC CGA TG-3`, L: 5`-TGG GCG ATC CCA ATT ACA CC-3`) GPX (U: 5`-GAA TGT GGC GTC CCT CTG AGG C-3`, L: 5`-GCA ACA TCG TTG CGA CAC ACC GG-3`), catalase (U: 5´- GCT GAT GTC CTG ACC ACT GGA GC-3´, L: 5´- GCT GAA CCC GAT TCT CCA GC-3´) IL1b (U: 5`-GGG CCT CAA GGA AAA GAA TC-3`, L: 5´-TTC TGC TTG AGA GGT GCT GA-3´) and TNFa (U: 5`-TGT GTT GTC CTT CCT GCA AC-3`, L: 5´-CTT GTA GGT GCC CAG GAG AG-3´) mRNA were quantified with real time PCR using specific primers (Light Cycler System, Roche Diagnostics Inc., Mannheim, Germany).3 The mRNA of the radical scavenger enzymes and the cytokines was expressed as ratio to 18S-rRNA (U: 5`-ATA CAG GAC TCT TTC GAG GCC C-3`, L: 5`-CGG GAC ACT CAG CTA AGA GCA T-3`), which was amplified as house keeping gene.3 The variability for triplicate measurements was <5%.
Quantification of protein expression
Briefly, 50 µg of skeletal muscle proteins were separated on a SDS-polyacrylamide-gel (SDS-PAGE) and electrotransferred to a polyvinylidene fluoride membrane (PVDF; Roth GmbH, Karlsruhe, Germany). Protein expressions were determined using an anti-MnSOD antibody (1:1000, StressGene, Victoria, Canada), an anti-catalase antibody (1:1000, Biomol GmbH, Hamburg, Germany), and an anti-CuSOD antibody (1:1000, Upstate Biotechnologies, Lake Placid, New York, USA). The bound primary antibody was detected by a peroxidase-coupled secondary antibody followed by a chemiluminescent reaction using luminol (SuperSignal West Pico, Pierce Rockford, IL USA). Protein expression of the radical scavenger enzymes was quantified applying a 1-D analysis software package (One-Dscan, Scanalytics, Billerica, MA, USA) as previously described.2 To compensate for blot to blot variations an internal standard was loaded twice on each SDS-PAGE, and the densitometry results are expressed as ratio between sample and standard intensity. The variability for duplicate measurements was <10%.
Quantification of enzymatic activities
The frozen biopsy samples were homogenized in buffer and protein content was determined as described previously.2 Activity of total SOD was measured monitoring the rate of cytochrome c reduction by superoxide radicals derived from the xanthine/xanthine oxidase system.4 GPX activity was determined measuring the NADPH oxidation in the presence of reduced glutathione and hydrogen peroxide (H2O2).5 Activity of catalase was measured spectrophotometrically at 240 nm detecting the H2O2 decay.6 Enzyme activities are expressed as U/mg protein or mU/mg protein. The variability for triplicate measurements was <10%.
Measurement of lipid peroxidation as a marker of oxidative stress
Homogenates of the vastus lateralis muscle were processed according to the instructions of the manufacture and lipid peroxide concentrations were determined photometrically using a commercially available kit (Immundiagnostik AG, Bensheim, Germany).7 The intra- and inter-assay variability was 3.1% and 5.1%, respectively.
Measurement of cytokine expression, Measurement of nitrotyrosine formation
In eight patients of each group, IL1b and TNFa expression were determined immunohistochemically using specific polyclonal antibodies (IL1b: Upstate Biotechnology, Lake Placid, New York, USA; TNFa: Sigma, St. Louis, Missouri, USA). Paraffin sections (3µm) of the skeletal muscle biopsies were dewaxed with xylene and rehydrated. Endogenous peroxidase activity was blocked by an exposure of the tissue section to 60% methanol supplemented with 0.3% hydrogen peroxide for 30 minutes followed by a rinse in phosphate buffered saline (PBS) and a 20 minute incubation in 0.2% Triton X100/PBS. Unspecific binding of antibodies was blocked by 1.5% horse serum diluted in 2% fat free milk/PBS for 30 minutes at room temperature (20°C). Afterwards, skeletal muscle section were incubated in primary antibody diluted in 2% fat free milk/PBS overnight at 4°C (IL1b: 1:50; TNFa: 1:100). The bound primary antibody was visualized using the alkaline phosphatase antialkaline phosphatase (APAAP) method.3 Three different tissue sections from each biopsy were analyzed.
Nitrotyrosine formation was assessed as a second maker of oxidative stress,8 representing a read-out of peroxynitrite production using a specific antibody as described by Bachmeier et al.9
All immunohistochemical slides for TNFa, IL1b and nitrotyrosine formation were prepared in pairs (i.e., three tissue sections of a skeletal muscle biopsy before and three tissue sections of a biopsy after the intervention) in order to avoid differences in staining intensity. The positive stained myocyte tissue area on each slide was quantified using an automatic image analysis software measuring the myocyte area with red staining (KS 300, Zeiss Inc, Oberkochen, Germany) by three different reviewers blinded for patient identity, assignment and status.3
In order to determined, which cell type is the predominant type in the skeletal muscle biopsies, tissue sections were treated as described above. Afterwards, tissue sections were incubated in an anti skeletal muscle actin antibody. The bound primary antibody was recognized by a TRITC-labeled secondary antibody. Endothelial cells were identified by an anti von Willebrand factor antibody staining. Binding of the primary antibody was visualized by a FITC-labeled secondary antibody (all antibodies from Dako, Hamburg, Germany). Nuclei were counterstained with Hoechst dye and tissue sections were evaluated using a fluorescence microscope (KS 300, Zeiss Inc, Oberkochen, Germany).
Measurement of cytokines in the serum
The serum concentration of TNFa and IL1b were determined in patients with CHF and healthy subjects using commercially available high-sensitive enzyme-linked immunosorbent assay kits (R&D Systems, Wiesbaden, Germany). All measurements were performed in duplicate. The sensitivity of the respective ELISA assays was 0.18 pg/ml for TNFa and 0.10 pg/mL for IL1b.
Detection of apoptosis
Apoptosis was determined applying the Apo Tag Peroxidase Oligo Ligation Apoptosis Detection Kit according to the instructions of the manufacture (Chemicon, Temecula, California, USA). The number of total skeletal muscle myocyte nuclei and apoptotic nuclei in skeletal muscle myocytes was counted in the tissue sections. The myocyte origin of the cells the apoptotic nuclei were belonging to was confirmed by an anti-skeletal muscle actin antibody staining using a commercially available antibody (HFF 35, Dako GmbH, Hamburg, Germany).10 An average number of 10350±690 skeletal muscle myocyte nuclei was analyzed in each biopsy for the presence of apoptosis. The data are expressed as number of apoptotic nuclei per 10000 skeletal muscle myocyte nuclei. Non-myocyte nuclei were not analyzed.
Statistical Analysis
Mean value±standard error was calculated for all variables. Data were tested for normal distribution and for homogeneity of variances. In case of a normal distribution of the data a two-sided t-test was used to compare patients with CHF and healthy subjects, in case of a non-normal distribution the Mann Whitney U test was applied. Inter- and intragroup comparisons (begin vs. 6 months, control vs. training) for CHF patients were performed using the two-way repeated measures ANOVA followed by the Tukey post-hoc test in case of a normal distribution of the data. Otherwise, a Mann Whitney U test or a Wilcoxon signed rank test was applied for the inter- and intragroup comparison, respectively. A p value of less than 0.05 was considered statistically significant.
References
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2. Hambrecht R, Adams V, Gielen S, Linke A, Mobius-Winkler S, Yu J, Niebauer J, Jiang H, Fiehn E, Schuler G. Exercise intolerance in patients with chronic heart failure and increased expression of inducible nitric oxide synthase in the skeletal muscle. J Am Coll Cardiol. 1999;33:174-179.
3. Gielen S, Adams V, Mobius-Winkler S, Linke A, Erbs S, Yu J, Kempf W, Schubert A, Schuler G, Hambrecht R. Anti-inflammatory effects of exercise training in the skeletal muscle of patients with chronic heart failure. J Am Coll Cardiol. 2003;42:861-868.
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9. Bachmaier K, Neu N, Pummerer C, Duncan GS, Mak TW, Matsuyama T, Penninger JM. iNOS expression and nitrotyrosine formation in the myocardium in response to inflammation is controlled by the interferon regulatory transcription factor 1. Circulation. 1997;96:585-591.
10. Adams V, Jiang H, Yu J, Mobius-Winkler S, Fiehn E, Linke A, Weigl C, Schuler G, Hambrecht R. Apoptosis in skeletal myocytes of patients with chronic heart failure is associated with exercise intolerance. J Am Coll Cardiol. 1999;33:959-965.
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