Published online before print July 9, 2009, doi: 10.1161/CIRCRESAHA.109.201251
© 2009 American Heart Association, Inc.
Molecular Medicine |
Nonsense-Mediated mRNA Decay and Ubiquitin–Proteasome System Regulate Cardiac Myosin-Binding Protein C Mutant Levels in Cardiomyopathic Mice
From the Institut National de la Santé et de la Recherche Médicale (N.V., B.F., K.S., L.C.), U582, U974, Paris, France; University Pierre et Marie Curie-Paris6, Unité Mixte de Recherche S974 (N.V., B.F., N.M., L.C.), Centre National de la Recherche Scientifique Unité Mixte de Recherche 7215, Institut de Myologie, IFR14, Paris, France; Institute of Experimental and Clinical Pharmacology and Toxicology (S.S., G.M., E.K., T.E., L.C.), Cardiovascular Research Center, University Medical Center Hamburg-Eppendorf, Germany; Commissariat à l’Énergie Atomique-Grenoble (H.P., J.G., M.V.), iRTSV, France; Commissariat à l’Énergie Atomique-Fontenay, iRCM Fontenay-aux-roses, France (M.V.); and Heinrich Pette Institute (R.R., H.H.), University of Hamburg, Germany.
Correspondence to Lucie Carrier, Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail l.carrier{at}uke.uni-hamburg.de
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Abstract |
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Rationale: Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive.
Objective: The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations.
Methods and Results: We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype.
Conclusions: These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin–proteasome system in lowering the level of mutant proteins.
Key Words: cardiomyopathy • hypertrophic cardiomyopathy • mRNA stability • transgenic mice • ubiquitin
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Introduction |
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Cardiac myosin-binding protein (cMyBP)-C is a major component of the A-band of the sarcomere, where it interacts with myosin, actin and titin (see elsewhere1,2 and reviewed previously3). It is exclusively expressed in the heart in humans and mice.4,5 Its role has been enigmatic for long, but accumulating recent evidence suggests that cMyBP-C is essential for normal diastolic relaxation by inhibiting actin-myosin interactions at low intracellular Ca2+ concentrations.6–10
Mutations in MYBPC3 encoding cMyBP-C cause hypertrophic cardiomyopathy (HCM) (reviewed previously3,11). HCM is an autosomal-dominant disease characterized by left ventricular (LV) hypertrophy, which predominantly involves the interventricular septum and is associated with myocardial disarray and interstitial fibrosis.12 HCM involves more than 450 mutations in at least 13 genes encoding sarcomeric proteins.11,13 Out of them, mutations in MYBPC3 are frequent.14 In contrast to other disease genes, in which the majority of the mutations are missense, 
70% of MYBPC3 mutations result in a frameshift creating a premature termination codon (PTC) and should produce C-terminal truncated proteins.11,14,15 The mechanism by which MYBPC3 mutations lead to HCM is not resolved. Truncated cMyBP-C were consistently undetectable in myocardial tissue of patients16–18 and found to be unstable in cardiac myocytes transfected with mutant cDNAs.19 Previous data have shown that truncated cMyBP-Cs, as well as a E344K cMyBP-C, are rapidly and quantitatively degraded by the ubiquitin–proteasome system (UPS) after gene transfer in cardiac myocytes, Hela, or COS cells.20,21
The goal of the present study was to gain insights into the molecular mechanisms controlling the expression of MYBPC3 mutations. We chose a G>A transition located on the last nucleotide of exon 6, which is associated with a severe phenotype and a bad prognosis in humans.14 The molecular consequences of this mutation were unknown, except that mutant mRNA deleted of exon 6 was detected in lymphocytes from one patient, suggesting exon skipping as a mechanism to produce a PTC and protein truncation.22 However, no data were obtained from myocardial tissue. The mutation was introduced in mice by gene targeting and represents the first targeted cMyBP-C knock-in mouse that carries a point mutation.
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Methods |
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The G>A transition on the last nucleotide of exon 6 was introduced in mice by gene targeting using the Cre/lox system, as detailed in the Online Data Supplement (available at http://circres.ahajournals.org) and depicted in Figure 1A. Two clones presenting the exact digestion pattern for both the 5'- and 3' polymerase chain reaction (PCR) fragments (Figure 1B) were used to obtain germline transmitting chimera. Cre-mediated recombination was obtained by crossing heterozygous cMyBP-C knock-in females with a Sycp1-Cre transgenic male and confirmed by PCR (Figure 1C). Pups were born in the expected Mendelian ratios of wild-type (WT), heterozygous (Het) and homozygous (KI) cMyBP-C knock-in mice. Both Het and KI appeared normal and were viable up to 2 years.
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See the expanded Methods section in the Online Data Supplement for a detailed description of RT-PCR, isolation of neonatal and adult mouse cardiac myocytes, protein fractionation, recombinant protein, Western blot, echocardiography, histology, immunofluorescence, ultrastructural analysis, and statistical analysis.
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Results |
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Low Levels of cMyBP-C mRNAs
We investigated the amount of total cMyBP-C mature mRNAs in ventricular tissue by RT-qPCR. Compared to WT, the amount of total cMyBP-C mature mRNAs was 50% and 80% lower in Het and KI, respectively (Figure 2A). To investigate whether this decrease already occurred at the level of the premRNA, RT-qPCR was performed using primers located in introns 1 and 2 (Figure 2B), in the middle or in the 3' portion of the gene (Online Figure I). PremRNA levels did not differ between the groups. No amplification was obtained without reverse transcription, excluding genomic contamination (data not shown). This suggests that the markedly lower mRNA levels in Het and KI were not the consequence of lower transcription efficiency.
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A Single Point Mutation Gives Rise to Three Different mRNA Isoforms
The G>A transition is located in the general consensus sequence for donor splice sites, which is AG| GTRAGT.23 It should produce either a missense mRNA, a nonsense mRNA deleted of exon 6, or both. Recent data reported the presence of nonsense, but not of missense mRNAs in lymphocytes from a patient with this mutation.22 To characterize the different mRNA isoforms, RT-PCR of exons 5 to 12 was performed. Whereas an expected 396-bp product was amplified in WT, 3 products of 396-bp (mutant-1), 278-bp (mutant-2), and 322-bp (mutant-3) were detected in KI (Figure 2C). Mutant-1 contains the G>A transition (missense) that should produce an E264K 150-kDa protein (Figure 2D). Mutant-2 and mutant-3 are both deleted of exon 6, inducing a frameshift. Mutant-2 contains a PTC in exon 9 (nonsense) and should produce a 32-kDa truncated cMyBP-C, including 41 novel amino acids. Mutant-3 retains 46 bp of intron 8 followed by a new cryptic acceptor splice site (deletion/insertion), which restores the reading frame and should produce a 147-kDa protein, containing an internal deletion and 41 novel amino acids. Mutant-2 and mutant-3 together represented 50% of total cMyBP-C mRNAs in KI mice (Figure 2C), and were hardly detected by classic RT-PCR in Het (data not shown).
Involvement of Nonsense-Mediated mRNA Decay
The low amount of total cMyBP-C mRNAs and the nondetection or low amount or of mutant-2 mRNAs in Het and KI suggested posttranscriptional regulation. Nonsense-mediated mRNA decay (NMD) specifically targets PTC-bearing mRNAs for degradation when the PTC lies >50 to 55 nt upstream of the last exon–exon junction.24 This rule applies to mutant-2, in which the PTC is located far upstream of the last junction between exons 34 to 35.15 In mammals, NMD requires a pioneer round of translation and can be prevented by translation inhibitors such as emetine or cycloheximide (CHX).25 To investigate this hypothesis, neonatal mouse cardiac myocytes (NMCMs) were cultured in the presence of emetine (300 µg/mL) for 4 hours. Similar to the data obtained in adult hearts, the levels of total cMyBP-C mRNAs were markedly lower in NMCMs from Het and KI than from WT (Figure 3A). Hydrolysis probes were designed to quantify specifically the levels of mutant-1 (Figure 3D and 3H), mutant-3 (Figure 3C and 3G), and mutant-2 plus mutant-3 ("frameshift"; Figure 3B and 3F). Emetine increased total mRNAs level in Het and KI 1.4- and 2-fold, respectively (Figure 3A), and increased 15- and 7.5-fold frameshift mRNAs level in Het and KI, respectively (Figure 3B). Surprisingly, mutant-3 mRNAs became undetectable after emetine in both groups (Figure 3C). This is likely attributable to competition with mutant-2 having an advantage by size and quantity after emetine. Interestingly, mutant-1 was not detectable in Het, suggesting its absence (Figure 3D). Emetine did not significantly affect mutant-1 mRNAs level in KI cells (Figure 3D). CHX induced similar effects in NMCMs (Online Figure II).
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To test whether NMD was also involved in vivo, CHX was subcutaneously injected in WT, Het and KI mice (120 mg/kg, 4 times, every hour). CHX did not result in LV hypertrophy (data not shown). The results almost exactly matched those with emetine in NMCMs (Figure 3E through 3H), except that mutant-3 mRNAs were still detectable after CHX treatment. It is not clear whether this is because of the overall slightly smaller effect of CHX in vivo or differences in action. Taken together, these experiments clearly revealed that NMD lowers the level of mutant-2 nonsense mRNAs, but not of WT and other mutant mRNAs in cells and in vivo in Het and KI.
Low Levels of Mutant Proteins
To evaluate whether all mutant mRNAs are translated, Western blots were performed. The abundance of cMyBP-C in crude protein fraction was quantified by Western blot using the C0C1 antibody, expected to recognize all cMyBP-C isoforms (Figure 4A). A single 150-kDa band was detected in WT, Het, and KI mice, but the expected 32-kDa mutant-2 was not. Whereas the 150-kDa band corresponds to the WT-cMyBP-C in WT mice, it could also correspond to mutant-3 in Het, and to mutant-1 and/or mutant-3 in KI mice. Compared to WT, the level of 150-kDa cMyBP-C was 21% and 90% lower in Het and KI, respectively (Figure 4A). Immunofluorescence of adult mouse ventricular myocytes using the C0C1 antibody showed classic alternation of cMyBP-C (A-band doublets) and 
-actinin (Z-band) in all groups, although cMyBP-C was hardly detected in KI myocytes (Figure 4B).
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To specifically evaluate whether mutant-2 and mutant-3 are detected in Het and KI, we developed a new cMyBP-C peptide antibody directed against novel amino acids produced by the frameshift. This antibody was tested by immunofluorescence in NMCMs from KI mice (Figure 4C). It revealed frameshift cMyBP-C in the A-band of the sarcomere, and, unexpectedly, in the nuclei as well. The frameshift antibody revealed a single 147-kDa band in KI and, to a lower extent in one Het, but not in WT mice (Figure 4D), underlying its specificity. The level of mutant-3 protein was 3-fold higher in KI than in Het, and the absence of detection in 2 Het was likely attributable to lower quantity of loaded proteins as shown by the Ponceau. In contrast to mutant-3, truncated mutant-2 was not detected in crude protein fraction (data not shown).
Involvement of UPS
The absence of detection of mutant-2 in Het and KI suggested that the UPS could be involved in its degradation. To investigate this hypothesis, mice were treated for 1 week with the proteasome inhibitor epoxomicin (0.5 mg/kg per day) via osmotic mini-pumps. Recombinant mutant-2 protein was produced in bacteria and used as a positive control, and proteins were separated into four different fractions by sequentially increasing the stringency of solubilization (Figure 5A): no detergent (cytosolic), triton, sodium dodecyl sulfate (SDS), and urea. The chymotrypsin-like activity of the β5 subunit of the proteasome, measured in the cytosolic fraction, was inhibited in all groups by 50% (data not shown). This was associated with the appearance of 2 β5 subunit bands in Western blots in all but mainly in cytosolic fraction (Figure 5A). The upper band likely corresponds to epoxomicin-bound (estimated 4 molecules) to β5 subunit; the lower band corresponds to free β5 subunit. The ubiquitinated proteins and cMyBP-C were detected only in SDS and urea fractions. Unexpectedly, the level of ubiquitinated proteins did not increase after proteasome inhibition in vivo (Figure 5A).
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All 150-kDa cMyBP-C isoforms were detected as a single band with the C0C2 antibody in SDS (Figure 5B) and urea fractions (not shown). Epoxomicin increased by 100% the level of 150-kDa cMyBP-C isoforms in KI, but not in WT and Het (Figure 5B), suggesting a major effect on mutant-1 and mutant-3, but not on WT-cMyBP-C. The frameshift cMyBP-C antibody detected mutant-3 as 3 bands of 150 kDa mainly in the SDS fraction in Het and KI (Figure 5C and 5D). Epoxomicin increased the intensity of each mutant-3 bands by 30% to 50% in Het and KI (Figure 5C and 5D). Truncated mutant-2 was detected only in the last urea fraction in the KI, but not in Het mice, and was not affected by proteasome inhibition (Figure 5E).
Myocyte and Eccentric Left Ventricular Hypertrophy in cMyBP-C KI Mice
To investigate whether the mutation results in a phenotype, echocardiography was performed at 3, 12, and 18 months of age in WT, Het, and KI mice. No major differences were found between Het and WT (Online Table I). In contrast, KI mice exhibited LV hypertrophy and reduced fractional shortening compared to WT (Figure 6A and Online Table I). The LV mass/body weight ratio, the LV posterior wall thickness, and the LV diameter (LV end-diastolic diameter and LV end-systolic diameter), but not the interventricular septum, were higher in KI compared to WT (Figure 6A).
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Longitudinal heart cross-sections and histological analysis of transversal ventricular sections confirmed enlargement of LV and showed accumulation of interstitial fibrosis in KI (Figure 6B through 6D). The heart/body weight ratio was 60% higher in KI (Figure 6E). Body weight was unchanged (data not shown). In contrast to KI, myocardial morphology and heart/body weight ratio were not affected in Het mice. KI myocytes were 27% longer and slightly wider than WT (Figure 6F). Myocyte area, calculated as myocyte lengthxwidth, was 41% greater in KI. Interestingly, Het myocytes were 10% wider than WT, which resulted in a 4% higher cell area. The mRNA amounts of β-myosin heavy chain, -skeletal actin, brain natriuretic peptide, and atrial natriuretic peptide were 8- to 14-fold higher in KI (Figure 6G). Het mice did not show any changes except a slight, not significant higher β-myosin heavy chain mRNA level. Electron microscopy of adult mouse ventricular myocytes showed a correct sarcomere ultrastructure, including a well-defined M-band in most of the sarcomeres in WT and KI (Figure 6H).
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Discussion |
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We developed a cMyBP-C knock-in mouse model to study the molecular mechanisms of HCM in a genomic context as close as possible to the human situation. We chose a MYBPC3 point mutation, which is associated with a severe phenotype and a poor prognosis in humans.14 The major findings of the present study are: (1) the single point mutation unexpectedly resulted in 3 different mutant mRNAs and proteins; (2) their concentration was markedly lower than the respective WT levels in WT mice; (3) the level of mutant-2 nonsense mRNAs markedly and selectively increased after acute inhibition of NMD; (4) the levels of mutant-1 and/or mutant-3 proteins increased after prolonged inhibition of the UPS; and (5) homozygotes exhibited myocyte and LV hypertrophy with reduced fractional shortening; heterozygotes had no major phenotype. Our findings provide the first in vivo evidence that NMD and UPS act in parallel to eliminate mutant mRNAs and proteins.
In both KI and Het mice, the level of total cMyBP-C mature mRNAs but not of premRNAs was markedly lower than in WT, suggesting a posttranscriptional regulation. Involvement of NMD in cardiac genetic diseases just starts to be recognized.26–28 Nonsense mRNAs resulting from lamin A/C or hERG mutations were detected at very low levels and were stabilized by CHX in cultured skin fibroblasts or after gene transfer in HEK cells.26–28 In the present study, NMD was investigated using CHX and emetine that act differently on the ribosomal subunits. CHX competes with the binding of ATP to the 60S subunit and prevents protein elongation,25 whereas emetine binds to the 40S subunit thereby preventing the EF-2–dependent translocation of the ribosomes.29 Both CHX and emetine markedly increased the level of mutant-2 mRNAs, but not of the other mutant mRNAs in Het and KI. Importantly, the CHX effect was similarly seen in vivo, indicating that NMD is operational in the whole animal. The absolute level of the remaining mutant-2 mRNAs could not be determined because the RT-qPCR assay did not differentiate between mutant-2 and mutant-3, but standard RT-PCR indicated a ratio of 1:1 in KI (Figure 2C). This indicates very low levels of mutant-2 mRNAs in Het (<2%; Online Table II) and, together with the dramatic increase after NMD inhibition, an almost complete effectiveness of NMD. This is supported by the absence of mutant-2 protein in any fraction in Het and the low level in KI (Figure 5C through 5E; Online Table II). In contrast to our initial hypothesis,20 the apparent absence of truncated cMyBP-C in Het mice was likely not attributable to UPS-mediated degradation but rather reflects the different sensitivities of RT-PCR and Western blot. In any case, it fully matches the few data obtained in myocardial tissue of HCM patients with other frameshift mutations.17,18 We estimate that 70% of the 165 known MYBPC3 mutations11,13 should result in a PTC-involving NMD, indicating that this could be a major mechanism involved in HCM.
In addition to the PTC-bearing mRNAs, which were detected in lymphocytes from 1 HCM patient,22 the G>A transition resulted also in 2 other mutant mRNAs. Whereas mutant-1 was only present in KI, mutant-3 was detected in both Het and KI mice. Our data provide evidence that the expression of mutant-1 and mutant-3 is not regulated by NMD but by the UPS in vivo. Interestingly, proteasome inhibition did not result in accumulation of ubiquitinated proteins in vivo (Figure 5A). The reason for this unexpected finding is not clear, but a similar observation was recently made in another mouse model.30 It may relate to the fact that epoxomicin only inhibits part of the proteasomal machinery.31
In Het mice, total cMyBP-C mRNAs levels amounted to 50% of WT, mutant-1 mRNA was absent, and frameshift mRNA represented less than one-third of KI (Online Table II). Because total cMyBP-C mRNA levels in KI represented only 10% of the level in WT, the concentration of all mutant mRNAs in Het must be very low (<2%; Online Table II), indicating that the majority of total mRNAs in Het is WT. This argues for a normal transcription rate of the remaining WT allele (50% total mRNAs) and some posttranscriptional compensation, because the total protein level was 80% of WT (Online Table II). Similar levels were found in heterozygous KO mice (as a model of pure haploinsufficiency32) and in patients with HCM,18 suggesting that reduced levels of cMyBP-C may play a role in the pathogenesis of HCM. In addition, mutant-3 was present at a low level in Het and appeared to be incorporated in the A-band of the sarcomere. Thus, it could act as a "poison peptide" on the function of the sarcomere. Our data do not allow to discriminate between these 2 mechanisms. Although Het mice did not develop an overt phenotype (only slightly higher myocyte width without LV hypertrophy), other preliminary results indicate alterations of sarcomere shortening and Ca2+ transient in Het myocytes (B Fraysse, L Carrier, unpublished data) and septal hypertrophy with proteasome impairment on adrenergic stress in heterozygotes.34
Homozygotes differ from Het in several aspects. Missense mutant-1 was present in KI (absent in Het), mutant-2 and mutant-3 mRNAs levels were 3-fold higher than in Het, and mutant-2 protein was readily detectable, albeit only in the SDS-insoluble, urea fraction. These data could be interpreted as indicating compensatory mechanisms toward a full-length, semifunctional protein (mutant-1) and saturation of the quality control system NMD in case of mutant-2. Functionally, KI exhibited marked myocyte and LV hypertrophy and reduced fractional shortening and dilatation at 3 months of age. This phenotype is similar to what was observed in other homozygous cMyBP-C mutant mice32,35,36 and in the few patients with homozygous mutations reported so far.14 The phenotype results from either reduced protein amount or expression of mutant proteins or both in homozygous mice. Interestingly, homozygous KI exhibited higher steady-state levels of ubiquitinated proteins and chymotrypsin-like activity of the proteasome than WT mice (Online Figure III). This underlines UPS alterations in KI mice as previously shown in human and experimental heart failure (reviewed elsewhere37).
In conclusion, these data unravel an unanticipated complexity of gene expression of a single point mutation that needs to be taken into account in further genetic studies. Furthermore, our findings support the view that the expression of the point mutation is regulated at the mRNA level involving NMD and at the protein level involving UPS. On the basis of our former,20,21 present, and other data,16–18 we propose that NMD and UPS act as parallel quality control systems to eliminate mutant cMyBP-C.
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Acknowledgments |
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We thank Minoo Rassoulzadegan (Nice, France) for providing Sycp1-Cre mice, Ju Chen (University of California at San Diego) for the LoxP-pGK-neo-HSVtk cloning vector, and Denise Juhr and Barbara Holstermann (Hamburg, Germany) for technical assistance.
Sources of Funding
This work was supported by the Sixth Framework Program of the European Union (Marie Curie EXT-014051), the French Research Government Department (ACI-191, Decision no. 02-2-0547), the Deutsche Forschungsgemeinschaft (FOR-604-CA 618/1-2), the Institut de la Santé et de la Recherche Médicale (PNRMC-A04048DS), and the Association Francaise contre les Myopathies (AFM-9471).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Deceased. Original received September 7, 2007; resubmission received May 22, 2009; revised resubmission received June 24, 2009; accepted June 29, 2009.
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