Andreas Schubert

Anti-inflammatory effects of exercise training in the skeletal muscle of patients with chronic heart failure

OBJECTIVES The aim of this study was to assess the effects of regular physical exercise on local inflammatory parameters in the skeletal muscle of patients with chronic heart failure (CHF). BACKGROUND Inflammatory activation with increased serum cytokine levels and expression of inducible nitric oxide synthase (iNOS) in the myocardium and peripheral skeletal muscles has been described in CHF. METHODS Twenty male patients with stable CHF (left ventricular ejection fraction 25 +/- 2%; age 54 +/- 2 years) were randomized to a training group (n = 10) or a control group (n = 10). At baseline and after six months, serum samples and vastus lateralis muscle biopsies were obtained. Serum tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1-beta levels were measured by enzyme-linked immunosorbent assay, local cytokine, and iNOS expression by real-time polymerase chain reaction. RESULTS Exercise training improved peak oxygen uptake by 29% in the training group (from 20.3 +/- 1.0 to 26.1 +/- 1.5 ml/kg. min; p < 0.001 vs. control group). While serum levels of TNF-alpha, IL-6, and IL-1-beta remained unaffected by training, local skeletal muscle TNF-alpha decreased from 1.9 +/- 0.4 to 1.2 +/- 0.3 relative U (p < 0.05 for change vs. control group), IL-6 from 71.3 +/- 16.5 to 41.3 +/- 8.8 relative U (p < 0.05 vs. begin), and IL-1-beta from 2.7 +/- 1.1 to 1.4 +/- 0.6 relative U (p = 0.02 vs. control group). Exercise training also reduced local iNOS expression by 52% (from 6.3 +/- 1.2 to 3.0 +/- 1.0 relative U; p = 0.007 vs. control group). CONCLUSIONS Exercise training significantly reduced the local expression of TNF-alpha, IL-1-beta, IL-6, and iNOS in the skeletal muscle of CHF patients. These local anti-inflammatory effects of exercise may attenuate the catabolic wasting process associated with the progression of CHF.

Antioxidative Effects of Exercise Training in Patients With Chronic Heart Failure Increase in Radical Scavenger Enzyme Activity in Skeletal Muscle

Background—In chronic heart failure (CHF), cross-talk between inflammatory activation and oxidative stress has been anticipated in skeletal muscle (SM). The role of the radical scavenger enzymes superoxide dismutase (SOD), catalase (Cat), and glutathione peroxidase (GPX), which remove oxygen radicals, has never been assessed in the SM in this context. Moreover, it remains unknown whether exercise training augments the activity of these enzymes in CHF. Methods and Results—Twenty-three patients with CHF were randomized to either 6 months of exercise training (T) or a sedentary lifestyle (C); 12 age-matched healthy subjects (HS) were studied in parallel. Activity of Cat, SOD, and GPX was assessed in SM biopsies before and after 6 months (6 months). Oxidative stress was determined by measuring nitrotyrosine formation. SOD, Cat, and GPX activity was reduced by 31%, 57%, and 51%, respectively, whereas nitrotyrosine formation was increased by 107% in SM in CHF (P<0.05 versus HS). In CHF, exercise training augmented GPX and Cat activity in SM by 41% (P<0.05 versus before and group C) and 42% (P<0.05 versus before and group C), respectively, and decreased nitrotyrosine production by 35% (from 3.8±0.4% tissue area before to 2.5±0.3% after 6 months; P<0.05 versus before). Conclusions—The reduced activity of major antioxidative enzymes in the SM of CHF patients is associated with increased local oxidative stress. Exercise training exerts antioxidative effects in the SM in CHF, in particular, due to an augmentation in activity of radical scavenger enzymes.

Regulation of the endothelin system by shear stress in human endothelial cells

1 In this study, the effect of shear stress on the expression of genes of the human endothelin‐1 system was examined. Primary cultures of human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress of 1, 15 or 30 dyn cm−2 (i.e. 0.1, 1.5 or 3 N m−2) (venous and two different arterial levels of shear stress) in a cone‐and‐plate viscometer. 2 Laminar shear stress transiently upregulates preproendothelin‐1 (ppET‐1) mRNA, reaching its maximum after 30 min (approx 1.7‐fold increase). In contrast, long‐term application of shear stress (24 h) causes downregulation of ppET‐1 mRNA in a dose‐dependent manner. 3 Arterial levels of shear stress result in downregulation of endothelin‐converting enzyme‐1 isoform ECE‐1a (predominating in HUVEC) to 36.2 ± 8.5%, and isoform ECE‐1b mRNA to 72.3 ± 1.9% of static control level. 4 The endothelin‐1 (ET‐1) release is downregulated by laminar shear stress in a dose‐dependent manner. 5 This downregulation of ppET‐1 mRNA and ET‐1 release is not affected by inhibition of protein kinase C (PKC), or tyrosine kinase. Inhibition of endothelial NO synthase (L‐NAME, 500 μm) prevents downregulation of ppET‐1 mRNA by shear stress. 6 In contrast, increasing degrees of long‐term shear stress upregulate endothelin receptor type B (ETB) mRNA by a NO‐ and PKC‐, but not tyrosine kinase‐dependent mechanism. 7 In conclusion, our data suggest the downregulation of human endothelin synthesis, and an upregulation of the ETB receptor by long‐term arterial laminar shear stress. These effects might contribute to the vasoprotective and anti‐arteriosclerotic potential of arterial laminar shear stress.

Reduction of insulin-like growth factor-I expression in the skeletal muscle of noncachectic patients with chronic heart failure.

OBJECTIVES We sought to assess the role of insulin-like growth factor-I (IGF-I) in muscle wasting in chronic heart failure (CHF), serum concentrations and local muscular IGF-I expression were determined in patients with severe CHF. BACKGROUND Chronic heart failure is associated with progressive muscle atrophy, leading to cardiac cachexia. Skeletal muscle disuse and inflammatory activation with elevated cytokine levels have been implicated; however, the pathomechanism involved remains largely unknown. METHODS Serum levels of IGF-I were measured by competitive solid phase immunoassay in 47 patients with severe CHF (left ventricular ejection fraction < or =30%) and 15 age-matched healthy subjects (HS). Insulin-like growth factor-I and IGF-I receptor expression were assessed in vastus lateralis biopsies by real-time PCR and Western blot analysis. RESULTS Although serum IGF-I was not significantly different (175 +/- 10 ng/ml in CHF vs. 170 +/- 12 ng/ml in HS, p = NS), local muscle IGF-I mRNA expression was reduced by 52% in CHF (6.7 +/- 0.4 vs. 14.0 +/- 0.9 arbitrary units in HS, p < 0.001). This was accompanied by an increase in IGF-I receptor mRNA expression (86.8 +/- 5.4 in CHF vs. 23.1 +/- 1.8 arbitrary units in HS, p < 0.001). Local IGF-I expression was significantly correlated with muscle cross-sectional area (R = 0.75, p = 0.01). Chronic heart failure patients with a body mass index of < 25 kg/m(2) showed signs of peripheral growth hormone (GH) resistance, as indicated by elevated serum GH, and reduced IGF-I levels. CONCLUSIONS In CHF patients, muscle IGF-I expression is considerably reduced in the presence of normal serum IGF-I levels, possibly contributing to early loss of muscle mass. These findings are consistent with a potential role of IGF-I for skeletal muscle atrophy in CHF.

Effects of exercise training on insulin-like growth factor-I expression in the skeletal muscle of non-cachectic patients with chronic heart failure:

Background Chronic heart failure (CHF) is associated with progressive muscle atrophy and reduced local expression of insulin-like growth factor I (IGF-I). Design The present study was designed to test the hypothesis that the local deficiency of IGF-I in the skeletal muscle of patients with CHF would respond to a 6-months aerobic training intervention. Therefore, 18 patients [mean age 52.4 (SD 4.8) years, left ventricular ejection function (LVEF) 27 (SD 6)%] were prospectively randomized to either 6 months of training or sedentary lifestyle. Methods Serum levels of growth hormone (GH) were measured by immunofluorometric assay, IGF-I by competitive solid phase immunoassay. IGF-I expression was assessed in vastus lateralis biopsies by real-time PCR. Results Exercise training led to a significant increase in peak oxygen uptake by 26% [from 20.3 (SD 3.3) ml/kg per min to 25.5 (SD 5.7) ml/kg per min, P=0.003 versus control]. Local expression of IGF-I increased significantly after exercise training by 81% [from 6.3 (SE 0.8) to 11.4 (SE 1.4) relative units, P=0.007 versus control] while IGF-I receptor expression was reduced by 33% [from 20.0 (SE 2.1) to 13.8 (SE 1.7) relative units, P=0.008 versus control]. Serum growth hormone (GH) rose modestly from 0.12 (SE 0.07) to 0.65 (SE 0.37) ng/ml in the training group (P=0.043 versus baseline), however, this change was not significant compared to the control group (P=0.848). IGF-I serum levels remained virtually unchanged. Conclusions Exercise training improves local IGF-I expression without significant changes of systemic parameters of the GH/IGF-I axis. These findings indicate that exercise training has the therapeutic potential to attenuate peripheral skeletal muscle alterations in particular with respect to local IGF-I expression in patients with moderate CHF.

Exercise training in chronic heart failure: correlation between reduced local inflammation and improved oxidative capacity in the skeletal muscle.

Background Chronic heart failure (CHF) is accompanied by an inflammatory activation which occurs both systemically and in the skeletal muscle. Exercise training has been shown to reduce the local expression of cytokines and inducible nitric oxide synthase (iNOS) in muscle biopsies of CHF patients. INOS-derived NO can inhibit oxidative phosphorylation and contribute to skeletal muscle dysfunction in CHF. Design To investigate the correlation between changes in local iNOS expression associated with regular exercise and changes in aerobic enzyme activities in the skeletal muscle of patients with CHF. Twenty male CHF patients [ejection fraction 25% (SE 2), age 54 (SE 2) years] were randomized to a training (n = 10) or a control group (C, n = 10). Methods At baseline and after 6 months skeletal muscle iNOS expression was measured by real-time polymerase chain reaction. INOS protein and protein nitrosylation were assessed by immunohistochemistry. Cytochrome c oxidase (COX) activity was quantified electrochemically using the Clark oxygen electrode. Results Exercise training led to a 27% increase in cytochrome c oxidase activity [from 21.8 (SE 3.2) to 27.7 (SE 3.5) nmol O2/mg per min, P=0.02 versus baseline]. Changes in iNOS expression and iNOS protein content were inversely correlated with changes in COX-activity (r= −0.60, P=0.01; r= −0.71, P<0.001). Conclusions The inverse correlation between iNOS expression/iNOS protein content and COX-activity indicates that local anti-inflammatory effects may contribute to improved muscular oxidative metabolism.

Endothelial Protection, AT1 Blockade and Cholesterol-Dependent Oxidative Stress The EPAS Trial

Background— Statins and angiotensin type 1 (AT1) receptor blockers reduce cardiovascular mortality and morbidity. In the Endothelial Protection, AT1 blockade and Cholesterol-Dependent Oxidative Stress (EPAS) trial, impact of independent or combined statin and AT1 receptor blocker therapy on endothelial expression of anti-atherosclerotic and proatherosclerotic genes and endothelial function in arteries of patients with coronary artery disease were tested. Methods and Results— Sixty patients with stable coronary artery disease undergoing elective coronary artery bypass grafting (CABG) surgery were randomized 4 weeks before surgery to: (A) control without inhibition of renin-angiotensin system or statin; (B) statin (pravastatin 40 mg/d); (C) AT1 blockade (irbesartan 150 mg/d); or (D) combination of statin and AT1 blocker in same dosages. Primary end point was a priori therapy-dependent regulation of an anti-atherosclerotic endothelial expression quotient Q including mRNA expression (in arbitrary units measured by real-time polymerase chain reaction) of endothelial nitric oxide synthase and C-type natriuretic peptide, divided by expression of oxidized low-density lipoprotein receptor LOX-1 and NAD(P)H oxidase subunit gp91phox in left internal mammary arteries biopsies obtained by CABG surgery; 49 patients completed the study. Statin therapy increased lnQ from 3.2±0.4 to 4.4±0.4 significantly versus control. AT1 blockade showed a trend to increase lnQ to 4.2±0.5. Combination of statin and AT1 blocker further increased lnQ to 5.1±0.6, but a putative interaction of both therapies in lnQ was not significant. Furthermore, preoperative therapy with statin, AT1 blocker and their combination improved endothelial function in internal mammary artery rings. Conclusions— Statin and AT1 blocker therapy independently and in combination improve an anti-atherosclerotic endothelial expression quotient and endothelial function.

Shear Stress–Dependent Regulation of the Human β-Tubulin Folding Cofactor D Gene

Abstract— The flowing blood generates shear stress at the endothelial cell surface. The endothelial cells modify their phenotype by alterations in gene expression in response to different levels of fluid shear stress. To identify genes involved in this process, human umbilical vein endothelial cells were exposed to laminar shear stress (venous or arterial levels) in a cone-and-plate apparatus for 24 hours. Using the method of RNA arbitrarily primed polymerase chain reaction, we cloned a polymerase chain reaction fragment representing an mRNA species downregulated by arterial compared with venous shear stress (shear stress downregulated gene-1, SSD-1). According to Northern blot analysis, corresponding SSD-1 cDNA clones revealed a similar, time-dependent downregulation after 24 hours of arterial shear stress compared with venous shear stress or static controls. Three SSD-1 mRNA species of 2.8, 4.1, and 4.6 kb were expressed in a tissue-specific manner. The encoded amino acid sequence of the human endothelial SSD-1 isoform (4.1-kb mRNA species) revealed 80.4% identity and 90.9% homology to the bovine &bgr;-tubulin folding cofactor D (tfcD) gene. Downregulation of tfcD mRNA expression by shear stress was defined at the level of transcription by nuclear run-on assays. The tfcD protein was downregulated by arterial shear stress. The shear stress–dependent downregulation of tfcD mRNA and protein was attenuated by the NO synthase inhibitor N&ohgr;-nitro-l-arginine methyl ester. Furthermore, the NO donor DETA-NO downregulated tfcD mRNA. Because tfcD was shown to be a microtubule-destabilizing protein, our data suggest a shear stress–dependent regulation of the microtubular dynamics in human endothelial cells.

Extracellular matrix gene expression correlates to left ventricular mass index after surgical induction of left ventricular hypertrophy

Abstract The cardiac extracellular matrix (ECM) is a dynamic entity maintaining the structural and functional properties of the myocardium. Little is known about alterations in ECM regulation during controlled induction of compensated left ventricular hypertrophy (LVH) using experimental aortic stenosis.Fifteen growing sheep received supra-coronary banding at an age of 7 ± 1 months whereas 10 age-matched sheep served as the control group (C). Explantation of the hearts was performed 8.3 ± 1 months after banding. Gene sequences for sheep matrix metalloproteinase (MMP)-1,-2,-3,-9 and tissue inhibitors (TIMP)-1,-2,-3 were isolated and cloned. Then mRNA and protein gene expression analyses were performed.Concentric LVH with no evidence of heart failure was diagnosed at explantation. Left ventricular mass index (LVMI) was 150 ± 33 g/m2 (LVH) versus 88 ± 23 (C) and 82 ± 21 (baseline) (p < 0.01 versus LVH). Parallel to LVH there was a significant increase in mRNA and protein expression for MMP-1,-2,-3,-9 and for TIMP-1,-2 whereas there was a significant decrease in TIMP-3 gene expression. A close correlation between changes in LVMI and ECM gene expression was found.Compensated LVH goes along with a significant modification of MMP and TIMP gene expression. Alterations in ECM gene expression may be part of the adaptive process during left ventricular remodeling.

Experimental Aortic Stenosis and Corresponding Left Ventricular Hypertrophy in Sheep

Left ventricular hypertrophy (LVH) is an independent cardiac risk factor. A simple standard experimental model of inducing LVH for further studies using experimental aortic stenosis in sheep was performed. The aim of this study is to describe animal-specific requirements as well as perioperative therapy, postoperative care, and the use of echocardiography for routine follow-up examinations. Supracoronary aortic banding was performed in 55 female sheep at an age of 6 to 8 months. General anesthesia and an antero-lateral thoracotomy were used. The objective was to achieve pressure gradients of 20 to 30 mm Hg. In addition a 4th intercostal space rib window was created to improve echocardiographic vision. The operations were completed successfully in all animals. Intraoperatively, little severe arrhythmia occurred. During the follow-up interval of 8 +/- 1.3 months, 8 animals died, due to incomplete perforation of the ascending aorta (3), chronic heart failure (2), pericardial cyst (1), and respiratory failure and infection (2). All remaining animals were amenable for further studies. Severe LVH was diagnosed with routine echocardiography on follow-up. Thus, experimental aortic stenosis in sheep is a safe and relatively simple technique to generate stable LVH. Echocardiography is an easy tool for follow-up evaluations. Due to low complication rates, the sheep model is well suited for further research in LVH.