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Cardiovascular Research 2005 66(1):33-44; doi:10.1016/j.cardiores.2005.01.004
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Copyright © 2005, European Society of Cardiology

Impairment of the ubiquitin–proteasome system by truncated cardiac myosin binding protein C mutants

Antonio Sarikasa,1, Lucie Carrierb,c,1, Carolus Schenkea, Daniela Dolla, Jeanne Flavignyb, Katrin S. Lindenbergd,2, Thomas Eschenhagenc and Oliver Zolka,*

aInstitute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-University Erlangen-Nuremberg, Germany
bINSERM U582, Pitié-Salpêtrière Hospitals, Paris, France
cInstitute of Experimental and Clinical Pharmacology, University Hospital Eppendorf, Hamburg, Germany
dDepartment of Neurology, University of Ulm, Ulm, Germany

* Corresponding author. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany. Tel.: +49 9131 8522783; fax: +49 9131 8522773. Email address: Zolk{at}pharmakologie.uni-erlangen.de

Received 23 July 2004; revised 2 December 2004; accepted 4 January 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHC patients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin–proteasome system (UPS).

Methods and results: By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHC patients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70 ± 4% and 11 ± 5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS.

Conclusion: Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC.

KEYWORDS Hypertrophic cardiomyopathy; Cardiac myosin binding protein C; Ubiquitin; Proteasome

This article is referred to in the Editorial by H.-P. Vosberg (pages 1–3) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Familial hypertrophic cardiomyopathy (FHC) is a cardiac disorder characterized by left ventricular hypertrophy (LVH) with predominant involvement of the interventricular septum in the absence of other causes of hypertrophy. The main histological features are disordered architecture of the hypertrophied cardiomyocytes and interstitial fibrosis [1]. Hypertrophic cardiomyopathy is associated with an autosomal-dominant mode of inheritance with an estimated prevalence of 1:500 in the general population [1]. To date, more than 200 mutations in 14 different genes have been identified in FHC and most of them encode cardiac sarcomeric proteins [2]. Most of the families present a mutation in the MYBPC3 gene [3], which encodes the cardiac myosin-binding protein C (cMyBP-C). MYBPC3 mutations most frequently result in a frameshift, and are expected to produce C-terminal truncated proteins lacking the titin and/or the major myosin-binding sites [3–5]. The MYBPC3 gene encodes a 144 kDa protein composed of 11 globular domains (numbered C0 to C10; Fig. 1). cMyBP-C is a major constituent of the thick filaments and is localized in doublets in the C-zone of the A-band of the sarcomere. cMyBP-C has both structural and regulatory roles. By binding to myosin [6,7], titin [8] and actin [9], cMyBP-C contributes to the structural integrity of the sarcomere and regulates cardiac contractility in response to adrenergic stimulation [10].


Figure 1
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  		Fig. 1 Expression of WT and truncated cMyBP-Cs in NRCM. (A) General domain organization of WT, M6t and M7t cMyBP-C. The WT full-length cMyBP-C contains 1273 amino acids, and is composed of eight immunoglobulin-I motifs, three fibronectin type III repeats and the MyBP-C motif containing three phosphorylation sites. Insertion/deletion in exon 33 results in a loss of the terminal 54 residues, which are replaced with 19 novel residues (mutation M6, truncation by about 3% of the weight length). Skipping of the exon 6 results in a loss of the terminal 1055 amino acid residues, including the MyBP-C motif; a frameshift encodes 41 novel residues followed by premature termination (mutant M7t, truncation by about 80% of WT length) Each cMyBP-C protein contained a myc-tag placed after the initiator methionine residue. (B) Western blot analysis with an anti-myc antibody detects adenoviral overexpressed cMyBP-Cs. Cell lysates were derived from NRCM infected with Ad.WT, Ad.M6t and Ad.M7t, respectively. As an internal control protein EGFP was quantified. The bar graph summarizes the results from 10 independent experiments. *p<0.05. (C) Northern blot analysis of human MyBP-C mRNA expressed in cardiomyocytes infected with Ad.WT, Ad.M6t and Ad.M7t, respectively. For control, EGFP mRNA levels were determined. Note that the Northern probe does not recognize endogenous rat cMyBP-C. Ctr, not infected control NRCM. The bar graph summarizes the results (n=3).

 
The molecular mechanisms by which frameshift cMyBP-C mutations lead to FHC are not completely elucidated. Western blots of myocardial tissue from patients carrying a frameshift cMyBP-C mutation did not reveal measurable expression of truncated cMyBP-C [11–13]. Overexpression of human truncated cMyBP-Cs in fetal rat cardiomyocytes or in transgenic mice showed markedly lower expression and sarcomere incorporation than wild type (WT) protein [14,15]. These data are compatible with the "null-allele" mechanism and suggest rapid degradation of truncated cMyBP-C, which may be promoted by the absence of biomolecular interaction between truncated forms of cMyBP-C and β-myosin heavy chain (β-MyHC) [7].

Eukaryotic cells are equipped with two main protein degradation systems: the lysosomes, which degrade membrane and endocytosed proteins, and the ubiquitin–proteasome system (UPS) [16]. Degradation of proteins via the latter system involves an enzymatic cascade through which multiple ubiquitin molecules are covalently attached to the protein substrate, which is then degraded by the 26S proteasome complex [17]. Protein degradation by the UPS seems to play an important role in a variety of fundamental cellular processes, including regulation of the cell cycle, modulation of cell surface receptors and ion channels and antigen presentation [18]. Moreover, the pathway has been implicated in several forms of malignancy, in the pathogenesis of several genetic diseases, in immune surveillance/viral pathogenesis and pathology of neurodegenerative disorders like Alzheimer or Parkinson [19,20]. In addition, it is becoming increasingly apparent that a dysfunctional UPS is also implicated in the initiation and progression of atherosclerosis [16].

In this experimental study, we investigated whether truncated cMyBP-Cs resulting from two different naturally occurring mutations are subject to lysosomal or proteasomal degradation. We demonstrate that these truncated cMyBP-Cs are rapidly and quantitatively degraded by the UPS and, more importantly, provide evidence that truncated cMyBP-C mutants also impair the proteolytic capacity of the UPS. We propose that this could contribute to the pathogenesis of FHC related to frameshift cMyBP-C mutations.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Isolation of neonatal rat cardiac myocytes
All care and treatment of animals were in accordance with the guidelines for the Care and Use of Laboratory Animals published by the National Institute of Health (NIH Publication 85-23, revised 1985) and subjected to prior approval by the local animal protection authority. NRCM were dissociated from the ventricles of 1–3-day-old Wistar rats (Charles River) by serial trypsin/DNase II digestion as described previously [21].

2.2. Generation of recombinant adenovirus expressing wild type and truncated cMyBP-Cs
Three different cDNAs encoding WT, M6t and M7t human cMyBP-Cs were used in this study (Fig. 1A). The WT cMyBP-C encodes the full-length human cMyBP-C. M6t is a 3% truncated cMyBP-C resulting from an insertion/deletion in exon 33 [5]. M7t is a 80% truncated cMyBP-C, which results from a G to A transition on the last nucleotide of exon 6 that leads to the skipping of exon 6 [3]. The procedure to construct WT and M6t cMyBP-C cDNAs has been described previously [7,14]. The M7t cDNA was generated by PCR mutagenesis from the WT cDNA with two complementary primers overlapping the end of exon 5 and the beginning of exon 7 (forward: 5'-CAGCTACGACCGCGCCAGCAAGAGGCCATGGGCACCGGA G-3' and reverse: 5'-CTCCGGTGCCCATGGCCTCTTGCTGGCGCGGTCGTAGCTG-3'). Each plasmid contains a c-myc epitope in the 5'-end of the cDNA. Adenovirus generation was performed as described [21]. Adenovirus was applied at a concentration of MOI 5–10, and adenovirus with the same parental genome carrying the enhanced green fluorescent protein (EGFP) gene was used as control.

2.3. Generation of recombinant adenovirus expressing ubiquitinG76V-DsRed
We established a red fluorescent protein (DsRed)-based reporter system (UbiG76V-DsRed) for quantifying proteasomal activity in living NRCM. The stable DsRed was converted into a substrate for proteasomal degradation by fusion to ubiquitinG76V, as described [22]. This fusion construct was subcloned into the monocistronic pShuttle vector and a recombinant adenovirus was generated as described earlier [21].

2.4. Immunofluorescence analysis
NRCM cultured on glass slides were infected with Ad-WT, Ad-M6t or Ad.M7t. 48 h later, cells were fixed in methanol/acetone (1:1) and immunofluorescence staining was performed using the following primary and secondary antibodies: anti-myc mAb (Invitrogen, 1:500), anti-ubiquitin polyclonal antibody (1:1000, Dako), anti-titin polyclonal antibody (1:1000, kindly donated by Dr. Siegfried Labeit, Heidelberg, Germany), anti-rabbit IgG antibody conjugated with Alexa 488 (1:1000, Molecular Probes), anti-mouse IgG antibody conjugated with Cy3 (Sigma, 1:1000) or Alexa 350 (1:1000, Molecular Probes).

2.5. Northern blot analysis
RNA was isolated from NRCM with Trizol (Gibco). Ten-microgram aliquots were subjected to electrophoresis, transferred onto Hybond-N membranes (Amersham). A 1519 bp fragment of the human cMyBP-C cDNA (including the myc-tag) and a 759 bp fragment of the EGFP cDNA, respectively, were used to generate specific probes.

2.6. Western blot analysis
NRCM were lysed in lysis buffer (30 mM Tris–HCl, pH 8.8, 3% SDS, 10% glycerol, 5 mM EDTA, 30 mM sodium fluoride, 2 mg/l aprotinin) in a glass/teflon homogenizer. SDS-PAGE followed by transfer of proteins onto nitrocellulose membranes and staining procedures have been described previously [21]. Polyclonal antibodies directed against the c-myc epitope (Sigma, 1:5000), EGFP (Santa Cruz, 1:2000) or DsRed (BD Biosciences Clontech, 1:2000) were used.

2.7. FACS analysis
NRCM (5 x 105 cells/dish) were harvested in 0.5% trypsin–EDTA for 5 min at RT, washed in PBS and stored on ice until analysis on a FACScan (Becton-Dickinson) flow cytometer. Data were analyzed by using CELLQUEST software (Becton Dickinson).

2.8. Statistical analysis
Data are expressed as mean ± S.E.M. Statistical analysis was made by ANOVA followed by post hoc analysis with the Bonferroni correction for multiple comparisons. A value of P<0.05 was considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Diminished amount of truncated cMyBP-C protein relative to WT despite similar mRNA levels
Three bicistronic recombinant adenoviruses were constructed, which allowed the concomitant expression of human WT, M6t or M7t cMyBP-Cs together with enhanced green fluorescent protein (EGFP). The Ad.WT encodes the full-length human cMyBP-C, while Ad.M6t and Ad.M7t encode a 3% and 80% truncated mutant cMyBP-C, respectively (Fig. 1A). In order to distinguish the expression of exogenous cMyBP-C from the endogenous rat one, exogenous proteins were N-terminally myc-tagged. The myc tag at this position does not interfere with the capacity of the recombinant cMyBP-Cs to incorporate into the sarcomere [14].

Fig. 1B illustrates expression levels of WT, M6t or M7t protein 48 h after adenoviral infection as determined by Western blotting. In lysates from Ad.WT-infected cells, the myc-antibody detected a major band of the expected molecular weight of 144 kDa. Infection with Ad.M6t and Ad.M7t showed the predicted truncated proteins of 140 and 34 kDa, respectively. Importantly, the amount of truncated cMyBP-C was markedly lower than WT, especially for M7t. Immunoreactive bands running below WT and M6t protein respectively most likely represent degradation products. To correct for minor inter-individual differences in transfection efficiency, cMyBP-C expression was related to EGFP levels. Densitometric analysis revealed that M6t and M7t were reduced by 30 ± 4% and 89 ± 5%, respectively, when compared to WT (Fig. 1B).

To investigate whether an instability of the corresponding mRNA transcripts accounts for the decrease in truncated cMyBP-C levels, Northern blot analyses were carried out (Fig. 1C). No differences between WT and truncated cMyBP-Cs mRNA levels were observed (Fig. 1C), indicating that mRNA stability was not affected.

3.2. Low levels of truncated cMyBP-C proteins were mainly due to proteasomal degradation
Specific inhibitors were used to test whether lysosomal and/or proteasomal degradation of truncated cMyBP-Cs accounts for their instability. Treatment of the cells with bafilomycin A1 (50 nM, 6 h), an antibiotic that inhibits the vacuolar type H+-ATPase responsible for generating the acidic environment and pH optimum of lysosomal enzymes [23], increased the level of M7t (+193%) and WT (+12%), but did not affect M6t (Fig. 2). The proteasome degradation pathway was analyzed with two selective inhibitors, lactacystin (25 µM, 24 h), an irreversible inhibitor of the chymotryptic- and tryptic-like activities of the proteasome, and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal, 50 µM, 2.5 h), a reversible inhibitor of the chymotrypsin-like activity of the proteasome [24]. Lactacystin raised the protein level of M6t and M7t by 135% and 737%, respectively (Fig. 3). MG132 increased protein levels of M6t and M7t by 54% and 1913%, respectively. Relative to changes observed for mutants both inhibitors induced only minor changes in WT levels.


Figure 2
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  		Fig. 2 Inhibition of the lysosomal degradation pathway. Western blot analysis of steady-state levels of WT and truncated cMyBP-Cs 48 h after infection of NRCM with Ad.WT, Ad.M6t or Ad.M7t, in the absence (-Baf) or after 6 h treatment with the lysosomal inhibitor bafilomycin at 50 nM (+Baf). As a control, EGFP protein levels were determined. The bar graph summarizes the results from six independent experiments. *p<0.05.

 

Figure 3
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  		Fig. 3 Inhibition of the proteasome degradation pathway. Western blot analysis of steady-state levels of WT and truncated cMyBP-Cs after infection of NRCM with Ad.WT, Ad.M6t and Ad.M7t, respectively, in the absence of after treatment with proteasome inhibitors lactacystin (25 µM, 24 h) or MG132 (50 µM, 2.5 h). EGFP protein levels were determined for control. Bar graphs summarize the results from five independent experiments. *p<0.05.

 
3.3. Altered incorporation of truncated cMyBP-C mutants in the sarcomere
To investigate the expression and sarcomeric localization of WT and mutant cMyBP-Cs, NRCM were infected with the different recombinant adenoviruses for 48 h (MOI 5) and co-immunostained for myc and the Z-band part of titin. Fig. 4A shows typical confocal microscopy and fluorescence profiles. The WT cMyBP-C was incorporated in doublets in the A-band of the sarcomere and the endogenous Z-band striations were conserved. The M6t mutant was also incorporated in the A-band of the sarcomere, but more diffusely than WT. In contrast to WT and M6t, the M7t mutant did not incorporate into the A-band but co-stained with the Z-band of titin.


Figure 4
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  		Fig. 4 Immunofluorescence staining and confocal microscopy of NRCM. (A) Cultured day 4 neonatal rat cardiomyocytes infected with Ad.WT, Ad.M6t or Ad.M7t were double-stained with an anti-myc monoclonal antibody (Alexa 488, green, to detect exogenous cMyBP-Cs) and an anti-titin polyclonal antibody (Cy3, red) directed against the Z1 domain (which marks the Z-band). Typical examples (left panels) show correct A-band incorporation of the WT cMyBP-C, diffuse A-band incorporation of M6t and Z-band localization of M7t. The endogenous Z-band striation pattern was not altered. Fluorescence profiles (right panels) visualize the localization of the cMyBP-C (green line) relative to the Z-lines (red lines). (B) The percentage of myc-positive NRCM was estimated 24 h after infection with Ad.WT, Ad.M6t or Ad.M7t. A total number of 934, 776 and 824 cells transfected with Ad.WT, Ad.M6t and Ad.M7t, respectively, were investigated.

 
The percentage of NRCM showing expression of exogenous cMyBP-Cs was estimated by scoring the number of myc-positive cells (Fig. 4B). This showed that 79% of NRCM infected with Ad.WT expressed the exogenous protein (742 out of 934 cells). In contrast, expression of M6t and M7t was observed in only 24.6% (191 out of 776 cells) and 3.5% (29 out of 824 cells), respectively, despite very similar EGFP fluorescence. After treatment with lactacystin (20 µM, 24 h), the number of myc-positive cells infected with Ad.M7t increased three-fold from 3.5% to 11.6%, while the phenotype of incorporation remained unchanged (data not shown).

3.4. Formation of aggregates by truncated cMyBP-C
Cells infected with Ad.M7t exhibit two different staining patterns: Some cells demonstrate sarcomeric (mis-)incorporation of the mutant protein (Fig. 4). Other cells demonstrate aggregates but few incorporation into the sarcomere (Fig. 5). In contrast, no aggregates were observed in Ad.WT or Ad.M6t infected cells even when the virus titer was increased (MOI 15) to augment WT and M6t protein expression (data not shown). This rules out the possibility that protein expression per se induces aggregate formation. To determine the composition of the aggregates, NRCM were co-stained for ubiquitin. Ubiquitin and cMyBP-C co-localized (Fig. 5A), suggesting that aggregate formation might be due to impaired proteasomal degradation of ubiquitin-conjugated proteins, e.g. ubiquitinated mutant cMyBP-C.


Figure 5
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  		Fig. 5 Aggregate formation in NRCM infected with truncated cMyBP-C. Immunofluorescence of 4-day cultured NRCM infected with Ad.M7t. Cells were double-stained with an anti-myc antibody (Alexa 488, green) and an anti-ubiquitin antibody (Cy3, red). Note the co-localization of M7t and ubiquitin fluorescence in multiple cytosolic and nuclear aggregates.

 
To test whether the formation of M7t aggregates result from its inability to correctly incorporate in the sarcomere, we investigated the effects of adenoviral expression of WT, M6t and M7t in HeLa cells, which are non-muscle cells. In the absence of MG132, the expression level of cMyBP-Cs was directly correlated to the size of the proteins, as shown before in NRCM. MG132 significantly increased the expression of M6t and M7t, but not that of WT (Fig. 6A). Two days after infection of HeLa cells with Ad.WT, Ad.M6t and Ad.M7t (MOI 5), respectively, cells stained positive for cMyBP-C (Fig. 6B). While WT cMyBP-C was homogeneously distributed in the cytosol, aggregates were observed in HeLa cells expressing M7t. The M6t mutant showed an intermediate phenotype, accumulating predominately in the perinuclear region with a grainy appearance. These data suggest that UPS-dependent degradation and aggregation are determined by the structure of the truncated protein per se and not by the absence or presence of sarcomeric incorporation.


Figure 6
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  		Fig. 6 Expression of cMyBP-Cs in HeLa cells: effect of proteasome inhibition. HeLa cells were infected with Ad.WT, Ad.M6t or Ad.M7t (MOI 5 each) and cultured for 2.5 h in the absence and presence of the proteasome inhibitor MG132 (50 µM). (A) Steady-state levels of cMyBP-C proteins were investigated by Western blots incubated with an anti-myc antibody. EGFP levels were quantified as an internal control. The bar graph summarizes the results from four independent experiments. *p<0.05. (B) Intracellular localization of cMyBP-C proteins was evaluated by immunofluorescence using the anti-myc antibody in the absence of proteasome inhibitors.

 
3.5. Impairment of the proteasome by truncated cMyBP-C mutants
To investigate whether mutant cMyBP-Cs might block proteasomal degradation of other cellular proteins, we established a red fluorescent protein (DsRed)-based reporter system for quantifying proteasomal activity in living cells. Normally, the UbG76V-DsRed fusion protein is rapidly degraded by the UPS and therefore not detectable, whereas impairment of the UPS function leads to the accumulation of UbiG76V-DsRed and thus increased fluorescence (Fig. 7). To test the method, NRCM were infected with the reporter virus for 24 h (MOI 5). With the exception of a few cells, no DsRed fluorescence was observed (Fig. 7B). In contrast, NRCM treated with MG132 (50 µM, 4 h) showed a marked increase in DsRed fluorescence, therefore validating the method. To analyze whether cMyBP-C mutants also impair proteasomal function, the UbG76V-DsRed fusion protein (MOI 5) was co-expressed with WT or truncated cMyBP-Cs (MOI 10) for 24 h. The green fluorescence of the bicistronically expressed EGFP served as a positive control for infection. Fig. 8A shows that expression of WT did not induce red fluorescence, indicating a normal function of the proteasome. In contrast, NRCM co-expressing the mutant M6t or M7t cMyBP-C exhibited much stronger DsRed fluorescence (Fig. 8A). Quantification by Western analysis revealed an increase in UbG76V-DsRed by 107% for M6t and 246% for M7t relative to WT (Fig. 8C). These results were confirmed by FACS analysis. Expression of M7t increased the number of DsRed-positive cells 3.6-fold compared to WT (Fig. 8B). DsRed fluorescence, indicative of UPS inhibition, was associated with M7t protein accumulation and aggregate formation (Fig. 8D). Moreover, Western analysis with an ubiquitin antibody (Fig. 9) demonstrated more directly that at least the M7t mutant impairs proteasomal degradation of other UPS substrates, which subsequently accumulate.


Figure 7
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  		Fig. 7 Generation and validation of a proteasome reporter system. (A) Schematic representation of the DsRed substrate UbG76V-DsRed, encoded by the Ad.Ub-DsRed adenovirus. The red fluorescence protein DsRed was fused to an ubiquitin molecule mutated on the last nucleotide (UbG76V). The uncleavable UbG76V moiety serves as a target for polyubiquitination. (B) Fluorescence analysis (immunocytochemistry and FACS analysis) of NRCM infected with the UbG76V-DsRed reporter (MOI 5) in the absence (control) or after 4 h treatment with the proteasome inhibitor MG132 (50 µM). Note the marked increase in fluorescence intensity (left panel) and number of cells with DsRed fluorescence above background level (FACS analysis, middle panel) after treatment with MG132. The right panel shows a scheme to illustrate the method.

 

Figure 8
Figure 8
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  		Fig. 8 Impairment of the proteasome activity by truncated cMyBP-C mutants. (A) NRCM co-expressing UbG76V-DsRed and either WT, M6t or M7t were monitored for their EGFP and DsRed fluorescence. Representative micrographs (left panel) demonstrate no difference in the EGFP fluorescence, indicating that transfection efficiency was similar for Ad.WT, Ad.M6t and Ad.M7t (MOI 10 each). In contrast, red fluorescence of the UbG76V-DsRed reporter was increased in cells expressing mutant MyBPC. (B) Corresponding FACS analysis of UbG76V-DsRed fluorescence. Percentages of DsRed positive cells are indicated. (C) Quantification of UbG76V-DsRed accumulation by Western blot analysis with an anti-DsRed antibody. Data were normalized to EGFP expression. *p<0.05 (n=5). (D) Representative micrographs of cardiomyocytes infected with Ad.M7t and Ad.UbG76V-DsRed. Shown is the EGFP (upper) and DsRed fluorescence (middle panel) in living cells. After acetone fixation, the same cells have been stained with a myc antibody to visualize M7t protein expression (lower panel). Note that DsRed fluorescence, indicative of UPS inhibition, is associated with M7t protein accumulation and aggregate formation (arrows).

 

Figure 9
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  		Fig. 9 Accumulation of polyubiquitinated proteins in cardiomyocytes expressing M7t. Western blot analysis of polyubiquitinated proteins after infection of NRCM with Ad.WT, Ad.M6t and Ad.M7t, respectively. To demonstrate basal (Ctr) and maximum levels of polyubiquitinated proteins, non-infected cells were treated or not with the proteasome inhibitor MG132 (MG, 10 µM for 48 h). The blots are representative of four independent experiments. *p<0.05.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
By using adenoviral transfection of NRCM, we demonstrated that human truncated cMyBP-C mutants are not well incorporated into the sarcomere, form ubiquitin-positive aggregates, and are subject to rapid and quantitative degradation mainly by the UPS. Importantly, the data also provide direct evidence that truncated cMyBP-Cs impair the function of the UPS.

Despite well established genetic etiologies of FHC [2], the pathogenetic process remains not fully elucidated, especially for cMyBP-C related FHC, which represents a major part of the disease-causing mutations [3]. Most of the cMyBP-C gene mutations result in a frameshift and are expected to lead to C-terminal truncated proteins lacking the major titin and/or myosin-binding sites [3,5]. At present, the most likely disease mechanism is that frameshift cMyBP-Cs act as "null alleles" leading to "haploinsufficiency" of cMyBP-C in the sarcomere. When myocardial tissue of four individual FHC patients carrying a cMyBP-C frameshift mutation were studied, no truncated cMyBP-C was detected although the aberrant mRNAs were present [11–13]. In addition, overexpression of human truncated cMyBP-Cs in fetal rat cardiomyocytes or in transgenic mice resulted in markedly lower protein levels and sarcomere incorporation than WT protein [14,15]. Our experimental data are consistent with previous findings and provide first evidence that truncated cMyBP-Cs are rapidly degraded mainly by the UPS. Taking into account that in the experimental setting the strong CMV promoter drives expression of mutant cMyBP-Cs, it is most likely that in the context of human FHC the mutants are indeed rapidly cleared.

Incorporation of the truncated cMyBP-Cs into the sarcomere was altered for both mutants investigated. While the mutant M6t showed a diffuse incorporation into the A-band, mutant M7t misintegrated into the Z-disc of the sarcomere. Previous interaction studies suggested that in particular the C-terminal domains are important to target cMyBP-C to the correct location in the sarcomere [6,7]. These include the myosin binding site in the C10 domain of cMyBP-C and sites involved in trimerization of cMyBP-C into a collar around the thick filament. On the basis of this previous work, the aberrant localization of the mutant M7t protein lacking the domains C2–C10 is not surprising. Its mis-incorporation into the Z-disks likely results from the presence of an actin-binding site in the C0 domain [27]. Although M6t interacts with myosin in vitro [7], integration of M6t into the sarcomeres of NRCM was clearly altered suggesting that full preservation of the C10 domain of cMyBP-C is essential for correct integration and spatial arrangement.

Infected NRCM exhibited peri- and intranuclear aggregates of M7t mutants. These aggregates were not observed with WT or M6t cMyBP-C. Notably, the aggregates were ubiquitin-positive suggesting that they are composed of mutant cMyBP-C that is marked for degradation, but not further processed. Formation of ubiquitin-positive aggregates is a prominent feature of most neurodegenerative disorders [19], and was observed as well in other human diseases such as cystic fibrosis or metabolic disorders [28]. Interestingly, in an animal model of cardiac hypertrophy, augmented ubiquitin-positive lipofuscin deposits have been reported [29]. More recently, hyperubiquitination of proteins and deposition of nuclear or cytosolic ubiquitin-positive aggregates have been observed in human dilated cardiomyopathy [30]. In the context of human FHC, however, no evidence for aggregate formation has been documented yet.

Degradation of truncated cMyBP-C by the UPS might depend either on its inability to incorporate into the sarcomere or on the structure of the mutant proteins itself, i.e. misfolding. To test the latter hypothesis, we performed an additional set of experiments in the non-muscle HeLa cells lacking a sarcomeric contractile apparatus. As in NRCM, both truncated cMyBP-Cs, M7t more than M6t, were degraded by the UPS and expression of M7t was associated with aggregate formation in HeLa cells. These results indicate that the degradation and aggregation are determined by the mutant protein structure per se rather than by the inability of truncated cMyBP-Cs to stably incorporate into the sarcomere.

A particularly important aspect of our findings is the demonstration that truncated cMyBP-Cs are not only substrates but also inhibitors of the UPS as shown with a UbG76V-DsRed reporter system. Inhibition of the proteasome by mutant protein is not unique to M7t. A recent example might be the UBB+1 protein, a mutant ubiquitin carrying a 19-amino acid C-terminal extension, which accumulates in patients with Alzheimer disease. Like M7t, UBB+1 was recognized as an UPS substrate and acts as a potent inhibitor of the UPS in living cells [26]. The mechanisms by which proteasome-function is inhibited remain unclear. Most likely, mutant cMyBP-Cs provide an unusually strong degradation signal and effectively compete with other degradation-prone proteins for the proteasome. Additionally, protein aggregation by itself–as observed with M7t–may impair the proteasome activity. This would initiate a vicious circle. Indeed, a similar mechanism has been postulated by Bence et al. who observed that ubiquitin-positive aggregates impair the proteasome function [25].

Our data highlight some general differences between wild type and truncated cMyBP-C concerning their stability, their capability to form aggregates, and their capacity to impair the UPS. Whether these findings can be translated to the in vivo situation remains speculative at present. One important limitation of our study may result from the high expression level of exogenous cMyBP-C. Nevertheless, several findings corroborate the belief that general mechanisms identified in the present study may have some relevance for the pathogenesis of FHC.

Recent studies suggest that functions of the UPS cover more than just protein disposal. For example, the functional state of the UPS can be an important determinant of cell survival. Indeed, in our cell culture model, we found an increased rate of apoptosis in NRCM expressing M7t compared to WT cMyBP-C (unpublished observations). In line with this reasoning, endomyocardial biopsies from patients with hypertrophic cardiomyopathy showed the highest rate of TUNEL-positive cells when compared to dilated or ischemic cardiomyopathy [31]. Another key issue regards the functional significance of the UPS for cells to exhibit a synthetic/anabolic phenotype. In fact, whether a muscle cell grows or atrophies is determined largely by the overall rate of proteasome-dependent proteolysis, as demonstrated at least for skeletal muscle [32]. Additional evidence support the view that, secondary to proteasome inhibition, accumulation of growth factors or their receptors may occur in some cell types [33]. Although the mechanisms are still a matter of debate, it is well established that proteasomes promote internalization and ultimately degradation of membrane bound receptor molecules, such as the growth hormone receptor or the β2-adrenergic receptor [33,34].

Taken into account that the capacity of the UPS declines with age and with oxidative stress [35,36], it is tempting to speculate that cMyBP-C mutants impose a continuous, lifelong additional workload on the protein degradation machinery in the heart. The consequence may be decreased degradation of abnormal proteins resulting from aging or oxidative stress, whose accumulation could be toxic, and of many regulatory proteins (e.g. regulators of apoptosis), whose short half-lives determine their activities. Failure to eliminate these proteins might disrupt cellular homeostasis and initiate a late onset cardiomyopathy, which is characteristic for FHC secondary to mutations of the MYBPC3 gene [37]. Although the specific effects of UPS remain to be investigated in detail, the present study raises the possibility that impairment of the UPS by truncated cMyBP-C mutants might contribute to the pathogenesis of FHC.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank Jonathan Jantsch (University of Erlangen-Nuremberg, Erlangen, Germany) for assistance on flow cytometric analysis and critical discussion and Siegfried Labeit (Institut für Anästhesiologie und Operative Intensivmedizin, Universitätsklinikum Mannheim, Germany) for the kind gift of the titin antibody. Sven Engman and Ingo Schubert provided expert technical assistance. This work was supported by the Fritz Thyssen Stiftung (O.Z.), the Deutsche Forschungsgemeinschaft (GRK 750, Zo 123/1–2), AFM (Association Francaise contre les Myopythies) grants (L.C.) and a grant from the Procope programme for scientific cooperation (T.E. and L.C.).


   Notes
 
1 AS and LC contributed equally. Back

2 Present address: Institute for Neurodegenerative Disease (MIND), Harvard Medical School, Boston, MA, USA. Back

Time for primary review 33 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Maron B.J., Casey S.A., Hauser R.G., Aeppli D.M. Clinical course of hypertrophic cardiomyopathy with survival to advanced age. J. Am. Coll. Cardiol. (2003) 42:882–888.[Abstract/Free Full Text]
  2. Bonne G., Carrier L., Richard P., Hainque B., Schwartz K. Familial hypertrophic cardiomyopathy: from mutations to functional defects. Circ. Res. (1998) 83:579–593.[Free Full Text]
  3. Richard P., Charron P., Carrier L., Ledeuil C., Cheav T., Pichereau C., et al. EUROGENE Heart Failure Project. Hypertrophic Cardiomyopathy: distribution of disease genes, spectrum of mutations and implications for molecular diagnosis strategy. Circulation (2003) 107:2227–2232.[Abstract/Free Full Text]
  4. Bonne G., Carrier L., Bercovici J., Cruaud C., Richard P., Hainque B., et al. Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat. Genet. (1995) 11:438–440.[CrossRef][ISI][Medline]
  5. Carrier L., Bonne G., Bahrend E., Yu B., Richard P., Niel F., et al. Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy. Circ. Res. (1997) 80:427–434.[ISI][Medline]
  6. Gruen M., Gautel M. Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin binding protein-C. J. Mol. Biol. (1999) 286:933–949.[CrossRef][ISI][Medline]
  7. Flavigny J., Robert P., Camelin J., Schwartz K., Carrier L., Berebbi-Bertrand I. Biomolecular interactions between human recombinant β-MyHC and cMyBP-Cs implicated in familial hypertrophic cardiomyopathy. Cardiovasc. Res. (2003) 60:388–396.[Abstract/Free Full Text]
  8. Freiburg A., Gautel M. A molecular map of the interactions between titin and myosin-binding protein C. Implications for sarcomeric assembly in familial hypertrophic cardiomyopathy. Eur. J. Biochem. (1996) 235:317–326.[ISI][Medline]
  9. Squire J.M., Luther P.K., Knupp C. Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identification of a possible actin-binding domain. J. Mol. Biol. (2003) 331:713–724.[CrossRef][ISI][Medline]
  10. McClellan G., Kulikovskaya I., Winegrad S. Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C. Biophys. J. (2001) 81:1083–1092.[Abstract/Free Full Text]
  11. Rottbauer W., Gautel M., Zehelein J., Labeit S., Franz W.M., Fischer C., et al. Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy. Characterization of cardiac transcript and protein. J. Clin. Invest. (1997) 100:475–482.[ISI][Medline]
  12. Moolman J.A., Reith S., Uhl K., Bailey S., Gautel M., Jeschke B., et al. A newly created splice donor site in exon 25 of the MyBP-C gene is responsible for inherited hypertrophic cardiomyopathy with incomplete disease penetrance. Circulation (2000) 101:1396–1402.[Abstract/Free Full Text]
  13. Vignier N., Perrot A., Schulte H.D., Richard P., Sebillon P., Schwartz K., et al. Cardiac myosin-binding protein C and familial hypertrophic cardiomyopathy: from mutations identification to human endomyocardial proteins analysis. Circulation (2001) 104:ll–1. (Suppl.)
  14. Flavigny J., Souchet M., Sébillon P., Berrebi-Bertrand I., Hainque B., Mallet A., et al. COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes. J. Mol. Biol. (1999) 294:443–456.[CrossRef][ISI][Medline]
  15. Yang Q., Sanbe A., Osinska H., Hewett T.E., Klevitsky R., Robbins J. In vivo modeling of myosin binding protein C familial hypertrophic cardiomyopathy. Circ. Res. (1999) 85:841–847.[Abstract/Free Full Text]
  16. Herrmann J., Ciechanover A., Lerman L.O., Lerman A. The ubiquitin–proteasome system in cardiovascular diseases–a hypothesis extended. Cardiovasc. Res. (2004) 61:11–21.[Abstract/Free Full Text]
  17. Ciechanover A., Orian A., Schwartz A.L. The ubiquitin-mediated proteolytic pathway: mode of action and clinical implications. J. Cell. Biochem. (2000) 77:40–51.[CrossRef]
  18. Hershko A., Ciechanover A. The ubiquitin system. Annu. Rev. Biochem. (1998) 67:425–479.[CrossRef][ISI][Medline]
  19. Ciechanover A., Brundin P. The ubiquitin proteasome system in neurodegenerative diseases: sometimes the chicken, sometimes the egg. Neuron (2003) 40:427–446.[CrossRef][ISI][Medline]
  20. Schwartz A.L., Ciechanover A. The ubiquitin–proteasome pathway and pathogenesis of human diseases. Annu. Rev. Med. (1999) 50:57–74.[CrossRef][ISI][Medline]
  21. Zolk O., Marx M., Jackel E., El-Armouche A., Eschenhagen T. Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase. Cardiovasc. Res. (2003) 59:563–572.[Abstract/Free Full Text]
  22. Dantuma N.P., Lindsten K., Glas R., Jellne M., Masucci M.G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat. Biotechnol. (2000) 18:538–543.[CrossRef][ISI][Medline]
  23. Yoshimori T., Yamamoto A., Moriyama Y., Futai M., Tashiro Y. Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, inhibits acidification and protein degradation in lysosomes of cultured cells. J. Biol. Chem. (1991) 266:17707–17712.[Abstract/Free Full Text]
  24. Lee D.H., Goldberg A.L. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. (1998) 8:397–403.[CrossRef][ISI][Medline]
  25. Bence N.F., Sampat R.M., Kopito R.R. Impairment of the ubiquitin–proteasome system by protein aggregation. Science (2001) 292:1552–1555.[Abstract/Free Full Text]
  26. Lindsten K., de Vrij F.M., Verhoef L.G., Fischer D.F., van Leeuwen F.W., Hol E.M., et al. Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation. J. Cell Biol. (2002) 157:417–427.[Abstract/Free Full Text]
  27. Kulikovskaya I., McLellan G., Flavigny J., Carrier L., Winegrad S. Effect of MyBP-C binding to actin on contractility in heart muscle. J. Gen. Physiol. (2003) 122:1–15.[Free Full Text]
  28. Golab J., Bauer T.M., Daniel V., Naujokat C. Role of the ubiquitin–proteasome pathway in the diagnosis of human diseases. Clin. Chim. Acta (2004) 340:27–40.[CrossRef][ISI][Medline]
  29. Van Vleet J.F., Ferrans V.J., Weirich W.E. Pathologic alterations in hypertrophic and congestive cardiomyopathy of cats. Am. J. Vet. Res. (1980) 41:2037–2048.[ISI][Medline]
  30. Kostin S., Pool L., Elsasser A., Hein S., Drexler H.C., Arnon E., et al. Myocytes die by multiple mechanisms in failing human hearts. Circ. Res. (2003) 92:715–724.[Abstract/Free Full Text]
  31. Kavantzas N.G., Lazaris A.C., Agapitos E.V., Nanas J., Davaris P.S. Histological assessment of apoptotic cell death in cardiomyopathies. Pathology (2000) 32:176–180.[ISI][Medline]
  32. Mitch W.E., Goldberg A.L. Mechanisms of muscle wasting. The role of the ubiquitin–proteasome pathway. N. Engl. J. Med. (1996) 335:1897–1905.[Free Full Text]
  33. van Kerkhof P., Govers R., Alves dos Santos C.M., Strous G.J. Endocytosis and degradation of the growth hormone receptor are proteasome-dependent. J. Biol. Chem. (2000) 275:1575–1580.[Abstract/Free Full Text]
  34. Shenoy S.K., McDonald P.H., Kohout T.A., Lefkowitz R.J. Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science (2001) 294:1307–1313.[Abstract/Free Full Text]
  35. Bulteau A.L., Szweda L.I., Friguet B. Age-dependent declines in proteasome activity in the heart. Arch. Biochem. Biophys. (2002) 397:298–304.[CrossRef][ISI][Medline]
  36. Okada K., Wangpoengtrakul C., Osawa T., Toyokuni S., Tanaka K., Uchida K. 4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules. J. Biol. Chem. (1999) 274:23787–23793.[Abstract/Free Full Text]
  37. Charron P., Dubourg O., Desnos M., Bennaceur M., Carrier L., Camproux A.C. Clinical features and prognostic implications of familial hypertrophic cardiomyopathy related to cardiac myosin binding protein C gene. Circulation (1998) 97:2230–2236.[Abstract/Free Full Text]

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