© 2002 European Society of Cardiology
Effect of thalidomide on the skeletal muscle in experimental heart failure
a Internal Medicine Adria Hospital, 45011 Adria, (RO), Italy
b CNR Unit for Muscle Biology and Physiopathology, Department of Biomedical Sciences University of Padova, Padova, Italy
c Cardiovascular Pathology University of Padova, Padova, Italy
d Cardiac Physiology Gussago (BS), Italy
ldl{at}civ.bio.unipd.it
* Corresponding author. Tel.: +39-0426-940-451; fax: +39-049-827-6040
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Background: Tumour Necrosis Factor
(TNF) has been shown to contribute to heart failure (CHF) progression.
Aims: We have tried to antagonise the detrimental effects of TNF on skeletal muscle apoptosis, by using thalidomide, a drug that inhibits its biosynthesis.
Methods: CHF was induced in 20 rats by injecting monocrotaline, which determines right ventricle (RV) failure. After 2 weeks, when CHF developed, 12 rats were treated with thalidomide 3.5.mg/kg per day for 2 weeks. Eight had saline and served as CHF controls.
Results: Thalidomide failed to decrease TNF and its second messenger sphingosine (SPH), but was able to prevent the shift toward the fast myosin heavy chains. In the Tibialis Anterior muscle of the thalidomide group, the degree of atrophy, the number of apoptotic nuclei and the levels of caspases, were similar to those of the CHF controls.
Conclusions: Thalidomide, at the doses used in this study, which are the same employed for the treatment of tubercolosis, leprosy, AIDS and cancer in humans, did not lower either TNF or SPH and only marginally influenced the apoptosis-induced muscle atrophy. Since other TNF blockers are under investigation for improving the clinical status of patients with CHF, the present data could be relevant in the design of randomised clinical trials in humans.
Key Words: Apoptosis • Cytokines • Heart failure • Skeletal muscle, TNF • Sphingosine
Received August 30, 2001; Revised October 31, 2001; Accepted January 17, 2002
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Since the first observations of elevated levels of Tumour Necrosis Factor
(TNF) in patients with heart failure (CHF) [1], there have been several speculations on the role played by this cytokine in worsening the clinical syndrome. TNF could have a cytotoxic effect on the heart by inducing myocyte apoptosis [2] or by decreasing cardiac contractility [3]. Enbrel (Etanercept), a soluble P75 TNF receptor fusion protein, can lower biologically active TNF and improve the functional status of CHF patients [4]. Several other substances have been used to block TNF in various diseases, such as Infliximab in rheumatoid arthritis, pentoxyfilline in dilated cardiomyopathies [5], and thalidomide in tuberculosis, AIDS and leprosy [6].
TNF may trigger apoptosis, the cause of skeletal muscle atrophy, one of the most important determinants of exercise capacity (EC) [7,8] in patients and animals with CHF. TNF may induce cardiac contractility depression [9] and skeletal muscle apoptosis through sphingosine (SPH) a second messenger released by cardiac myocytes when TNF binds to their membrane [2,10]. SPH is responsible for many of the cytotoxic effects of TNF. TNF also plays a pleiotropic cytotoxic role in cardiac cachexia, which is characterised by muscle waste and extreme weight loss [10,11]. In this study, we tested the hypothesis that thalidomide, an inhibitor of TNF production [6,12–14], could prevent skeletal muscle apoptosis and atrophy in an animal model of CHF, forming the pathophysiological rationale for using thalidomide in humans with CHF. TNF inhibition may in fact improve EC, not only by acting on heart function, but also preventing skeletal muscle waste.
We tested this hypothesis in the monocrotaline (M) model of heart failure. This model mimics the CHF syndrome in man, in terms both of skeletal muscle changes and neuro-hormonal activation [7,10,15,16].
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.1. Animals
Four groups of animals were studied. (A) Five controls; (B) 12, 80–100 g Sprague Dawley rats, injected with 30 mg/kg monocrotaline (M) intraperitoneally, an alkaloid able to produce pulmonary hypertension, followed by right ventricular (RV) hypertrophy and failure, without itself producing changes in skeletal muscle myosin heavy chain (MHC) composition and apoptosis [7,10,15,16]. They received, from the 3rd week of treatment, thalidomide [3.5 mg/kg per day, dissolved in dimethyl sulfoxide–ethanol (1:1.4) delivered by Alzet (Palo Alto, CA, USA) osmotic minipumps]. (C) Eight animals injected with the same dosage of M that received, from the 3rd week, a saline infusion (CHF group). In addition, since dimethyl sulfoxide is known to be a free radicals scavenger, we studied five control animals (D) in which the same amount of dimethyl sulfoxide–ethanol was administered with minipumps. After 4 weeks, all the animals were killed. The Tibialis Anterior (TA) muscle which, beyond the fact that it is one of the biggest hind limb muscles, has been well characterised in our previous works in CHF, was excised, together with the heart. TA was immediately frozen in liquid nitrogen and stored at –80 °C until used. RV Mass/LV Mass and RV Mass/RV Volume Index were calculated with a computerised planimeter on transverse sections of the heart, stained with ematoxylin-eosin [7]. Experiments were approved by the Ethical Committee of the Interdepartmental Biological Building of the University of Padova and were carried out according to the Italian laws, which is in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH).
2.2. Electrophoretic separation of myosin heavy chains (MHCs)
MHCs [MHC2a (fast oxidative) and MHC2b (fast glycolytic)] of TA, were separated using the electrophoretic method previously described [8] and their percent distribution determined by densitometric scan with a Jandel Scientific computerised system.
2.3. Degree of fiber atrophy
The degree of fiber atrophy was assessed on histologic slides, by measuring the fibers cross-sectional area with an image analyser Zeiss IBAS 2000 [7].
2.4. Assessment of apoptosis
In situ DNA nick-end labelling (TUNEL) (Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling) of fragmented DNA was performed as previously described [17].
2.5. Western blot for pro-Caspase 3, activated Caspases 3 and 9, and Bcl-2
Western blot was performed as previously described [17]. Anti-pro-Caspase 3 (34 kDa), anti-Bcl-2 (29 kDa) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-cleaved Caspase-3 (17 kDa) and anti-cleaved Caspase-9 (37 kDa) Cell Signaling Technology (Beverly MA, USA) were used. The absolute values were calculated on blot bands with the above described densitometric method and expressed as percent of controls.
2.6. DNA isolation, PCR amplification and analysis (DNA-ladder)
DNA was extracted from TA muscle and the ApoAlert LM-PCR Ladder assay (Clontech, Palo Alto, CA, USA) was used to amplify the detection of nucleosomal ladder on 1.8% agarose gel [17].
2.7. Confocal microscopy immunofluorescence
Frozen sections were incubated with anti-activated-Caspase-3 antibody, diluted 1:50, incubated with anti-rabbit Cy3-conjugated antibody and then analysed with a Bio-Rad (Hercules, CA, USA) Confocal Microscopy [17].
2.8. Angiotensin II (AngII) assay
AngII was measured on serum using an enzyme-immunometric assay kit (SPI-BIO, Massy, France) [17].
2.9. TNF and SPH
TNF was measured with a solid phase sandwich ELISA (Euroclone, UK). SPH was measured with the HPLC method described by Dalla Libera et al. [10].
2.10. Statistical analysis
Mean±S.D. are reported. Student t-test and ANOVA were used.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.1. Occurrence of CHF in the monocrotaline and thalidomide animals (Table 1)
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After 4 weeks, all the MCHF rats showed, at post-mortem examination, the presence of pericardial, pleural and peritoneal effusions. LVM/RVM was 2.7±0.6 vs. 3.5±0.6 in the control group (P<0.04), and 3.0±0.3 in the thalidomide group (P=0.04 vs. controls). The right ventricular cavity was markedly dilated in all the CHF rats, as reflected by the RVM/V index that was 0.25±0.15 in MCHF vs. 1.12±0.7 in C (P=0.019) and 0.35±0.09 in thalidomide (P=0.04 vs. CHF and P=0.014 vs. control). Dimethyl sulfoxide animals behaved as the control animal treated with saline (LVM/RVM 3.4±0.4, RVM/V 1.1±0.5).
3.2. Degree of muscle atrophy
The cross-sectional area of the TA fibres was 1370±430 µm2 in the CHF, 2110±300 in the control animals (P=0.019) and 1590±240 in the thalidomide group (P=0.05 vs. controls). In the dimethyl sulfoxide group, the cross-sectional area was similar to that of control group.
3.3. MHCs pattern
The electrophoretic pattern of the TA in the CHF animals showed a significant shift toward the fast glycolitic isoform. MHC2a decreased from 26.0±1.7% (C) to 18.0±3.0 (P=0.005). MHC2b increased from 74.0±1.7% to 81.0±3.0 (P=0.005). In the thalidomide group, MHC2a was 27.0±5.0 (P=0.04 vs. CHF) and MHC2b 72.0±3.0 (P=0.04 vs. CHF). Dimethyl sulfoxide rats showed the same electrophoretic pattern of control.
3.4. Count of in situ DNA nick-end labelling (TUNEL) positive nuclei (Table 2)
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The count of the total TUNEL positive nuclei in the TA of C was 1.2±2.8 mm3 vs. 39.6±42.7 (P=0.004) in the CHF group. In the thalidomide group, we found 26.2±25.2 TUNEL positive nuclei (P<0.03 vs. control) (Fig. 1 C,C’). Myocyte TUNEL positive nuclei count was 0.4±0.8 in the control group, 12.6±11.9 in the CHF group and 9.0±8.1 in the thalidomide group. In dimethyl sulfoxide animals, the number of TUNEL positive nuclei was 1.1±2.1.
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3.5. Bcl-2
Bcl-2 was 97±12 in the control group, which was similar to that of the thalidomide animals ( 96±17, P=N.S.). In the CHF rats, Bcl-2 was 70±16 (P=0.02 vs. both the control group and P=0.007 vs. thalidomide).
3.6. Pro-Caspase-3
Pro-Caspase-3 was significantly increased in the CHF rats when compared to the control animals (206±80 vs. 100±8, P=0.05). In the thalidomide group, though lower than that of the CHF group, Pro-Caspase-3 was still significantly higher than that of the control animals (140±27, P=0.03).
3.7. Activated Caspases-3 and 9 (Fig. 1A,B)
We confirmed the occurrence of apoptosis in the CHF and thalidomide animals that showed a high number of TUNEL positive nuclei, by revealing, with immunoblotting, the presence of activated Caspase-9 and 3 (the mitochondrial regulatory and executioner). They were negative in the control and dimethyl sulfoxide groups. We also demonstrated the positivity of activated Caspase-3 with confocal microscopy in the thalidomide rats.
3.8. DNA ladder (Fig. 1D)
In our model of CHF, where the percentage of apoptotic cells is low, the genomic DNA ladder is very often not visible. For this reason, we used a PCR assay to specifically amplify the nucleosomal ladder. We found a positive DNA ladder in the CHF and thalidomide groups. The ladder was negative in the control and dimethyl sulfoxide groups (results not shown).
3.9. TNF-
There was a rise in plasma TNF in the CHF animals (270±10 vs. 105±9 pg/ml, P=0.001). In the thalidomide group it did not differ from CHF, but it was significantly higher than the controls (280±30, P=0.004). In the dimethyl sulfoxide animals, it was 101±15.
3.10. Sphingosine
SPH was significantly increased in the CHF rats (1161±452 vs. 743±148 pmol/ml, P=0.04 vs. control) and did not show any changes after thalidomide treatment (1215±550, P=N.S. vs. CHF, P=0.04 vs. control).
3.11. AngII
Plasma AngII in MCHF and thalidomide was 89±8 pg/ml and 85±7, respectively. In the control group it was 26±5 (P=0.008 vs. thalidomide and CHF) and it was similar in the dimethyl sulfoxide group (22±4).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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This is the first report on the effect of thalidomide, used as a blocker of TNF
synthesis, on the skeletal muscle in CHF. Although thalidomide and its analogues have a broad spectrum of properties, ranging from anti-inflammatory [13,18] to anti-angiogenetic [18] and anti-tumoral activities [19], which may induce several dichotomous variation in Interleukins and Interferons secretion, its major action is to inhibit TNF synthesis [6,12–14]. The rationale for blocking TNF with thalidomide, was to prevent the well-known detrimental cardiovascular effects of TNF itself. It has, in fact, been demonstrated that TNF is elevated in CHF, together with other pro-inflammatory cytokines [20,21] and in patients with cardiac cachexya and muscle waste [22].
We have also demonstrated a link between the degree of muscle atrophy and the magnitude of skeletal muscle apoptosis [23], suggesting that apoptosis could be one of the major causes of skeletal muscle waste. Preventing muscle atrophy could be one of the targets for improving exercise capacity in patients with CHF, since muscle trophism is an independent predictor and determinant of it [8,23–25]. At the same time we have previously shown that TNF plasma levels and skeletal muscle apoptosis can be lowered, preventing the development of muscle atrophy, even by intervening on other mechanisms, such as angiotensin II receptors [17].
The effects of thalidomide were studied in a well established model of CHF, the monocrotaline-treated rat [7,15,17]. Monocrotaline is able to produce pulmonary hypertension, followed by RV hypertrophy and failure, without inducing changes in MHCs composition or skeletal muscle apoptosis [7,16]. In this study, monocrotaline indeed induced RV hypertrophy and failure, and skeletal muscle apoptosis, confirmed by different techniques, including TUNEL, immunoblotting of activated caspases and the DNA ladder. TNF was significantly elevated in MCHF rats, confirming that this cytokine is increased in CHF [1,7,10,17]. While in the heart a cause–effect relationship between TNF and apoptosis is well established [2], the same is not demonstrated for skeletal muscle. The result of the cytotoxic effect of TNF on skeletal muscle could be due to its second messenger, SPH that is released by cardiac myocytes when TNF binds to their surfaces [2,10]. In fact, SPH is able to induce skeletal muscle apoptosis both in vivo and in vitro [10].
The working hypothesis for blocking TNF is therefore to prevent skeletal muscle apoptosis and muscle atrophy, for further improving EC and symptoms. Recently, Enbrel, a specific p75 TNF receptor fusion protein, by reducing the biological activity of TNF by 50%, was able to improve EC [4], indicating that TNF is an important therapeutic target in CHF. In the present study, where we used thalidomide to block the synthesis of TNF, the plasma levels of this cytokine, as well as SPH, were substantially unchanged. At the same time, apoptosis was still ongoing, as demonstrated by the persistence of elevated TUNEL positivity, the presence of DNA ladder and activated Caspases. Dimethyl sulfoxide-treated controls did not show any difference with control rats, excluding the possibility that this free radicals scavenger may have influenced skeletal muscle MHCs composition, apoptosis and cytokine levels.
In thalidomide rats, there are two aspects that cannot be explained by our present knowledge: (a) the normalisation of the MHCs patterns; and (b) the slightly reduced degree of muscle atrophy. As for MHCs, we know that their composition is influenced by factors other than apoptosis, which is not selective for fast or slow fibre type [7,15] and is perhaps influenced by adaptive mechanisms to the relative hypoxia at muscle level in CHF [7]. The possibility that this may be due to different haemodynamics cannot be ruled out, although it seems unlikely, because the circulating levels of AngII were similar in both the thalidomide and CHF groups. We can only speculate that some effects of CHF on the skeletal muscle could not be TNF-mediated. Thalidomide was unable to block TNF, but it may have acted through some other mechanisms that were not explored in the present study, such as the expression of other cytokines or angiogenetic factors [13,14,18]. It looks as if simply blocking the reactive inflammatory response occurring in CHF does not entirely lead to a prevention of skeletal muscle myopathy. Some other mechanisms may play a role in the progression of the syndrome.
In a previous paper, we have, in fact, shown that Ang II receptors blockade can prevent apoptosis, muscle atrophy and MHCs shift [17], together with a reduction in TNF and SPH plasma levels.
Thalidomide, in this study, was used at the same doses employed, and shown to be efficacious for the treatment of TB, AIDS and leprosy in humans [6]. There are no studies, at present, indicating that higher doses may be effective in experimental models of CHF and we cannot therefore exclude the fact that higher doses or a longer period of treatment may be required in CHF, although the second hypothesis seems unlikely, since in this model, 2 weeks treatment with other drugs was able to produce improvements in skeletal muscle changes [17]. The results of the present study, with all the limitations of animal investigations, should be taken into account pathophysiologically and clinically, now that the cytokine hypothesis is under investigaton in large randomised clinical trials, and lively discussed because of contradictory results with TNF blockers such as Enbrel (RENAISSANCE), and because some other molecules are going to be tested.
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Acknowledgements |
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We thank Mr Valerio Gobbo for the skillful technical assistance.
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