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Cardiovascular Research 2007 73(1):120-129; doi:10.1016/j.cardiores.2006.10.026
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Copyright © 2006, European Society of Cardiology

Myocardial expression of Murf-1 and MAFbx after induction of chronic heart failure: Effect on myocardial contractility

Volker Adamsa,1,*, Axel Linkea,1, Ulrik Wisloffb, Christian Döringa, Sandra Erbsa, Nicolle Kränkela, Christian C. Wittc, Siegfried Labeitc, Ursula Müller-Werdand, Gerhard Schulera and Rainer Hambrechta

aUniversity Leipzig-Heart Center Leipzig, Department of Cardiology, Leipzig, Germany
bDepartment of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway
cInstitute of Anesthesiology and Operative Intensive Medicine; University Mannheim, Germany
dDepartment of Medicine III, Martin-Luther University Halle-Wittenberg, Halle, Germany

* Corresponding author. Universität Leipzig, Herzzentrum, Klinik für Innere Medizin / Kardiologie, Strümpellstrasse 39, D-04289 Leipzig, Germany. Tel.: +49 341 865 1671; fax: +49 341 865 1461. Email address: adav{at}medizin.uni-leipzig.de

Received 29 June 2006; revised 26 October 2006; accepted 30 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: In chronic heart failure (CHF) the myocardial expression of the inflammatory cytokine tumor necrosis factor alpha (TNF-{alpha}), which is thought to contribute to myocardial remodeling, was found to be increased. However, it is unknown whether the E3-ubiquitin ligases MAFbx and Murf-1 are involved in this remodeling process and whether their expression is regulated by TNF-{alpha}.

Methods: Rats underwent ligation of the left coronary artery to induce CHF or were sham-operated. The expression of MAFbx/Murf-1 and troponin I was analyzed by RT-PCR and Western blotting in the non-infarcted area of the left ventricle. In cell culture experiments the potency of TNF-{alpha} to stimulate Murf-1/MAFbx expression, the intracellular signaling pathway, and the involvement of the E3-ligases for the impairment of contractility were assessed.

Results: In CHF the myocardial expression of TNF-{alpha} was elevated 3.1-fold as compared to control. This was associated with a 4.5-fold and 2.7-fold increase in MAFbx and Murf-1 expression, respectively. A positive correlation between TNF-{alpha} and the expression of MAFbx or Murf-1 was evident. In neonatal rat cardiomyocytes, TNF-{alpha} induced the expression of MAFbx through p38MAPK-dependent pathways, whereas the induction of Murf-1 required the activation of the p42/44 MAPK pathway. Exposure of cardiomyocytes to TNF-{alpha} resulted in troponin I ubiquitinylation, subsequent degradation, and a decline in contractility. This was completely abrogated by siRNAs against Murf-1/MAFbx.

Conclusion: TNF-{alpha}, which is increasingly expressed in CHF, induces troponin I degradation through a MAFbx/Murf-1-dependent pathway. This was associated with an impairment of contractility and might be one mechanism involved in the adverse remodeling process in CHF.

KEYWORDS Heart failure; Gene expression; Cytokines; Myocytes; Protein catabolism; Ubiquitin proteasome system


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Chronic heart failure often develops secondary to myocardial infarction. The death of myocytes either by apoptosis or necrosis results in cardiac remodeling, which is characterized by left ventricular dilatation and pathological hypertrophy of surviving myocytes. The functional competence of these myocytes was found to be impaired, partially due to alterations of contractile proteins and increased expression of inflammatory cytokines [1,2]. In particular tumor necrosis factor-alpha (TNF-{alpha}) produced locally in cardiomyocytes, has been shown to reduce contractility, induce pathologic hypertrophy and promote apoptosis of cardiomyocytes which finally accelerates left ventricular remodeling [3,4]. Preliminary data suggest, that JNK and Akt activation are involved in these processes [5], but the exact pathways involved are only partially understood. Clearly, myocyte remodeling involves reorganization of the complete intracellular contractile apparatus [1,6] and a switch to a fetal gene expression profile [7,8]. During this process of reorganization structural proteins as well as transcription factors are degraded through the activation of the ubiquitin–proteasome system (UPS) [9,10]. The UPS is an ATP requiring multienzymatic process. Proteins degraded by the UPS are first conjugated to ubiquitin. This reaction requires the activation of ubiquitin by the ubiquitin activating enzyme (E1), transfer to an ubiquitin conjugating enzyme (E2), and subsequent linkage to the lysine residue of proteins destined for degradation (E3) [11]. Expression of the E3-ligases Murf-1 (for Muscle Ring Finger 1) and atrogin1/MAFbx (for Muscle Atrophy F-box) [12] is restricted to heart and skeletal muscle tissue. Murf-1 shows ubiquitin ligases activity [13], binds the sarcomeric protein titin [14], and degrades cardiac troponin I [13]. MAFbx interacts with calcineurin A, {alpha}-actinin-2 [15], and degrades MyoD [16], respectively. At least in skeletal muscle cells TNF-{alpha} is able to stimulate the expression of MAFbx via the activation of p38MAPK [17].

Although, the involvement of the UPS and the E3 ligases Murf-1 and MAFbx in the turnover of skeletal muscle proteins is clearly established [18–21], nothing is known so far about the contribution of the these E3 ubiquitin ligases to the cardiac remodeling in heart failure. Therefore, aim of the present study was to elucidate whether MAFbx and Murf-1 are involved in the degradation of structural proteins of the heart in CHF, whether their expression is upregulated by TNF-{alpha} and whether this has any functional consequences. Our data show for the first time that, TNF-{alpha}, which is increasingly expressed in CHF, induces troponin I degradation in a p38MAPK/MAFbx and p42/44MAPK/Murf-1 dependent manner. This was linked to a decline in contractility and might represent one mechanism involved in adverse remodeling in CHF.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Study design
Wistar Kyoto rats underwent LAD ligation to induce CHF (n=9) or sham operation (n=8) as described previously [22]. The LAD ligation procedure had a mortality rate of 30% and the average infarct size in the surviving animals was 36±1% of the left ventricular circumference. After 7 weeks the myocardium was removed and the remote area of the anterior wall of the left ventricle in the infarcted animals and the corresponding area of the left ventricle in the sham-operated animals was collected. The harvested tissue was immediately snap frozen in liquid nitrogen and stored at –80 °C. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The local Council for Animal Research approved experimental protocols.

2.2. Echocardiography
After 7 weeks, echocardiographical measurements were performed using a 12-MHz transducer connected to a Hewlett–Packard Sonos-5500 echocardiograph. A short-axis two-dimensional image-guided M-mode view of the left ventricle was acquired. Left ventricular end-diastolic and end-systolic dimensions, wall thickness of the anterior and posterior wall in diastole and systole respectively, were measured in the M-mode tracing in the parasternal short axis according to the leading-edge technique. Measurements from 5 adjacent cardiac cycles were averaged and used for further analysis. Fractional shortening (FS) was calculated according to this formula: FS=[(LVEDD–LVESD)/LVEDD]x100. Left ventricular end-diastolic and end-systolic volumes as well as ejection fraction were calculated in a parasternal long axis view using the disk method.

2.3. RNA-isolation and quantification of mRNA-expression
Total RNA was isolated from heart muscle tissue using RNeasy (Qiagen, Hilden, Germany). 100 ng of total RNA was reverse transcribed into cDNA using random hexamers and Sensiscript reverse transcriptase (Qiagen, Hilden, Germany). An aliquot of the cDNA was used for quantitative RT-PCR applying the Light Cycler system (Roche Diagnostics Inc). The expression of specific genes was normalized to the expression of 18S-rRNA. The following primers and conditions were used 18S rRNA: 5'-ATACAGGACTCTTTCGAGGCCC-3' and 5'-CGGGACACTCAGCTAAGAGCAT-3' at 61 °C annealing; MAFbx: 5'-GGTCCAGAGAGTCGGCAAGT-3' and 5'-GGAGCAGCTCTCTGGGTTGT-3' at 62 °C annealing; Murf-1: 5'-TCCAAGGACAGAAGACTGAACT-3' and 5'-TGGAAGCTTCTACAATGCTCTT-3' at 59 °C annealing.

2.4. Quantification of protein expression
Frozen tissue samples were homogenized in lysis buffer [23] and Western blot analysis was performed as previously described [24,25]. Murf-1 and Murf-2 protein expression was quantified using specific antibodies [26]. Protein concentration of troponin I was detected by a commercially available antibody (Santa Cruz, Heidelberg, Germany). To compensate for blot to blot variations, an internal standard was loaded on each SDS-polyacrylamide-gel, and the densitometry results were expressed as ratio between sample and standard intensity. Loading differences were controlled by re-probing the blot with an antibody against GAPDH (Hytest, Turku, Finland). TNF-{alpha} expression was quantified by ELISA (R&D Systems, Germany) in tissue homogenates. All samples were analyzed in duplicate.

2.5. Quantification of ubiquitin-modified proteins
Neonatal rat cardiomyocytes or frozen tissue samples were homogenized in lysis buffer and Western blot analysis was performed using a specific antibody only detecting ubiquitin-modified proteins, whereas free ubiquitin is not recognized (MBL, Woburn, CA). Quantification of the modified proteins was performed using densitometry with a 1-D analysis software package (One-Dscan, Scanalytics, Billerica, MA, USA).

2.6. Proteasome activity assay
The peptidase activities of the proteasome in the cytosolic fraction was determined as recently described [27]. Chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities were assayed using the fluorogenic peptides Suc-LLVY-AMC, Bz-VGR-AMC and Z-LLE-AMC, respectively (Biomol, Hamburg, Germany). 20 µg of cytosolic proteins were incubated with reaction buffer (0.05 mol/L Tris–HCl, pH 8.0, 0.5 mmol/L EDTA) and the respective labeled peptide (40 µmol/L). The kinetics of the reaction was recorded using a spectrophotometer (Tecan safir2, Tecan, Crailsheim, Germany) with excitation at 380 nm (Ex) and emission at 440 nm (Em). Only the proportion of the reaction that could be inhibited by MG132 (20 µM, Sigma, Deissenhofen, Germany) was regarded as proteasomal activity. For the calculation of enzymatic activity a calibration curve of free 7-amino-4-methylcoumarine (Sigma) was recorded.

2.7. Cell culture studies
Isolated rat neonatal cardiomyocytes [28], were cultured in Dulbecco's modified Eagle's medium (DMEM) (Biochrom KG, Berlin, Germany) supplemented with 10% fetal calf serum (Biochrom KG), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells, 60–80% confluent were incubated with TNF-{alpha} (10 ng/ml) (R&D Systems, Heidelberg, Germany) in serum-free media for up to 24 h. Gene expression of Murf-1 and MAFbx was determined by qRT-PCR, whereas Murf-1 protein expression was quantified by Western blot. To elucidate signal transduction pathways involved in TNF-{alpha} mediated activation of MAFbx/Murf-1, the cells were pre-incubated for 1 h with the following specific inhibitors: SB203580 (10 µM; Calbiochem, La Jolla, CA, USA) a highly specific, cell-permeable inhibitor of p38MAPK [29]; PD98059 (50 µM; Calbiochem), a specific blocker for the activation of p42/44MAPK [30].

2.8. Immunoprecipitation studies
Ubiquitinylation of troponin I after TNF-{alpha} stimulation was assessed by immunoprecipitation using an anti-ubiquitin antibody (MBL, Woburn, CA) and protein G agarose followed by Western blot with an anti-cardiac troponin I antibody (Santa Cruz, Heidelberg, Germany) as recently described [31].

2.9. Knockdown of Murf-1 and MAFbx expression by small interfering RNA (siRNA)
To suppress Murf-1 and MAFbx expression specific siRNA (Qiagen, Hilden, Germany were used: Murf-1-1: 5'-GAUGUGCAAGGAACACGAA-3'; Murf-1-2: 5'-GAAGCAAUAUGGAUUAUAA-3'; MAFbx-1:5'-GGAAGAAGAUGUACUUUAA-3'; MAFbx-2: 5'-GCGCUUCUUGGAUGA GAAA-3'. Briefly, cells were transfected with 5 nmol of siRNA duplex in 12-well plates using HiPerFect transfection reagent according to the recommendation of the manufacturer (Qiagen, Hilden, Germany) for 6 h before TNF-{alpha} was added to the cells. Twenty-four hours after TNF-{alpha} stimulation cells were harvested for RT-PCR analysis, immunoprecipitation studies, or functional measurements. The transfection efficiency was conveniently monitored by FACS analysis and fluorescent microscopy using a FITC-labeled control siRNA (Qiagen, Hilden, Germany) for transfection. Transfection efficiency was around 80%.

2.10. Contractility measurements
Isolation of adult cardiomyocytes, plating on laminin, loading with Fura-2, electrical field stimulation, [Ca2+]i imaging and measurement of fractional shortening was performed as described in detail previously [32]. To avoid biases due to the isolation procedure 30 cells were obtained from each of 4 different animals. Freshly isolated cardiomyocytes from left ventricle were plated (~ 3±103 cells/cm2) on laminin and stored in serum-free medium 199 (DM 199, Sigma) mixed with 2 mM DL carnitine, 5 mM creatine, 5 mM taurine, 0.1 mM insulin, 10–10 M triodothyronine (T3) (all from Sigma), 100 U mL–1 penicillin, and 100 µg mL–1 streptomyocin (both from Life Technologies, Gaitherburg, MO, USA) equilibrated with 5% CO2 and 95% O2 (37 °C, pH 7.4). After transfection with siRNA and stimulation with TNF-{alpha} as described above, isolated myocytes were placed in a cell chamber on an inverted microscope (Diaphot-TMD, Nikon, Tokyo, Japan), and stimulated electrically by bipolar pulses (5 ms duration, 5 Hz, 37 °C) using platinum electrodes on either side of the chamber. During stimulation the cells were superfused at 2 ml min–1 with DM199 at 37 °C.

2.11. Statistical analysis
Values are given as mean±SEM for all variables. Intergroup comparisons were performed with Mann–Whitney U test or a one-way ANOVA, where appropriate. A probability value of <0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Echocardiography
End-diastolic diameter (EDD), end-systolic diameter (ESD), end-diastolic volume (EDV) and end-systolic volume (ESV) were significantly enlarged in CHF, consistent with severe left ventricular dilation as a result of the remodeling process. In contrast fractional shortening and ejection fraction were severely impaired in CHF, indicative of a blunted contractile function (Table 1).


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  		Table 1 Echocardiographic characteristics of the animals

 
3.2. Myocardial expression of MAFbx and Murf-1 after induction of heart failure
In CHF the myocardial mRNA transcription of Murf-1 (Fig. 1A) and MAFbx (Fig. 1B) was 2.7-fold (control: 2.1±0.4 vs. CHF: 5.6±0.9 arbitrary units; p<0.01) and 4.5 fold (control: 13.2±2.3 vs. CHF: 60.5±11.4 arbitrary units; p<0.001) increased as compared to controls, respectively. In parallel, protein expression of Murf-1 was elevated by 123% in animals with CHF as compared to controls (control: 0.63±0.09 vs. CHF: 1.41±0.14 arbitrary units; p<0.01) (Fig. 1C). However, MAFbx protein expression could not be detected due to a lack of a specific antibody. In contrast, the protein expression of Murf-2, a close homologue of Murf-1, did not differ between the groups (control: 1.02±0.09 vs. CHF: 1.11±0.22 arbitrary units; p=NS).


Figure 1
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  		Fig. 1 Quantification of Murf-1 (A) and MAFbx (B) mRNA expression as well as Murf-1 protein expression (C) in the myocardium of sham-operated animals (controls, n=8) and rats with CHF (n=9). The values are depicted as ratio over the expression of 18S-rRNA or GAPDH. Results are expressed as mean±SEM. *p<0.05 vs. control.

 
3.3. Influence of heart failure on protein modification by ubiquitin
To elucidate the cross talk between MAFbx and Murf-1 expression and ubiquitinylation of proteins, Western blot analysis was performed. A significant higher amount of ubiquitin-modified proteins was evident in the heart of animals with CHF as compared to control animals (control: 0.75±0.06 vs. CHF: 1.31±0.22 arbitrary units; p<0.05) (Fig. 2).


Figure 2
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  		Fig. 2 Detection (A) and quantification (B) of ubiquitinated proteins in the myocardium of sham-operated animals (control, n=8) and rats with CHF (n=9). Representative Western blot using an ubiquitin specific antibody is depicted and the molecular weight is given in kDa (A). To control for equal loading of the lanes the blot was re-probed with a GAPDH specific antibody. Protein labeling was quantified by densitometry (B). Results are expressed as mean±SEM. *p<0.05 vs. control.

 
3.4. Proteasomal activity
The assessment of proteasomal activity revealed no difference between the sham-operated animals and the CHF group with respect to chemotrysin-like activity (control: 718±55 nmol/min/mg, CHF: 627±97 nmol/min/mg; p=NS), trypsin-like activity (control: 139±13 nmol/min/mg, CHF: 141±36 nmol/min/mg; p=NS), and peptidylglutamyl hydrolyzing activity if the myocardium (control: 216±19 nmol/min/mg, CHF: 200±41 nmol/min/mg; p=NS).

3.5. Myocardial concentration of TNF-{alpha}
In CHF, myocardial TNF-{alpha} content was 216% higher as compared to control (control: 325±42 pg/mg, CHF: 1027±129 pg/ml; p<0.0001 vs. control). (Fig. 3A). The increase in myocardial TNF-{alpha} expression was associated with an elevated expression of Murf-1 protein (r=0.75; p<0.05) and MAFbx m-RNA (r=0.82; p<0.05), respectively (Fig. 3B and C).


Figure 3
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  		Fig. 3 The myocardial TNF-{alpha} concentration (A) and the correlation between the concentration of TNF-{alpha} and Murf-1 protein (B), and MAFbx mRNA (C) expression in animals of the CHF group is depicted. Results are expressed as mean±SEM.

 
3.6. Impact of TNF-{alpha} on the expression of MAFbx and Murf-1
To determine if TNF-{alpha} stimulates Murf-1 and MAFbx expression, neonatal rat cardiomyocytes were exposed to TNF-{alpha} in cell culture. Incubation of cardiomyocytes with TNF-{alpha} for a period of 24 h resulted in a 2.7-fold and 2.0-fold increase in MAFbx and Murf-1 mRNA expression (Fig. 4A,B), and a 1.7-fold increase in Murf-1 protein content (Fig. 4C), respectively.


Figure 4
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  		Fig. 4 TNF-{alpha} induces the expression of MAFbx (A) and Murf-1 (B,C) in cardiomyocytes. Cells were pre-incubated for 1 h with inhibitors of different signaling cascade molecules before TNF-{alpha} (10 ng/ml) was added to the medium. After 24 h the cells were harvested and the expression of MAFbx and Murf-1 mRNA was determined by qRT-PCR (A,B). The protein expression of Murf-1 was evaluated by Western blot (C). The expression in the untreated cells was set as 1. Results are expressed as mean±SEM. An example of the Western blot analysis is shown on top of the graph (C). *p<0.05 vs. TNF-{alpha} treated cells (n=3 per data point). PD=PD98059, SB=SB203580.

 
To elucidate the signaling pathway involved in the TNF-{alpha}-mediated activation of Murf-1 or MAFbx expression, cardiomyocytes were stimulated with TNF-{alpha} in the presence or absence of MAPK-inhibitors-SB203580 (10 µM) an inhibitor of p38MAPK or PD98059 (50 µM) a selective inhibitor of p42/44 MAPK. The MAPK pathway was chosen as target since in skeletal muscle cells Li and co-workers could demonstrate the relevance of p38MAPK for stimulation of MAFbx [17]. Pre-incubation of the cardiomyocytes with SB203580 blunted the TNF-{alpha} induced up-regulation of MAFbx, but did not have an impact on the TNF-{alpha}-mediated increase of Murf-1 expression (Fig. 4A). On the other hand, the selective p42/44 MAPK inhibitor PD98059 suppressed the TNF-{alpha} mediated increase in Murf-1 expression, whereas the induction of MAFbx expression was not affected (Fig. 4B,C).

3.7. Impact of TNF-{alpha} on ubiquitin-modified proteins
TNF-{alpha} significantly increased the amount of ubiquitin-modified proteins in neonatal rat cardiomyocytes by 110%. To elucidate, whether this TNF-{alpha} mediated increase in protein ubiquitinylation was the result of Murf-1/MAFbx activation, rat neonatal cardiomyocytes were transfected with siRNA against Murf-1 and MAFbx before they were exposed to TNF-{alpha}. Preincubation of the cells with the specific siRNA's blunted the TNF-{alpha} induced increase of the respective E3-ligase (Fig. 5A) and attenuated the protein ubiquitinylation (Fig. 5B).


Figure 5
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  		Fig. 5 TNF-{alpha} induced expression of Murf-1/MAFbx (A) and amount of ubiquitinated proteins in the presence or absence of siRNA against Murf-1/MAFbx in neonatal rat cardiomyocytes. Cells were transfected with either a mixture of siRNA against Murf-1/MAFbx (spec. siRNA), an unspecific or a scrambled siRNA for 6 h before the cells were stimulated with TNF-{alpha} (10 ng/ml) for 24 h. Cells were harvested and the expression of Murf-1 and MAFbx was quantified by qRT-PCR (A). A representative Western blot using an ubiquitin specific antibody is depicted and the molecular weight is given in kDa (B). To control for equal loading of the lanes, the blot was re-probed with a GADPD specific antibody. To quantify the protein labeling densitometry was performed (B). Results are expressed as mean±SEM or x-fold increase vs. control. *p<0.05 vs. untreated cells, $ p<0.05 vs. cells stimulated with TNF-{alpha}, § p<0.05 vs. TNF-{alpha} treated cells (n=4 per data point).

 
3.8. Decreased expression and Murf-1/MAFbx mediated ubiquitination of troponin I
Troponin I was suggested as a potential target of Murf-1 mediated ubiquitinylation. In order to determine, whether a TNF-{alpha}-mediated activation of Murf-1 is associated with a breakdown of troponin I Western blot analysis was performed. Indeed, troponin I protein expression was significantly reduced by 49% in animals with CHF as compared to controls (control: 1.57±0.26 vs. CHF: 0.81±0.09 arbitrary units; p<0.05) (Fig. 6A). The close correlation between Murf-1 protein and troponin I expression (r=0.52, p<0.05) (Fig. 6B) in conjunction with results from yeast two hybrid studies [13,26], suggests that troponin I, as a protein of the contractile apparatus, may be a specific target of Murf-1. In contrast, the protein expression of myosin binding protein C is not affected (control: 2.43±0.70 vs. CHF: 2.64±0.55 arbitrary units; p=NS). To further confirm that troponin I is a target of Murf-1 in cardiomyocytes an immunoprecipitation using anti-troponin I antibody was performed. As documented in Fig. 6C the treatment of cardiomyocytes with TNF-{alpha}, which increases expression of Murf-1/MAFbx as shown above, augmented the amount of ubiquitinated troponin I. This effect was partially blocked by pretreatment of the cells with siRNA against Murf-1/MAFbx.


Figure 6
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  		Fig. 6 Troponin I expression in the myocardium (A) and correlation analysis between the expression of Murf-1 and troponin I (B) in the myocardium of sham-operated animals (control, n=8) and rats with CHF (CHF, n=9). Immunoprecipitation (IP) analysis using an antibody against troponin I followed by Western blot analysis with an ubiquitin specific antibody was performed from cell extracts treated with TNF-{alpha} or from cells transfected with siRNA against Murf-1/MAFbx before TNF-{alpha} stimulation. A representative IP and quantitative evaluation is depicted (C). Results are expressed as mean±SEM (n=3 per data point). *p<0.05 vs. control, $ p<0.05 vs. TNF-{alpha} treated cells.

 
3.9. Impact of Murf-1, MAFbx expression on myocyte contractility
To investigate if the TNF-{alpha} induced expression of Murf-1 and MAFbx has a physiological consequence, cell contractility was measured in adult rat cardiomyocytes isolated from the left ventricle. In comparison to control cardiomyocytes, TNF-{alpha} induced a significant reduction in fractional shortening and a prolongation of contractile kinetics (Table 2). Transfection of myocytes with siRNA against Murf-1 and MAFbx, prevented the TNF-{alpha} mediated decline in fractional shortening and kinetics (Table 2). In comparison to control cells TNF-{alpha} reduced significantly the amplitude of the calcium transient in electrically stimulated cardiomyocytes, which was recovered when the cells where transfected with a mixture of siRNA against Murf-1/MAFbx (Table 2).


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  		Table 2 Effects of 24 h of TNF-{alpha} stimulation on kinetics of cytosolic [Ca2+]i and cell shortening of electrically stimulated (5 Hz, 37 °C) cardiomyocytes treated with either placebo siRNA or siRNA for Murf-1 and MAFbx

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Left ventricular dysfunction after myocardial infarction is the result of cardiac remodeling, which involves pathologic hypertrophy and alterations of the contractile apparatus. A key feature of this remodeling process is the switch to a fetal gene expression profile, which is the consequence of de novo protein synthesis and elevated degradation of existing proteins [33]. However, the molecular mechanisms mediating these effects are less clear and were investigated in this report. Four important messages emerge from this study. First, Murf-1 and MAFbx are significantly upregulated in the myocardium in CHF. Second, the proinflammatory cytokine TNF-{alpha} has the potency to induce both the expression of Murf-1 and MAFbx in cardiomyocytes involving the MAP kinase pathway (via the activation of p38MAPK in the case of MAFbx or via the activation of p42/44 MAPK in the case of Murf-1). Third, the activation of Murf-1/MAFbx leads to an increased ubiquitination and degradation of troponin I. Forth, in cardiomyocytes the TNF-{alpha}-induced contractile dysfunction seems to be the result of Murf-1 and MAFbx activation.

Taken together, these results suggest that the two E3-ubiquitin ligases Murf-1 and MAFbx are involved in the myocardial remodeling process.

4.1. Chronic heart failure and the myocardial expression of Murf-1 and MAFbx
Despite the major role of the ubiquitin–proteasome system and the E3 ligases Murf-1 and MAFbx in the turnover of skeletal muscle protein, and despite of the evidence that the activity of the entire pathway is increased in a variety of conditions associated with muscle wasting [18–21] nothing was known so far about the regulation of Murf-1 and MAFbx in the myocardium in chronic heart failure. The observation that MAFbx and Murf-1 are significantly up-regulated in CHF is in favor of the notion of increased protein degradation via the ubiquitin–proteasome pathway during myocardial remodeling. Expression of these E3 ligases is highly correlated with protein breakdown by the UPS system [34,35]. In the present study we observed elevated levels of ubiquitinated proteins, which could either be due to a drop in specific activity of the proteasome, as reported by Tsukamoto [27] or by an increased expression of the respective E3 ubiquitin ligases and unchanged proteasome activity as reported here. The difference concerning both reports with regard to the proteasomal activity may be due to different heart failure models – pressure overload model vs. LAD ligation model. The rapid degradation of specific proteins permits adaptation to new physiological conditions and changes in cell composition [36]. The importance of the ubiquitin–proteasome system is further supported by proteomic analysis of hearts from animals [37] or patients with dilated cardiomyopathy (DCM) [38]. From all these studies it is evident that the activation of key components of the ubiquitin–proteasome system in the myocardium in dilative cardiomyopathy leads to a marked increase in protein ubiquitination and hence degradation.

Especially postinfarction failure and the subsequent development of heart failure is associated with elevated circulating levels of angiotensin II and inflammatory cytokines [39,40]. In this regard our data provide first time evidence, that TNF-{alpha}-as an inflammatory cytokine – is a potent activator of Murf-1 and MAFbx expression not only in the skeletal muscle [17] but particular in the heart in CHF.

What are cellular targets ubiquitinylated by MAFbx and Murf-1? Yeast two hybrid screening with Murf-1 as bait identified myofibrillar proteins like troponin-I, titin, nebulin, myosin light chain 2, as well as metabolic enzymes involved in energy production as binding partner [13,26]. The observation that the TNF-{alpha}-induced ubiquitination of troponin I is mediated by Murf-1/MAFbx and that troponin I is significantly decreased in CHF, may provide a mechanistic link to the impairment of contraction. The Murf-1-mediated degradation of troponin-I seems to be a specific event, since structural proteins such as myosin, actin, or MyBP-C, are not down-regulated. The impact of troponin I degradation on force generation of single cardiomyocytes is supported by a negative correlation between troponin I degradation and maximal force generation [41]. However, alternatively it is conceivable that the decrease in troponin I protein content is also at least partially related to a blunted expression of the protein. However, the change in protein synthesis as a result of remodeling was not the focus of this study.

Furthermore, incubation of cardiomyocytes with TNF-{alpha} significantly reduced the amplitude of the calcium transient in electrically stimulated cells, which was recovered when the cells were transfected with a siRNA against Murf-1/MAFbx. It remains to be seen if important proteins responsible for calcium handling are degraded via the proteasome system upon TNF-{alpha} stimulation which could explain the decreased contractility seen in CHF.

4.2. TNF-{alpha} and the induction of Murf-1 and MAFbx
It has been described that serum levels as well as the local concentration of inflammatory cytokines [3], especially TNF-{alpha}, are significantly increased in patients and animals with chronic heart failure and that they correlate with the degree of functional impairment as assessed by NYHA functional class [42,43] or 6-min walk test [44]. Since TNF-{alpha} seems to mediate muscle wasting [45] and is involved in cardiac remodeling, it is tempting to speculate that TNF-{alpha} may induce Murf-1 and MAFbx in the myocardium. This hypothesis is supported by the results of the present study, demonstrating a positive correlation between the myocardial concentration of TNF-{alpha} and the expression of Murf-1 and MAFbx. Additionally, these data are in accordance with a study reporting the induction of MAFbx expression by TNF-{alpha} in the skeletal muscle [17]. Concerning the intracellular signaling pathways involved in the TNF-{alpha} induced increase in Murf-1 and MAFbx expression our data suggest that the two E3 ligases are regulated differently. TNF-{alpha} induced the expression of MAFbx through an activation of p38 MAPK, which is in accordance with the data recently reported for the skeletal muscle [17], whereas the activation of Murf-1 is mediated by p42/44 MAPK.

In summary, TNF-{alpha} induces the expression of Murf-1 and MAFbx by a p38 or p42/44 dependent pathway in cardiomyocytes. The enhanced expression of TNF-{alpha} in CHF might account for the Murf-1/MAFbx mediated degradation of proteins from the contractile apparatus, in particular troponin I. This might implicate that a modulation of Murf-1/MAFbx might represent a novel strategy to attenuate cardiac remodeling in CHF.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We would like to thank Angela Kricke and Claudia Weiss for their excellent technical assistance, and acknowledge the generous financial support by the Deutsche Forschungsgemeinschaft (DFG).


   Notes
 
1 Both authors contributed equally to this work. Back

Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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