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European Journal of Heart Failure 2005 7(6):997-1002; doi:10.1016/j.ejheart.2004.11.010
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© 2005 European Society of Cardiology

Inflammation and perturbation of the L-carnitine system in heart failure

Giorgio Vescovoa, Barbara Ravarab, Valerio Gobbob and Luciano Dalla Liberab,*

a Internal Medicine 1, S. Bortolo Hospital Vicenza, Italy
b CNR Institute of Neurosciences, Unit for Neuromuscular Biology and Pathophysiology, Department of Biomedical Sciences, University of Padua Viale G. Colombo 3, 35100 Padova, Italy

* Corresponding author. Tel.: +39 49 8276031; fax: +39 49 8276040. E-mail address: ldl{at}bio.unipd.it


   Abstract
Top
 Abstract
1. Introduction
 2. Patients and methods
 4. Discussion
 References
 
Background: Heart failure (HF) is accompanied by elevated levels of pro-inflammatory cytokines. Skeletal muscle myopathy with atrophy of fibres, decreased oxidative metabolism and preferential synthesis of fast myosin heavy chains (MHCs) occurs, which contributes to the worsening of symptoms. L-Carnitine has been shown to be protective against the apoptosis-induced atrophy of fibres and fast MHCs shift.

Aims: To investigate the interrelationship between TNF{alpha} and sphingosine (SPH), which induce muscle wastage, and plasma levels of L-carnitine.

Methods: We studied 18 heart failure patients and correlated NYHA class and ventricular function with the plasma concentration of these molecules.

Results: TNF{alpha} and SPH levels were raised and correlated with the severity of HF. L-Carnitine levels were increased in HF patients, but decreased according to the severity of cardiac decompensation.

Conclusions: The increased levels of L-carnitine are likely due to release from the damaged muscle, reduced urinary excretion, decreased dietary intake and liver synthesis (malnutrition). It is possible that the cytokine-induced muscle wastage is not counterbalanced by the beneficial metabolic effects of L-carnitine, the metabolism of which is profoundly perturbed in CHF. L-Carnitine supplementation may produce positive effects on the skeletal muscle, as has been shown in animal models of HF.

Key Words: Heart failure • L-carnitine • TNF{alpha} • Sphingosine • Inflammation

Received September 15, 2004; Revised November 24, 2004; Accepted November 25, 2004


   1. Introduction
Top
 Abstract
 1. Introduction
2. Patients and methods
 4. Discussion
 References
 
Heart failure (HF) is a clinical syndrome characterized by decreased exercise capacity [1,2]. The reason for this decreased exercise tolerance requires further investigation. Skeletal muscle has been shown to be involved in the origin of symptoms and changes in the skeletal muscle leading to decreased muscle performance have been described [3–5]. These changes include muscle atrophy, switch from slow type I to fast type IIa and IIb fibres, increased synthesis of fast myosin heavy chains (MHCs), decreased oxidative metabolism with decreased mitochondria and cytochrome c [6,7]. The cause of these changes is not entirely understood, but a link between inflammatory status, with increased levels of pro-inflammatory cytokines, has been suggested [8]. Oxidative stress has been proposed as an upstream mechanism for production of NF-{kappa}B and proinflammatory cytokines [9]. Increased levels of TNF{alpha} and its second messenger sphingosine have been shown to produce skeletal muscle apoptosis [8], muscle wastage through the proteasome-ubiquitine cascade [10] and changes in fibre type probably acting on the NF-{kappa}B-calcineurine-PGC1{alpha} cascade [11], or antagonizing the insulin/IGF1pathway [12,13]. GH and L-carnitine have been shown to prevent the cardiomyopathy by both blocking apoptosis and by preventing the shift from slow to fast fibre type [7,14]. In particular, L-carnitine has been shown to have a protective effect on the skeletal muscle of animals with heart failure [14]. L-Carnitine has in fact shown antiapoptotic properties by blocking sphingomyelinase and preventing oxidative stress and contractile protein oxidation. At the same time in an animal model of heart failure, we have been able to demonstrate the ability of L-carnitine to restore the MHCs pattern by inducing the preferential synthesis of the slow isoforms [14].

In this paper, we investigate the possible relationship between levels of inflammatory cytokines and molecules with a protective effect on the skeletal muscle, in patients with heart failure.


   2. Patients and methods
Top
 Abstract
 1. Introduction
 2. Patients and methods
4. Discussion
 References
 
We studied 18 patients with NYHA classes I to IV heart failure. The demographics of these patients are summarized in Table 1. Eight healthy subjects were used as controls.


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  		Table 1 Demographic data from 18 heart failure (HF) patients and 8 controls (CON)

 
All the patients underwent echocardiography, with the evaluation of ejection fraction (EF). NYHA class was determined as well as Diuretic Score, which is an index of diuretic consumption and HF severity [15] according to frusemide dosage.

The study was approved by the hospital Ethics Committee and all patients gave informed consent.

2.1. Assessment of urinary and serum levels of L-carnitine
L-Carnitine was measured in urine and in serum as described by Longo et al. [16] and detailed in Vescovo et al. [14].

Carnitine clearance was measured with the following equation:


Formula

2.2. Assessment of serum levels of TNF{alpha}
TNF{alpha} was measured with a solid-phase sandwich ELISA, using a monoclonal antibody specific for human TNF{alpha} (Biosource International).

2.3. Assessment of serum levels of sphingosine
SPH was determined with high-performance liquid chromatography according to the method previously described by Dalla Libera et al. [8].

   2.4. Statistical analysis
Mean±S.D. are reported. Differences between groups were studied by means of Student's t-test for unpaired data and analysis of variance. A 5% difference was considered statistically significant. Linear regression between variables was also used.

3. Results
Patient demographics are summarized in Table 1.

HF patients had significantly higher levels of pro-inflammatory cytokines. SPH was significantly higher in HF patients than in controls (p<0.05). A significance was also seen for TNF{alpha} (p<0.003) (see Table 2).


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  		Table 2 Pro-inflammatory cytokine levels in HF patients and controls

 
L-Carnitine was slightly higher in HF patients (p<0.001).

There was however a clear trend for SPH (p=0.04) and TNF{alpha} (p=0.13) to be significantly elevated in patients with higher NYHA classes.

L-Carnitine, when analysed for individual NYHA classes, tended to be lower in patients with more severe breathlessness (p=0.09).

There was no difference in BMI between the four NYHA classes (24.5±4.8 NYHA I, 25.2±3.0 NYHA II, 25.4±1.8 NYHA III, 25.1± 0.6 NYHA IV, p=0.75 ANOVA).

Carnitine clearance was 4.2±2.7 nmol/ml/min in CHF patients vs. 5.4±4.5 (p<0.03) in controls.

There was no correlation between diuretic score and L-carnitine levels, r2=–0.6, p=NS.

3.1. Correlation between cytokines and EF
There was a significant positive correlation between the plasma levels of SPH and EF (r2=0.31, p<0.024) and a similar correlation between TNF{alpha} and EF (r2=0.3, p=0.06).

3.2. Correlation between TNF{alpha} and SPH
The plasma levels of SPH and TNF{alpha} were positively and significantly correlated (r2=0.25, p<0.04).

   3.3. Correlation between cytokines and L-carnitine
Within the heart failure patients, there was a negative but statistically highly significant correlation between the plasma levels of L-carnitine and SPH (r2=0.66, p<0.0001), while there was also a trend to significance for L-carnitine and TNF{alpha} (r2=0.16, p<0.06).

   3.4. Correlation between L-carnitine and EF
Within the group of heart failure patients, there was a significant correlation between EF and plasma levels of L-carnitine (r2=0.31, p=0.011).

3.5. Correlation between 24-h excretion of L-carnitine and EF We found a significant correlation between EF and 24 h urinary excretion of L-carnitine (r2=0.32, p=0.027). When carnitine clearance was correlated with NYHA class, we found a highly significant negative correlation between these two parameters (r2=0.546, p=0.002).

3.6. Correlation between creatinine and L-carnitine We could not find any correlation between L-carnitine and creatinine levels (r2=0.0013, p=0.37).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Patients and methods
 4. Discussion
References
 
The data from this present study, showing elevation of circulating cytokines in HF patients, confirm those previously observed in man and in animal models of heart failure [8,17]. We have clearly demonstrated that both SPH and TNF{alpha} are increased in HF patients when compared to controls. Moreover, the levels of these cytokines are increased for more severe NYHA classes. There was also a significant correlation between EF and SPH and TNF{alpha} plasma levels, confirming that those patients with more depressed EF have higher degree of inflammation both in terms of TNF{alpha} and its second messenger SPH. The role of inflammation is gaining importance in the clinical context. Inflammation has been linked to many processes leading to the deterioration of the heart failure patient [9,18]. The effect of inflammation on the heart itself with the negative inotropic effect of cytokines is well known, but even more important are the effects of the pro-inflammatory cytokines on other organs [19,20]. In the kidney, haemodynamics and function are profoundly altered by pro-inflammatory cytokines, which produce a further deterioration in renal function. The skeletal muscle is also influenced by inflammation. Inflammation can produce muscle wastage through several mechanisms: skeletal muscle apoptosis which is triggered by TNF{alpha} and SPH, protein degradation which occurs via the TNF{alpha}-ubiquitine-proteasome pathway, loss of appetite and perturbation of the balance between anabolic and catabolic hormones (see for review [18]). Cytokines can therefore lead to loss of muscle mass, which by itself influences exercise capacity [2,3], but they also play an important role in the genesis of fatigue [21]. Moreover, TNF{alpha} by interfering with NF-{kappa}B may act on the GH-IGF1-calcineurine-PGC1{alpha} pathway [12,10] and therefore influence the expression of slow skeletal muscle fibres, by shifting production towards the fast, glycolytic component, with the well-known negative consequences on muscle performance.

In this paper, in addition to TNF{alpha}, we also measured plasma levels of SPH, a sphingolipid which is considered a second messenger of TNF{alpha}. SPH has been shown to be produced in several organs, including the heart, by stimulation of TNFR1 which activates FAN (factor activating neutral sphingomyelinase) and NSMase (neutral sphingomyelinase) [22]. SPH is then released into the circulation, producing effects that mainly consist of activation of the apoptotic cascade, which in skeletal muscle has been shown to occur both in vivo and in vitro.

Patients with heart failure have increased levels of plasma L-carnitine, as if there was an attempt by the body to retain L-carnitine, maybe to prevent cellular apoptosis [14]. Levels of L-carnitine do not seem to be influenced by diuretic therapy, in fact there is no correlation between diuretic consumption (diuretic score) and L-carnitine levels, nor ACEI treatment (all the patients in our study were on ACEIs or ARBs). In addition, BMI index does not seem to influence L-carnitine levels, in our study BMI was the same in all the four NYHA classes. This is not the first reported observation that plasma levels of L-carnitine are raised in CHF patients. El-Aroussy et al. [23] showed that plasma concentrations of L-carnitine were increased in patients with dilated cardiomyopathy, as was urinary L-carnitine excretion. They postulated that the increased levels may be due to leakage from damaged muscle tissue or to deficient L-carnitine transport into cells. In our study, we confirmed that plasma L-carnitine was increased in HF patients when compared to controls; however, we observed that with the progression of the severity of the disease, plasma levels of L-carnitine decreased as shown by the correlation between EF, NYHA class and L-carnitine. In other words, our results suggest that levels of L-carnitine are lower the more severe the heart failure and the higher the degree of inflammation. This latter hypothesis is confirmed by the fact that plasma levels of L-carnitine are inversely correlated with both the plasma SPH and TNF{alpha}. The finding of perturbation of L-carnitine metabolism with the progression of heart failure is not new. In fact beyond the alteration in plasma levels, we previously found, in rats with heart failure, that the muscle content of L-carnitine is decreased [14]. There may be other explanations why L-carnitine decreases with the severity of HF, including reduced dietary intake, reduced renal reabsorption or reduced liver synthesis [24]. First of all, the 24 urinary excretion of L-carnitine was similar in the Control and HF patients, although the HF patients had a trend to lower values. Moreover, there was no excessive renal loss of L-carnitine in the sicker HF patients, in that we found a positive correlation between cardiac function and L-carnitine renal excretion, in fact patients with lower EF also had lower urinary output of L-carnitine. It looks as if patients with lower EF had a tendency to retain L-carnitine, maybe because of the decreased renal blood flow and the more impaired kidney function. The reduced intake and the reduced liver synthesis may occur in patients with HF, in whom the severity of the disease in accompanied by progressive malnutrition and worsening of liver function because of venous congestion. It is however clear that heart failure patients have muscle atrophy, reduced levels of muscle L-carnitine and, as shown in this report, progressively lower levels of circulating and muscle L-carnitine. It may be speculated on the role of the decreased levels of L-carnitine, since we know that it has an antiapoptotic effect, being a potent inhibitor of sphingomyelinase [22,25], a trophic action on skeletal muscle and protective activity on the synthesis of slow MHCs and fibres [14], perhaps mediated by the preferential activation of the mytochondrial and oxidative metabolism [26]. We have previously demonstrated in an animal model of heart failure that L-carnitine can prevent skeletal muscle apoptosis by blocking sphingomyelinase and therefore the production of SPH, or by inhibiting the cleavage of caspases at mitochondrial level [14]. Similarly, L-carnitine, by stimulating fatty acids β-oxidation and energy production at mitochondrial level, may produce slow MHCs synthesis via PGC1{alpha} and Nrt1 interplay [27].

It is clear that in these patients there is a vicious circle that leads to progressive deterioration of muscle function. Although we acknowledge that the number of patients of this study was small and the correlations were only modest, we have proposed a mechanism which is illustrated in Fig. 1. HF determines inflammation, malnutrition and kidney failure. All these pathophysiological alterations have a negative effect on the skeletal muscle that develops atrophy through apoptosis and protein wastage and changes in fibres and MHCs type and metabolism. Possible protective mechanisms on the muscle are impaired or lost. One of these, which is operated by L-carnitine is blunted. L-Carnitine muscle content is decreased and plasma levels [14] of L-carnitine are perturbed because of malnutrition, which causes reduced dietary intake and reduced liver production, kidney abnormalities, and muscle damage which tend to make the plasma levels of L-carnitine higher. However, as HF progresses, the L-carnitine system perturbation worsens and this can contribute to the decline in the clinical status of the patient, with worsening symptoms, deterioration in exercise capacity and cardiac cachexia [28]. It may be possible, if the source of increased plasma L-carnitine is the release from the damaged muscle, that in patients with more severe HF, the release from the muscle is reduced because muscle bulk is less. This is also paralleled by a more severe malnutrition and impaired liver function and endogenous synthesis of L-carnitine. However, it is important to note that levels of L-carnitine decrease for increasing levels of SPH and TNF{alpha}, as the more severe the inflammation the lower the availability of L-carnitine.


Figure 1
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  		Fig. 1 Proposed mechanism of effect of heart failure on L-carnitine.

 
Whether L-carnitine treatment might arrest the development of HF myopathy, as has been shown in rats with heart failure [14], with consequent improvement of symptoms, such as fatigue, may be worthy of further investigation.


   Acknowledgements
 
The financial support of Telethon-Italy for the project "Inhibition of apoptosis of skeletal muscle fibers in heart failure as a therapeutic tool to antagonize muscle atrophy" (Grant no. GGP02077 to L.D.L.) is gratefully acknowledged, as well as PRIN 2003–2005. We thank Dr. Pierluigi Di Loreto for expert advice.


   References
Top
 Abstract
 1. Introduction
 2. Patients and methods
 4. Discussion
 References
 

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