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European Heart Journal Advance Access originally published online on March 12, 2007
European Heart Journal 2007 28(7):821-828; doi:10.1093/eurheartj/ehl541
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© The European Society of Cardiology 2007. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Secretory sphingomyelinase is upregulated in chronic heart failure: a second messenger system of immune activation relates to body composition, muscular functional capacity, and peripheral blood flow

Wolfram Doehner1,2,*,{ddagger}, Alexander C. Bunck3,{ddagger}, Mathias Rauchhaus1,2, Stephan von Haehling2, Frank M. Brunkhorst3,{dagger}, Mariantonietta Cicoira2, Carsten Tschope4, Piotr Ponikowski5, Ralf A. Claus3,{dagger} and Stefan D. Anker1,2

1 Division of Applied Cachexia Research, Department of Cardiology, Charité Medical School, Campus Virchow-Klinikum, Berlin, Germany
2 Department of Clinical Cardiology, National Heart and Lung Institute, Imperial College, London, UK
3 Department of Anesthesiology and Intensive Care, University of Jena, Germany
4 Department of Cardiology, Charité Medical School, Campus Benjamin Franklin, Berlin, Germany
5 Cardiology Department, Clinical Military Hospital, Wroclaw, Poland

Received 16 April 2006; revised 4 January 2007; accepted 25 January 2007; online publish-ahead-of-print 12 March 2007.

* Corresponding author. Tel: +49 30 450 553507; fax: +49 30 450 553951. E-mail address: wolfram.doehner{at}charite.de

See page 777 for the editorial comment on this article (doi:10.1093/eurheartj/ehm025)


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Aims: Sphingomyelinases (SMase) are key regulating enzymes of the intracellular and paracrine ceramide second messenger system that mediates immune response to inflammatory cytokines and oxidative stress. Vascular endothelial cells are a rich and regulatable source of secretory SMase (S-SMase). Chronic heart failure (CHF) is a state of endothelial dysfunction and latent immune activation. The significance of S-SMase has not been studied in CHF in detail. The aim of the present study is to characterize S-SMase activity in patients with CHF in relation to disease severity and to pathophysiological characteristics such as immune activation, vasodilator capacity, and skeletal muscle function and body composition.

Methods and results: S-SMase activity was assessed by a fluorimetric method in 112 patients with CHF (age, 63 ± 11 years; NYHA class I/II/III/IV, 9/48/46/9; LVEF, 30 ± 15%; peak VO2, 18.6 ± 6.7 mL/kg/min) and in two control groups (healthy, n = 13 and hypertensive controls, n = 11). S-SMase activity was similar in both control groups (healthy, 150 ± 121 pmol/mL h; hypertensive, 157 ± 134 pmol/mL h) but was increased by >90% in CHF patients (299 ± 283 pmol/mL h; P = 0.004). S-SMase elevation was not different between ischaemic and non-ischaemic CHF and increased stepwise with NYHA class (I, 206 ± 202; II, 284 ± 242; III, 306 ± 212; IV, 440 ± 665 pmol/mL h; P = 0.003). S-SMase correlated with peak VO2 (R = –0.33, P = 0.0007) and with cytokine activation [tumour necrosis factor-{alpha} (TNF-{alpha}) R = 0.22, P = 0.02; sTNF-R1 R = 0.39, P < 0.0001]. S-SMase further correlated with reduced skeletal (quadriceps) muscle strength (R = –0.46, P < 0.0001) as well as impaired peripheral vasodilator capacity (R = –0.34, P = 0.02). In detailed body composition analysis (DEXA scan), S-SMase activity was highest in patients with cardiac cachexia (405 ± 357 vs. non-cachectic patients: 233 ± 202 pmol/mL h; P = 0.0007) and related to reduced lean tissue parameters but not to fat tissue parameters. In Cox proportional hazard analysis, elevated SMase related to impaired survival, independent of age, NYHA class, and mean BP (hazard ratio 2.92; 95% confidence interval 1.035–8.24; P = 0.04).

Conclusion: S-SMase is upregulated in CHF, independent of aetiology. The association of S-SMase with clinical status, tissue amount, functional capacity of skeletal muscle tissue, and vasodilator capacity suggests that S-SMase-mediated signalling may contribute to regulatory processes of CHF pathophysiology.

Key Words: Chronic heart failure • Inflammation • SMase • Cachexia • TNF-{alpha}

{dagger} Co-corresponding author. Tel: +49 3641 9325860; fax: +49 3641 9325862. E-mail address: ralf.claus{at}med.uni-jena.de Back

{ddagger} These authors contributed equally to this article. Back


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
The sphingomyelin/ceramide pathway is a ubiquitous signalling system in which ceramide functions as an important second messenger of intracellular and paracrine-signalling processes.1,2 The enzyme sphingomyelinase (SMase; EC 3.1.4.12 [EC] ) is a key regulatory step in the ceramide-signalling cascade, as it promotes the initial step of ceramide formation by hydrolysis of membrane-bound sphingomyelin, followed by further metabolism to sphingosine and sphingosin-1-phosphate, all of which carry signalling capacity. There are several isoforms of SMase distinguished by different pH optima, localization, and cation dependence.3 Recent studies have shown, however, that lysosomal and secretory SMase (S-SMase) derive from the same gene (acidic SMase or ASM gene) via differential protein trafficking of a common protein precursor.4,5 Opposite to other SMase isoforms, the role of S-SMase in pathophysiological and physiological processes is less well understood. Previous work has shown that the vascular endothelium is an abundant source of S-SMase.6 So far, S-SMase has been the only enzyme held responsible for sphingolytic activity observed in plasma.5 S-SMase activity is increased by several agents of inflammatory stimulation, including pro-inflammatory cytokines such as tumour necrosis factor-{alpha} (TNF-{alpha}) and interleukin (IL)-1ß,6 as well as lipopolysaccharides (LPS) and oxidative stress.7 In a recent report, we demonstrated that increased activity of S-SMase can be found in plasma of patients with septic shock and may contribute to the pathophysiology of organ dysfunction.8

The contribution of SMases as mediator of inflammation in cardiovascular disease such as atherosclerosis,9 ischaemia/reperfusion damage,10 and acutely impaired myocardial contractility11 has been demonstrated (for a review, see Levade et al.12). Immune activation has been established as a characteristic feature of the pathophysiology of chronic heart failure (CHF),13 being involved in myocardial damage14 as well as in impaired peripheral blood flow15 and vasodilator capacity,16 a hallmark of endothelial dysfunction in CHF. Moreover, TNF-{alpha} and other cytokines are involved in the pathogenesis of muscle wasting and cardiac cachexia17 and predict impaired survival in CHF.13 Although the sphingomyelin-signalling pathway has been suggested to mediate the response to TNF-{alpha} in various tissues,18,19 the significance of endothelium derived circulating S-SMase in CHF has not been investigated in detail.

We hypothesized that S-SMase activity is upregulated in CHF and that it relates to symptomatic status of CHF. We aimed to assess S-SMase in relation to clinical characteristics such as cardiopulmonary exercise capacity, to skeletal muscle function, and to peripheral vasodilator capacity. Detailed body composition including total amount and distribution of lean and fat tissue and the development of cardiac cachexia were taken into account.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Conclusion
 Acknowledgements
 References
 
Study population and follow-up
We studied 112 male ambulatory CHF patients with ischaemic and non-ischaemic aetiology. The diagnosis of CHF was based on clinical evidence of heart failure, with a disease history of at least 6 months. In all patients, evidence of impaired left ventricular systolic function by angiogram, radionuclide ventriculography, and/or echocardiography was present. When no evidence of previous myocardial infarction or coronary heart disease was found, non-ischaemic aetiology of heart failure was diagnosed. All patients were clinically stable and were on standard medical treatment as clinically indicated with diuretics, angiotensin-converting enzyme inhibitors or angiotensin II receptor antagonists, ß-blockers, digitalis, aspirin, and/or warfarin in varying combinations. Cardiac cachexia was defined as non-intentional, non-oedematous weight loss of 6% of body weight within 6 months.20 Patients with acute cardiac decompensation, neuromuscular disorders, peripheral vascular disease, signs of acute infection, malignancy, and cachexia of other reasons were excluded from the study. All CHF patients were consecutively recruited between 1997 and 2001 from the outpatient clinic of the Royal Brompton Hospital, London. All patients who agreed to take part in the study and who deemed eligible according to the criteria described earlier were enrolled in this study. A number of patients refused to some of the tests owing to strenuous procedures (strength tests) or discomfort (plethysmography), and reduced numbers of respective study data are indicated appropriately. Data from CHF patients were compared with a control group of healthy subjects (n = 13) that was recruited non-consecutively and randomly from a routine health-screening programme. Only male volunteers with no disease history or current clinical or biochemical sign of any cardiovascular disease or other chronic disease and none of the mentioned exclusion criteria were enrolled as healthy controls. A second control group of male patients with known hypertension but with no history and no clinical or biochemical indication of cardiovascular disease and none of the exclusion criteria was also recruited from the hospital's outpatient clinic (n = 11). Hypertensive controls were recruited randomly during the last 10 months of the recruitment period. Antihypertensive medication included ACE-inhibitors, AT II receptor blockers, diuretics, ß-receptor blockers, Ca antagonists, and {alpha}-receptor antagonists. Follow-up of CHF patients for survival status was available from the hospital information system and from the Office of National Statistics, where all patients had been flagged for death as part of the Royal Brompton Hospital heart failure registry. All patients gave written informed consent and the study was approved by the local Ethics Committee.

Assessments
Blood samples were collected in the morning, following overnight fasting and after 20 min of supine rest. All samples were processed immediately after withdrawal and serum was stored at –80°C until analysis. Enzymatic activity of S-SMase was determined by the hydrolysis of fluorescently labelled sphingomyelin (NBD-SM, Molecular-Probes), followed by chromatographic product separation and image analysis as previously described21 with the following modifications: the final composition of the reaction mixture was 10 µL serum, 10 µL zinc sulfate 2 mM, 1.0 nmol substrate in a final volume of 200 µL of 0.1% NP-40/62 mmol Na-acetate, pH 5.0. Incubation was performed at 37°C for 2 h. Product separation was completed using silica gel TLC plates 60 F254 (Merck, Darmstadt, Germany) and a mixture of chloroform/methanol/ammonium-hydroxide (2 M) 65/25/4 (v/v/v). The resulting NBD-labelled ceramide was assessed with an FLA-5000 imaging system using the LBP-filter set at 300 V/100 µm resolution (Fuji-Raytest, Berlin, Germany). Specific activity of S-SMase was calculated by comparison with a calibration curve which was linear for values corresponding to an activity of up to 2000 pmol/mL h. Samples were measured in duplicates, and the mean from each pair of values was used for further analysis. The intra-assay coefficient of variation was <6% and the interassay coefficient of variation was <13%. Activities determined by the use of fluorescent substrate were 85% of the values that were analysed using the radio-labelled substrate for the determination of SMase activity.21

TNF-{alpha} and soluble TNF receptor 1 (sTNF-RI) levels were determined using the human TNF-{alpha} and sTNF-RI immunoassay from R&D systems (Minneapolis, MN, USA). Other biochemical parameters including haemoglobin, sodium, creatinine, uric acid, and cholesterol were analysed using standard hospital analysis procedures.

Blood flow
Forearm blood flow was determined in the dominant arm, using strain-gauge venous occlusion plethysmography (EC4, Hokanson, Bellevue, USA), in 46 patients, as described previously.22 Resting flow (given in mL 100 mL–1 min–1) was assessed in the supine position after a resting period of at least 15 min. Stimulated peak blood flow during reactive hyperaemia was assessed after 3 min of total ischaemia (induced by external proximal suprasystolic compression of the respective limb). The highest flow result after cuff deflation was recorded as post-ischaemic peak blood flow. The ratio peak/resting flow was calculated as an estimate of vasodilator capacity.

Body composition
Body mass index (BMI) was calculated as the ratio of weight (kg) and squared height (m2). Total and regional fat and lean tissue mass were assessed by dual energy X-ray absorptiometry (DEXA) employing a Lunar DPX (Lunar Corp., Madison, WI, USA), as described previously.23 Total body scans were analysed using the extended research mode (version 3.6z software; Lunar Corp.) to obtain total and regional (trunk, arms, and legs) measurements of fat and lean tissue. The measurement error was <2% for lean tissue and <5% for fat tissue. Fat mass of the trunk, termed ‘central fat mass’, includes both visceral and subcutaneous fat of this anatomical region. The sum of tissue mass of the legs and arms was termed ‘peripheral tissue mass’. The distribution of tissue mass was calculated as the ratio of trunk/leg tissue mass.

Spiroergometry
All patients underwent symptom-limited cardiopulmonary treadmill exercise testing (modified Bruce protocol) using a respiratory mass spectrometer (Amis 2000, Odense, Denmark) and a standard inert gas dilution technique for the assessment of peak oxygen consumption (peak VO2), as described previously.24

Muscle strength
Maximal isometric muscle strength of the quadriceps muscle (expressed in N) was measured in 89 patients, as described previously.25 The freely hanging legs of the sitting patients were connected at the ankle with a pressure transducer, and maximal strength was assessed from the best of three contractions on each leg, with a resting period of at least 1 min in between (Multitrace 2, Lectromed, Jersey, Channel Islands). In order to adjust muscle functional capacity for muscle size, we measured the cross-sectional area of the quadriceps muscle in 39 patients, using ultra-fast computed tomography, as described previously (Imatron, San Francisco, USA26). Muscle size-adjusted strength was expressed in Newton/quadriceps cross-sectional area (N/cm2), providing a measure of muscle strength per unit muscle tissue.

Statistical analyses
All results are presented as mean value ± SD. Unpaired Student's t-test and ANOVA, followed by Fisher's post hoc test, were used as appropriate to compare mean values between groups. A P-value < 0.05 was considered significant. Distribution for biochemical variables was evaluated for normality, using the Kolmogorov–Smirnov test, and logarithmic transformation was applied where necessary to allow a parametric statistical approach. To analyse relationships between variables, univariable linear regression (least square method) was performed. As this study has been designed as an observational pilot study, power calculation was not performed. Association of SMase with NYHA classes was analysed using Spearman rank correlation. Cox proportional-hazard analysis was performed using baseline values to assess the association between variables and all-cause mortality. Hazard ratio (HR) and 95% confidence interval (95% CI) are presented. The variables selected for univariable analyses, besides the parameter of interest, i.e. S-SMase, were those which are generally accepted as being of prognostic value in heart failure, such as age, blood pressure, peak VO2, ejection fraction, creatinine, sodium, uric acid, haemoglobin, TNF{alpha}, sTNF-R1, and anthropometric parameters. In order to assess whether S-SMase was an independent prognosticator, multivariable analyses have been performed subsequently with all parameters that were significant in univariable analysis. Related variables were not included in the same model. The proportional hazard assumption of the model was assessed by the inspection of the log time–log hazard plot for all covariates. For statistical analysis, standard statistical software packages were used (SPSS version 13.0; StatView version 4.5, Abacus Concepts, Berkeley, USA).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Conclusion
 Acknowledgements
 References
 
Aetiology of CHF was ischaemic heart disease in 66% of the patients and non-ischaemic heart disease in 34%. Most of the patients were in NYHA class II and III. Within the CHF population, 43 patients (38%) presented with signs of cardiac cachexia. Main clinical characteristics are shown in Table 1. Patients with CHF had impaired exercise capacity compared with healthy controls and impaired central haemodynamic characteristics such as systolic cardiac function and mean blood pressure. Biochemical markers such as sodium, uric acid, and haemoglobin were abnormal in CHF compared with healthy controls. In the hypertensive controls, higher systolic and diastolic blood pressures and BMI were observed (Table 1). Tissue distributions of fat and lean tissues were not different between CHF and both control groups.


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  		Table 1 Clinical, biochemical, and anthropometric characteristics of 112 patients with CHF healthy controls and hypertensive controls

 
S-SMase in CHF in relation to clinical characteristics, biochemical markers, and pro-inflammatory cytokines
Serum S-SMase activity was similar in healthy controls and hypertensive controls (150 ± 121 vs. 157 ± 134 pmol/mL h; P = 0.89). In CHF patients, S-SMase activity was increased by >90% (299 ± 283 pmol/mL h) in comparison with healthy controls (P = 0.004) and with hypertensive patients (P = 0.01; Figure 1). No difference in elevated S-SMase activity was found between patients with ischaemic cardiomyopathy and non-ischaemic cardiomyopathy (283 ± 217 vs. 326 ± 383, respectively, P = 0.98; Figure 2). When subgrouping patients according to NYHA class, a stepwise increase in parallel to disease severity was found (NYHA class I: 206 ± 202, class II: 284 ± 242, class III: 306 ± 212, class IV: 440 ± 665 pmol/mL h; P = 0.0028, {rho} = 0.27, Figure 3).


Figure 1
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  		Figure 1 S-SMase activity in CHF compared with healthy controls and hypertensive controls. HTN, hypertensive.

 

Figure 2
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  		Figure 2 S-SMase activity in CHF patient subgroups according to ischaemic or non-ischaemic aetiology and in CHF patients with and without cardiac cachexia compared with healthy controls. IHD, ischaemic type heart failure; DCMP, non-ischaemic heart failure; cCHF, cachectic CHF; non-cCHF, non-cachectic CHF.*P-values vs. controls; §§, cCHF vs. non-cCHF; P = 0.0007.

 

Figure 3
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  		Figure 3 S-SMase activity in CHF patients according to NYHA functional class (P = 0.0028, {rho} = 0.27).

 
In univariable regression analysis (Table 2), S-SMase activity related to main clinical parameters such as peak VO2 (R = –0.33, P = 0.007; Figure 4A), creatinine (P = 0.0003), uric acid (P = 0.0005), as well as to pro-inflammatory cytokine markers such as TNF-{alpha} (R = 0.22, P = 0.02) and sTNF-R1 (R = 0.39, P < 0.0001). No correlation, however, was found between S-SMase activity and central haemodynamic parameters such as LV ejection fraction and mean arterial blood pressure (Table 2).


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  		Table 2 Univariable correlation analysis of SMase with clinical and biochemical parameters in patients with CHF

 

Figure 4
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  		Figure 4 S-SMase activity in CHF relates to skeletal muscle functional capacity: (A) vs. peak VO2 (mL/kg/min); (B) vs. maximum isometric muscle strength (N); (C) vs. muscle strength/unit muscle tissue (N/cm2).

 
S-SMase and body composition
Subgroup analysis according to the presence of cachexia showed that S-SMase activity was highest in cachectic CHF patients compared with non-cachectic CHF patients and controls (cachectic CHF: 405 ± 357 vs. non-cachectic CHF: 233 ± 202 pmol/mL h; P = 0.0007, Figure 2). The relation of SMase activity to the global and regional parameters of body composition are shown in Table 3. S-SMase activity correlated negatively with BMI (R = –0.25, P = 0.009), with lean tissue total mass and regional distribution (total lean tissue: R = –0.34, P = 0.001; peripheral lean tissue: R = –0.37, P = 0.0003). However, no relation was found between S-SMase activity and global or regional fat tissue parameters.


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  		Table 3 Univariable correlation analysis of SMase with parameters of body composition, skeletal muscle strength, and peripheral vasodilator capacity in patients with CHF

 
S-SMase and skeletal muscle functional performance and blood flow
Maximum isometric muscle strength of the quadriceps muscle correlated negatively with S-SMase activity (R = –0.46, P < 0.0001; Table 3, Figure 4B). When the maximum quadriceps muscle strength was normalized for the muscle cross-sectional area (i.e. strength per unit muscle), the correlation between S-SMase activity and muscle performance was even stronger (R = –0.67, P < 0.0001; Figure 4C). Further, S-SMase activity was correlated with impaired forearm post-ischaemic peak blood flow (R = –0.34, P = 0.02; Figure 5A) and with the ratio peak/resting blood flow (R = –0.32, P = 0.039; Figure 5B).


Figure 5
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  		Figure 5 S-SMase activity in CHF relates to markers of peripheral vasodilator capacity (n = 46). (A) vs. peak post-ischaemic blood flow (mL/100 mL/min); (B) vs. ratio peak/resting blood flow.

 
S-SMase and mortality
The mean follow-up period was 40 months; 54 fatal events were recorded during this period. No patient was lost with regard to survival status during follow-up. The cumulative 1 year mortality of the study population was 19% (95% CI 12–26). In univariable Cox proportional hazard analysis, S-SMase significantly related to mortality (HR 3.05; 95% CI 1.21–7.73; P = 0.019). Further, age (P = 0.006), NYHA class (P = 0.0004), peak VO2, (P = 0.0002), mean BP (P = 0.0002), uric acid (P < 0.0001), and sTNF-R1 (P < 0.0001) predicted survival in our study population but EF, sodium, haemoglobin, BMI, and TNF-{alpha} did not. In multivariable analyses including those parameters that were significant in univariable analysis, S-SMase remained a significant predictor of impaired survival, independent of age, NYHA class, and mean BP (HR 2.92; 95% CI 1.035–8.24; P = 0.04). When peak VO2, sTNF-R1, and uric acid were added to the model, S-SMase lost its independent predictive power (P = 0.37).


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Conclusion
 Acknowledgements
 References
 
This study demonstrates that S-SMase activity is elevated in patients with CHF compared with subjects with no heart failure. The elevation of S-SMase occurs similarly in ischaemic and non-ischaemic CHF and is associated with disease severity of CHF as indicated by NYHA functional class and by peak oxygen uptake. SMase activity was highest in cachectic patients. Elevated S-SMase activity was related to impaired vasodilator capacity and to structural and functional loss of skeletal muscle tissue. Furthermore, S-SMase emerged as marker of increased mortality, independent of age and body composition parameters, NYHA, LVEF, and TNF-{alpha}. To the best of our knowledge, this is the first study to characterize S-SMase in CHF patients.

S-SMase is secreted by endothelial cells upon stimulation with LPS and pro-inflammatory cytokines such as TNF-{alpha}6,27 forming ceramide from sphingomyelin. Ceramide may act as second messenger in various signalling pathways or is further metabolized to form other bioactive sphingolipids. In accord with this, substantial amounts of sphingomyelin in the outer membrane leaflet provide an abundant source of substrate for enzymatic metabolism by circulating S-SMase. Current evidence suggests a role for S-SMase in paracrine signalling involved in subendothelial lipoprotein aggregation during atherogenesis,27 in apoptosis, cellular differentiation, and senescence.28 Our findings extend the available data on immune activation regulatory processes in CHF. Elevated levels of cytokines (especially of TNF-{alpha}),29 LPS,30 and increased oxygen radical load31 are characteristic findings in CHF, and accordingly, we have found a correlation between S-SMase and TNF-{alpha} and the sTNF-R1. As we have shown previously, increased oxidative stress may directly contribute to activation of S-SMase.8 This suggests that immune activation in CHF may be mediated at least in part via the sphingomyelin/ceramide pathway.

Previous studies have shown that increased plasma cytokine levels in CHF patients are strongly correlated with impaired peripheral blood flow.15 TNF-{alpha} has been described to impair stimulated endothelium-dependent vasorelaxation. Interestingly, although the TNF-{alpha}-inhibiting therapeutic concept with etanercept, a TNF-R2 fusion protein, ultimately failed to exert clinical benefit in CHF,32 etanercept has been observed to improve both patients' functional status and endothelium vasodilator capacity in vivo as measured by forearm blood flow.33 Moreover, Zhang et al.34 found the inhibitory effect of TNF-{alpha} on the vasoreactivity of coronary arteries to depend on an increased SMase activity. Desipramine, a potent inhibitor of acidic SMase, nearly completely abrogated the effect of TNF-{alpha}. In our study, we show that elevated S-SMase activity correlated with the impairment of peripheral blood flow and vasodilator capacity in CHF. Accordingly, on the basis of our present data, we extend the concept of the outlined studies for CHF and hypothesize that the sphingomyelin/ceramide second messenger system represents a signalling link between cytokine accumulation and impaired vasodilator capacity—a hallmark of CHF pathophysiology.

Notwithstanding the observed association between SMase and impaired blood flow in CHF, S-SMase activity in the hypertensive control group was not different to the healthy control group. Endothelial dysfunction in hypertension, however, appears to be less dependent of the sphingomyelin pathway. Previous studies have shown that modulation of vascular tone by sphingomyelin metabolites is not uniform in all conditions35 and it is still a matter of debate, whether metabolites of sphingomelin favour vasoconstriction in chronic hypertension. So far, only data from animal studies with spontaneously hypertensive rats exist. In a recent study, Ryu et al.36 observed an increased vasoconstriction in mesenterial arteries of spontaneously hypertensive rats in response to sphingosylphosphorylcholine. Conversely, other groups showed ceramide signalling to be impaired in these animals37 and observed a less pronounced vasoconstrictive response to various sphingomyelin metabolites such as sphingosin-1-phosphate, sphingosylphophorylcholine, and ceramide.38 This might be due to an altered sphingomyelin metabolite receptor expression.39

Previously, S-SMase secretion was linked to the pathogenesis of atherosclerosis. However, it is the basolateral-directed secretion of acid SMase which is relevant for plaque formation.27,39 In the present study, the plasmatic activity of S-SMase did not relate to atherosclerotic disease underlying ischaemic CHF, as S-SMase activity was not different between ischaemic and non-ischaemic aetiology of CHF.

Cardiac cachexia has been recognized as an important feature of advanced CHF, indicative of severe symptomatic status and particular poor prognosis.17 Consistent with evidence of raised TNF-{alpha} plasma levels in cachectic patients,40 we found S-SMase, one of its mediators, to be highest in the subgroup of cachectic patients. Cachectic CHF patients are known to fatigue earlier, which has been attributed to both reduced skeletal muscle mass and impaired muscle quality. Although cardiac cachexia is characterized by a general loss of fat tissue, lean tissue, and bone tissue, it should be noted that muscle loss starts early in the process of wasting, being significant long before cachexia would become apparent.41 In our study, elevated S-SMase activity correlated with global parameters of body composition, such as weight and BMI. In detailed analysis, however, S-SMase activity was found to negatively correlate with lean tissue parameters (total mass and regional distribution) only, whereas no association was found for fat tissue parameters. This is in line with the observations of ceramide being involved in catabolic pathways of skeletal muscle, leading to muscle wasting. Ceramide has recently been shown to be the key downstream messenger for cytokine-induced impairment of muscle protein synthesis by blocking IGF-1 receptor-dependent signalling.18 Growth hormone resistance with inadequate IGF-1 response has previously been reported to occur in CHF,42 being part of the catabolic/anabolic imbalance that ultimately progresses to cardiac cachexia. In a recent ex vivo study by Hyde et al.,43 a significant diminution of the intracellular amino acid pool in response to ceramide was observed with ceramide negatively affecting both protein synthesis and amino acid transport in the skeletal muscle. Skeletal muscle is the largest compartment for the storage of unbound and protein-bound amino acids, with central importance for the body's amino acid economy. Hence, the impact of ceramide on skeletal muscle metabolism may affect the whole-body protein and energy turnover. Thus, the observed elevation in S-SMase activity in CHF suggests that S-SMase may contribute as an regulatory pathway to the pathogenesis of cardiac cachexia.

Besides the association of S-SMase with body composition and specifically with muscle tissue amount, we observed a strong negative correlation between S-SMase activity and functional skeletal muscle performance. Not only total strength but also muscle size-corrected strength, i.e. strength per unit as a measure of functional quality, related inversely to S-SMase levels. In accord, physical training to improve exercise capacity in CHF patients44 also results in decreased ceramide concentration in skeletal muscle with subsequent downregulation of the sphingomyelin-signalling pathway.45

The study was designed as an observational pilot study to characterize S-SMase for the first time in the setting of CHF in relation to key pathophysiological characteristics of the disease. Those data are associative and our observations alone may not allow a causal interpretation. The pilot characteristic of our study is also apparent from the small control groups, which may be a limitation of the study. Our data, however, complement previous studies on the role of S-SMase and ceramide-signalling in immune regulation and muscle structural and functional regulation. Viewed in this context, it may be hypothesized that S-SMase may be involved in pathophysiological processes in CHF. Being secreted into plasma and catalysing the initial and pivotal step in the metabolic cascade of sphingomyelin, the increased levels of S-SMase may be interpreted as an indicator of a systemic activation of the sphingomyelin pathway in CHF patients. This signalling pathway is characterized by complex interactivity and functional diversity of ceramide and the downstream sphingomyelin metabolites. Differing effects of ceramide and other metabolites such as sphingosine or sphingosine-1-phosphate on the heart have been described, and vasculature and even cardioprotective properties have been observed for sphingosine-1-phosphate (for review, see Alewijnse et al.35). Whether ceramide or other related metabolites dominate the pathophysiological processes depends on the expression and regulation of additional enzymes of sphingomyelin metabolism and may vary among different tissues. The net effect results from changes in the balance of sphingomyelin metabolites and is far from being understood. One could speculate that the unexpected neutral results of the etanercept trials in CHF may result in part from the imbalanced impact of etanercept on negative and protective cytokine effects mediated by the sphingomylin metabolites. Further studies are needed for a more comprehensive understanding of the role of the different sphingomyelin metabolites in CHF following activation of this pathway.


   Conclusion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
Acknowledgements
 References
 
We have shown that S-SMase activity is elevated in CHF, independent of aetiology. S-SMase activity in CHF is associated with clinical status and disease severity and relates to functional and structural impairment of skeletal muscle tissue and to impaired peripheral vasodilator capacity. Our study provides further data to support the concept of S-SMase-dependent signalling to contribute to CHF pathophysiology as a link between immune activation and wasting in CHF. Further studies are needed to test whether S-SMase-dependent signalling may represent a potential target for intervention to improve vasoreactivity and to impede muscular wasting in CHF.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
References
 
W.D. was supported by the ‘Verein der Freunde und Förderer der Berliner Charité’, Germany and the NHLI London, UK. R.A.C. and A.C.B. acknowledge financial support from IZKF Jena, TP2.2. S.v.H. is supported by the German Heart Foundation, Frankfurt, Germany. S.D.A. is supported with a Vandervell Fellowship and a donation from Hubert Bailey. The Division of Applied Cachexia Research is supported with a grant from the Charite Medical School.

Conflict of interest: none declared.


   Footnotes
 
{dagger} Co-corresponding author. Tel: +49 3641 9325860; fax: +49 3641 9325862. E-mail address: ralf.claus{at}med.uni-jena.de Back

{ddagger} These authors contributed equally to this article. Back


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusion
 Acknowledgements
 References
 

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