Cardiovascular Research

Cardiovascular Research 2004 63(2):293-304; doi:10.1016/j.cardiores.2004.04.009
© 2004 by European Society of Cardiology

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Copyright © 2004, European Society of Cardiology

Asymmetric septal hypertrophy in heterozygous cMyBP-C null mice

Lucie Carrier^*,a,b,c,1, Ralph Knöll^d,1, Nicolas Vignier^a,b, Dagmar I Keller^a,b, Pedro Bausero^a,b, Bernard Prudhon^a,b, Richard Isnard^b,e, Marie-Lory Ambroisine^a,b, Marc Fiszman^a,b, John Ross, Jr.^d, Ketty Schwartz^a,b and Kenneth R Chien^d

^aINSERM U582, Institut de Myologie, Bâtiment Babinski, GH Pitié-Salpêtrière, 47 boulevard de l'Hôpital, 75651 Paris, France
^bINSERM IFR14, GH Salpêtrière, Paris, France
^cInstitute of Experimental and Clinical Pharmacology, University Hospital Eppendorf, Hamburg, Germany
^dInstitute of Molecular Medicine, UCSD, La Jolla, CA, USA
^eService de Cardiologie, GH Pitié-Salpêtrière, Paris, France

^* Corresponding author. INSERM U582, Institut de Myologie, Bâtiment Babinski, GH Pitié-Salpêtrière, 47 boulevard de l'Hôpital, 75651 Paris, France. Tel.: +33-1-4216-5715; fax: +33-1-4216-5700. Email address: l.carrier{at}myologie.chups.jussieu.fr

Received 22 September 2003; revised 26 March 2004; accepted 14 April 2004

	Abstract

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

 
Objective: Cardiac myosin-binding protein C (cMyBP-C) gene mutations are involved in familial hypertrophic cardiomyopathy (FHC). Many of these mutations produce truncated proteins, which are unstable in the cardiac tissue of patients, suggesting that haploinsufficiency could account for the development of the phenotype. However, existing mouse models of cMyBP-C gene mutations have represented hypomorphic alleles without evidence of asymmetric septal hypertrophy, a key FHC phenotypic feature. In the present study, we generated a new model of cMyBP-C null mice and characterized the phenotype in both homozygotes and heterozygotes at different ages. Methods: The mouse model was based upon the targeted deletion of exons 1 and 2, which contain the transcription initiation site, and the phenotype was determined by molecular, functional and morphological analyses. Results: Herein, we demonstrate that inactivation of one or two mouse cMyBP-C alleles leads to different cardiac disorders at different post-natal time windows. The homozygous cMyBP-C null mice do not express the cMyBP-C gene, develop eccentric left ventricular hypertrophy with decreased fractional shortening at 3–4 months of age and a markedly impaired relaxation after 9 months. This is associated with myocardial disarray and an increase of interstitial fibrosis. The heterozygous cMyBP-C null mice present a slight but significant decrease of cMyBP-C amount and develop asymmetric septal hypertrophy associated with fibrosis at 10–11 months of age. Conclusion: These data provide evidence that heterozygous cMyBP-C null mice represent the first model with a key feature of human FHC that is asymmetric septal hypertrophy.

KEYWORDS Sarcomere; Targeted transgenesis; Cardiomyopathy; Hypertrophy

	1. Introduction

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

 
Cardiac myosin binding protein C (cMyBP-C) is a component of the A-band of the sarcomere, which specifically interacts with myosin, actin and titin (Fig. 1; [1–4]). It is exclusively expressed in the heart during human and murine development [5,6] and plays important structural and functional roles during cardiac contraction in health and disease (for review, see Ref. [7]).

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Structure of cardiac myosin-binding protein C. It features eight IgC2-like domains (empty box), three FN3 domains (hatched box), a MyBP-C motif containing three phosphorylation sites (dotted box). The protein-binding domains are indicated.

 
Mutations in the cMyBP-C gene have been shown to be involved in familial hypertrophic cardiomyopathy (FHC) (for reviews, see Refs. [8–11]). FHC is an autosomal-dominant disease characterized by left ventricular hypertrophy (LVH), which predominantly involves the interventricular septum, and is associated with myocardial disarray and interstitial fibrosis [12,13]. Most of the pure forms of FHC involve mutations in 12 different genes encoding proteins of the sarcomere and most of the families present mutations in the cMyBP-C gene [14]. In contrast to the other genes involved in FHC in which the majority of the mutations are missense and encode mutated proteins, most of the cMyBP-C gene mutations result in a frameshift and are predicted to produce C-terminal truncated proteins [15–17]. The molecular mechanism by which the frameshift cMyBP-C gene mutations lead to FHC is not elucidated. It has been shown that truncated proteins resulting from different cMyBP-C gene mutations are unstable in myocardial tissue of patients [18–20], in fetal rat cardiomyocytes after transfection with human mutated cMyBP-C cDNAs [21], and finally also in two mouse models of FHC obtained by additional or targeted transgenesis [22,23]. Altogether these data suggest that the "null allele" mechanism leading to protein haploinsufficiency could be involved in the pathogenesis of FHC. Therefore, a good model of human FHC would be the heterozygous cMyBP-C null mice carrying only one functional allele. Two targeted cMyBP-C mutant mouse models have been developed, they both resulted in hypomorphic alleles without evidence of asymmetric septal hypertrophy, a key phenotypic feature of human FHC [23,24]. The functional homozygous cMyBP-C knockout mice exhibit profound LVH whereas heterozygous mice do not develop any phenotype at 4–5 months of age [24].

In the present paper, we inactivated the mouse cMyBP-C gene by creating a targeted gene deletion which includes the transcription initiation site. This leads to a transcriptional knockout, and molecular analyses document that this targeting event has led to the development of the first cMyBP-C null mice carrying one or two null alleles. We analyzed the cardiac phenotype at different post-natal time windows in both homozygous and heterozygous cMyBP-C null mice. The homozygous cMyBP-C null mice develop eccentric LVH with decreased fractional shortening (FS) at 3–4 months of age and a markedly impaired relaxation at 9 months. Heterozygous mice develop asymmetric septal hypertrophy at 10–11 months of age. These data provide evidence that heterozygous cMyBP-C null mice represent the first model with a major feature of human FHC that is asymmetric LVH, predominantly involving the interventricular septum.

	2. Material and methods

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

 
The investigation conforms with the guide for the care and use of laboratory animals published by the NIH (Publication No. 85-23, revised 1985).

2.1. Gene targeting
A 15-kb fragment of the mouse MYBPC3 gene containing exons 1 to 19 was cloned from a FIX II genomic library (Stratagene) derived from a 129/SvJ mouse strain using a ³²P-labeled 325-bp probe starting at the ATG and ending within exon 2. A 3-kb SacI/EcoRI and a 3-kb BstUI/SnaBI fragments were cloned into the EcoRI and the XhoI sites of pPNT vector [25] containing the neomycin (Neo) resistance gene (Fig. 2a). The NotI linearized vector (20 µg) was electroporated in embryonic stem (ES) cells and proceeded for homologous recombination. Genomic DNA was extracted from G418-resistant ES clones with the Wizard Genomic DNA Purification kit (Promega). G418-resistant clones were screened for homologous recombination by PCR-digestion (Fig. 2b). Long-range PCR were performed with the Expand Long template PCR kit (Roche) to check for 5' homologous recombination (5'PCR, 4.326 kb) and 3' one (3'PCR, 3.9 kb). Primers used were the following: 5F, 5'-CCC TTA TGG ACT TCA CGC CTG TTC TTC-3'; 5R, 5'-GCA TCG CCT TCT ATC GCC TTC TTG ACG-3'; 3F, 5'-CGG TGG ATG TGG AAT GTG TGC GAG GC-3'; and 3R, 5'-CCT TGT TTG GCA GTG CTG TTG GGA GAA T-3'. PCR reaction mix (20 µl) included 1 µl of ES cells genomic DNA. PCR was performed at 94 °C/3 min, followed by 30 cycles of 94 °C/30 s, 65 °C/30 s, 68 °C/4 min, 72 °C/10 min, and final extension at 68 °C/15 min. PCR products were digested in 20 µl at 37 °C for 1 h with EcoRI/NcoI for the 5'PCR fragment and HindIII for the 3'PCR fragment, and then loaded on a 1% agarose gel. One targeted clone was injected into C57/B6 blastocysts and implanted into pseudopregant C57/B6 females, and gave rise to seven chimeric founder mice. Chimeric males were tested for germ-line transmission by crossing with Black Swiss female breeders. Genomic DNA was extracted from mouse tails with the Wizard Genomic DNA Purification kit (Promega). Mouse genotypes were determined by PCR on genomic DNA with the Platinium Taq DNA polymerase kit (Invitrogen) (Fig. 2c). Primers used to amplify the wild type (WT) allele (1.2 kb) were the following: WT-F, 5'-AGC CTT CTC TTC CAG CCC CAG-3' and WT-R, 5'-AGG GAT GGG GAA TGA AGC AGA GA-3'. Primers used to amplify the knockout (KO) allele (0.8 kb) were the following: KO-F, 5'-AGC CTT CTC TTC CAG CCC CAG-3' and KO-R, 5'-GCA TCG CCT TCT ATC GCC TTC TTG ACG-3'. PCR reaction mix (50 µl) included 80 ng genomic DNA, 100 µmol/l dNTPs, 1.5 mmol/l MgCl₂, 0.4 µmol/l of each primer, 5 µl 10 x buffer, and 1 U of platinium Taq polymerase. Touchdown PCR was performed at 94 °C/5 min, followed by 11 cycles of 94 °C/30 s, 70–65 °C/30 s, 72 °C/1 min, followed by 24 cycles of 94 °C/30 s, 65 °C/30 s, 72 °C/ 1 min, and final extension at 72 °C/3 min. PCR products were loaded on a 1% agarose gel.

View larger version (45K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Targeting the mouse MYBPC3 gene. (a) Targeting strategy. A restriction map of MYBPC3 genomic region of interest is shown on top, the pKO targeting construct is shown in the center, and the mutated locus after homologous recombination is shown at the bottom. The 5'PCR and 3'PCR used to check ES cells genomic DNA are indicated, as well as the ATG translational start site. (b) Detection of homologous recombinant ES clones by PCR. Long-range 5'PCR (2,3) and 3'PCR (6,7) were performed as shown in (a). This shows a negative clone (lanes 2 and 6) and a positive recombinant clone (lanes 3 and 7). Confirmation of the positive recombinant clone was performed by digestion of both PCR products with the following restriction enzymes: EcoRI/NcoI for the 5'PCR fragment (4) and HindIII for the 3'PCR fragment (8). Lanes 1, 5 and 9 correspond to the MW markers. (c) PCR genotyping strategy used to detect wild type (WT, 1.2 kb) and knockout (KO, 0.8 kb) alleles in genomic DNA from tail biopsies of wild type (+/+) and heterozygous (+/–) mice and from recombinant heterozygous ES cells (rES). This shows the amplification of only the WT allele in the wild type mice and both WT and KO alleles in the heterozygous mice.

 
2.2. RNA analyses
Total RNA was extracted from mouse ventricles using the SV Total RNA Isolation kit (Promega) according to the manufacturer's instructions. Concentrations were determined by spectrophotometry. Reverse transcription (RT) was performed on 1 µg total RNA using the SuperSriptTM First-Strand Synthesis System (Invitrogen). Serial dilutions of RT were performed (1/2, 1/20, 1/200, 1/2000). Quantitative PCR was performed in the LightCycler instrument (Roche Diagnostics). PCR reaction mix (20 µl) included 2 µl of each diluted RT, 4 mmol/l MgCl₂, 300 nmol/l of each primer in 1 x LightCycler DNA Master SYBR Green 1. Specific primers for cardiac cDNAs were chosen with the LightCycler program according to Genbank accession numbers: -skeletal actin (skAct, accession number M12347 [GenBank] ), brain natriuretic peptide (BNP, accession number D16497 [GenBank] ), cMyBP-C (accession number AF059576 [GenBank] ), β-MyHC (accession number L07306 [GenBank] ), and hypoxanthine guanine phosphorybosyl transferase (HPRT, accession number M-013556). PCR was performed using the following thermal settings: denaturation and enzyme activation at 95 °C/8 min and cycling at 95 °C/10 s, 64 °C/10 s and 72 °C/10 s. Amplification was followed online and the PCR stopped after the logarithmic phase. Additionally, a melting curve analysis was performed after PCR to check specificity of the reaction. PCR products were sequenced with the dGTP BigDyeTM Terminator (Applied Biosystems). The reaction products were then submitted to electrophoresis on an automated laser fluorescent DNA sequencer 377 (Applied Biosystems). The amount of each target mRNA (cMyBP-C, -skAct, BNP, β-MyHC) relative to the amount of the reference (HPRT) was determined in logarithmic phase by dilution series used to determine the fit coefficients of the relative standard curve. The PCR efficiency was similar for the different targets in order to compare individuals.

2.3. Antibodies
Polyclonal anti-cMyBP-C antibody (C0–C1 domains) and polyclonal anti-titin antibodies (Z-line or M-band) were kindly given by M. Gautel (London, UK). Polyclonal cMyBP-C antibody (MyBPC motif) was kindly given by C. Witt & W. Linke (Heidelberg, Germany). Monoclonal antibody directed against the myosin heavy chain (MyHC) was purchased from Biocytex (1050-S) and Polyclonal anti-calsequestrin antibody from Affinity Bioreagents.

2.4. Western blot analysis
Total cardiac proteins were extracted from mouse hearts. About 5 mg of tissue was homogenized in 5% SDS, 50 mmol/l Tris–HCl, pH 7.5, 250 mmol/l sucrose, 75 mmol/l urea, 1 mmol/l DTT at 4 °C and centrifuged at 1300 rpm for 2 min. The supernatant was collected and its concentration was determined using the BCA Protein Assay Kit (Pierce). Total proteins were loaded on 10% acrylamide/bisacrylamide (29/1) mini gels (Xcell SureLock^TM, NOVEX). Proteins were electro-transferred on a nitrocellulose membrane, incubated with different antibodies and revealed by chemiluminescence. The antibodies used were directed against calsequestrin (dilution 1/2000) and cMyBP-C (dilution 1/2000). The secondary antibody was coupled to HRP (dilution 1/6000).

2.5. Echocardiography
Mice were anesthetized with Avertin^R (125 mg/kg, IP) or isoflurane. Echocardiography was performed as described previously [26,27]. Thickness of the interventricular septum (IVS) and posterior wall (PW), and left ventricular diameter (LVD) were measured. LVM was calculated as follow: 1.055*((LVDd+PWd+IVSd)³-LVDd³) [26]. Heart rate (HR), the percent of fractional shortening (FS) and left ventricular mass/body weight ratio (LVM/BW) were calculated.

2.6. Hemodynamic measurements
Cardiac function of the left ventricle (LV) was measured by high fidelity microsonometry in mice after 9 months of age as described previously [27]. Heart rate (HR), maximum left ventricular pressure (LVP_max), maximum positive first derivative of LVP (contractility; dP/dt_max), maximum negative first derivative of LVP (relaxation, dP/dt_min), and the time constant of relaxation (using an exponential function, Exp. Tau) were calculated. The effects of the β-adrenergic agonist dobutamine (0.75, 1.25, 2 and 4 µg/kg/min) were also determined. Systolic meridional wall stress (dyn/cm²) of the LV was calculated as PRi/2h(1+h/2Ri), where P is LVP, Ri is inner diameter and h is the mean end-systolic wall thickness (h=(IVS+PW)/2) [28].

2.7. Histology and immunohistochemistry
Mouse ventricles were rapidly frozen in isopentane cooled in nitrogen liquid and 6-µm sections were performed. For histology, sections were stained with Hematoxylin and Eosin (HE), collagen-specific Sirius red F3B or modified Gomori's Trichrome. Sections were analyzed by light microscopy with Leica DMR, equipped with 10 x or 40 x objectives, and the pictures were acquired with a Sony DXC-950P TriCCD-color camera. For immunohistochemistry, sections were incubated with antibodies directed against cMyBP-C (dilution 1/200), MyHC (dilution 1/10), and titin (M-band and Z-line, dilution 1/200). Secondary antibodies were goat Cy3-conjugated anti-rabbit and Cy3-conjugated anti-mouse (Jackson Immuno-Research laboratory). Sections were analysed by fluorescence microscopy with Axiophot or Axioplan Zeiss microscopes, equipped with 40 x or 100 x oil objectives, and the pictures were acquired with a Sony DXC-950P TriCCD-color camera.

2.8. Statistical analysis
Data are mean±S.D. Statistical analyses were performed with the unpaired Student's t-test or using the nonparametric test of Mann–Whitney in small groups. A value of P<0.05 was considered significant.

	3. Results

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

 
3.1. Generation of cMyBP-C null mice
The cMyBP-C gene targeting strategy is depicted in Fig. 2a. The transcription initiation site and exons 1–2 were replaced by a Neo resistance gene, which results in the complete transcriptional inactivation of cMyBP-C gene expression. The linearized targeting construct was electroporated into J1 ES cells, and then selected with G418. A total of 96 colonies were screened for homologous recombination by two different long-range PCR (5'PCR and 3'PCR) on genomic DNA (Fig. 2a,b). Fig. 2b shows typical examples of long-range PCR followed by digestion with specific restriction enzymes. No amplification was obtained for the 5'PCR (lane 2) and the 3'PCR (lane 6) in one clone, indicating that this clone is not recombinant. This was the case for most of the clones. In contrast, one positive recombinant clone shows the expected 5'PCR fragment (lane 3) and 3'PCR fragment (lane 7), which were both confirmed by specific digestion (lanes 4 and 8, respectively). This clone was injected into C57BL/B6 blastocysts, which resulted in the generation of seven chimeras, three of which displayed germ-line transmission. PCR analyses were used to specifically amplify the WT and the KO alleles and therefore genotype the offsprings (Fig. 2c). Heterozygous offsprings were crossed to generate the three genotypes, wild-type (WT), heterozygous (cMyBP-C^+/–) and homozygous knock-out (cMyBP-C^–/–) mice. Expected Mendelian ratios of WT, cMyBP-C^+/– and cMyBP-C^–/– mice were obtained, indicating no embryonic lethality. Both cMyBP-C^+/– and cMyBP-C^–/– mice appeared normal in all respects and were viable.

3.2. Molecular analyses of cMyBP-C null mice hearts
To analyze the consequences of inactivation of cMyBP-C gene at the mRNA level, ventricular mRNAs were subjected to quantitative RT-PCR using the LightCycler system with specific primers for cMyBP-C, β-MyHC, -skAct, and BNP. No cMyBP-C mRNA was amplified in the cMyBP-C^–/– mice therefore validating the gene inactivation at the transcriptional level (Fig. 3a). In addition, the mRNAs for markers of hypertrophy were significantly up-regulated in the cMyBP-C^–/– mice (Fig. 3b). Heterozygous mice showed a decrease of cMyBP-C mRNA level at different post-natal windows (Fig. 3a). The hypertrophic markers were not up-regulated in cMyBP-C^+/– mice at 6 months of age (Fig. 3b) but at 10.5 months of age β-MyHC mRNAs significantly increased when compared to WT mice (Fig. 3c).

View larger version (36K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Molecular analyses in the cMyBP-C+/–, cMyBP-C–/– and in WT mice. Quantitative RT-PCR analyses were performed on ventricular RNA using the LightCycler system for cMyBP-C mRNA (a) and markers of hypertrophy at 6 months (b) and 10.5 months of age (c). Results were obtained on four to six different mice in WT (empty box), HET (hatched box) and KO (black box). Western blot analysis was performed on total ventricular proteins extracted at 4 months of age (d) and 10–11 months of age (e, f). Membranes were incubated with antibodies directed against cMyBP-C and calsequestrin. Quantitative protein analysis (f) was performed in HET (n=14, hatched box) and WT (n=14, empty box). Abbreviations used are: WT, wild type mice; HET, cMyBP-C+/– mice; KO, cMyBP-C–/– mice; CSQ, calsequestrin; -skAct, -skeletal actin; BNP, brain natriuretic peptide; β-MyHC, β-myosin heavy chain. Values are expressed as mean±S.D. Significantly different vs. WT mice with *P<0.05; **P<0.01; and ***P<0.001.

 
The amount of cMyBP-C protein was determined by Western blot performed on 20–60 µg of total ventricular proteins. No cMyBP-C protein was detected in the cMyBP-C^–/– mice using an antibody directed against either the C0C1 domains of cMyBP-C (Fig. 3d) or the MyBP-C motif (data not shown). This excludes the presence of a N-terminal truncated cMyBP-C in the cMyBP-C^–/– mice heart. At 10–11 months of age, the cMyBP-C^+/– mice exhibited about 25% decrease of cMyBP-C protein when compared to WT mice, without significant decrease of the calsequestrin amount (Fig. 3e,f).

3.3. Cardiac function on echocardiography
Transthoracic echocardiography was used to assess LV dimensions in mice at different ages. At 3–4 months of age, cMyBP-C^–/– mice exhibited significant eccentric LVH (increased IVSd, PWd, and LVDd), a significant decrease of FS and increase of LVM/BW when compared to WT mice (Table 1). These differences remained when males and females were separated. In contrast, no significant differences were found between cMyBP-C^+/– and WT mice (data not shown).

View this table: [in this window] [in a new window]   Table 1 Echocardiography summary data at 3 months of age

 
At 10–11 months of age, the profound LVH remained in cMyBP-C^–/– mice and the altered FS decreased below 20% (data not shown). At that age, the heterozygous cMyBP-C^+/– mice exhibited significant LVH when compared to WT mice, which is asymmetric and predominantly involves the IVSd (IVSd/PWd=1.31±0.22; Table 2). The LVM/BW ratio increased but no decrease of FS was found in cMyBP-C^+/– mice when compared to the WT mice. These differences remained when males and females were separated.

View this table: [in this window] [in a new window]   Table 2 Echocardiography summary data at 10–11 months of age

 
3.4. Hemodynamic measurements
Hemodynamic function was assessed after 9 months of age in the absence or presence of graded doses of dobutamine. Under basal conditions, there was an impaired relaxation in the cMyBP-C^–/– mice (decreased dP/dt_min and increased Exp. Tau) when compared to WT mice but surprisingly, no significant decrease of the contractility was observed (Table 3). In the presence of graded doses of dobutamine, the relaxation deficit (decreased dP/dt_min and increased Exp. Tau) was accentuated in the KO mice, indicating that the hastening effect of dobutamine on relaxation was blunted (Fig. 4d and e). In contrast to the KO mice, the cMyBP-C^+/– mice did not exhibit any LV dysfunction with or without dobutamine.

View this table: [in this window] [in a new window]   Table 3 Basal cardiac function of WT, HET and KO mice at 9.3–11.7 months of age assessed by heart catheterization

 

View larger version (33K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Hemodynamic measurements under basal conditions and in response to graded doses of dobutamine. Experiments were performed in WT (n=5), HET (n=6) and KO (n=6) mice between 9.3 and 11.7 months of age. Abbreviations used are: HR, heart rate; LVP; maximum left ventricular pressure; dP/dtmax, maximum positive first derivative of LVP (contractility); maximum negative first derivative of LVP (relaxation, dP/dtmin); Exp. Tau, exponential Tau. Symbols are: circles, WT mice; triangles, HET mice; squares, KO mice. *Significantly different vs. WT mice (P<0.05).

 
3.5. Cardiac morphology and structure in cMyBP-C null mice
The cMyBP-C^–/– mice exhibited a significant enlargement of the ventricles when compared to cMyBP-C^+/– and WT mice at 9 months of age (Fig. 5a). Histological examination showed myocardial disarray, increase of interstitial fibrosis and calcification in the fibrotic areas in cMyBP-C^–/– mice (Fig. 5b). In contrast, no major change in gross morphology was found in cMyBP-C^+/– mice when compared to WT at 9 months of age (Fig. 5a). However, at 10–11 months of age, the heterozygous mice exhibited a slight but significant increase of the ventricular weight/body weight ratio when compared to the WT (+19%; P<0.01; Fig. 5c). Histological examinations performed at that age showed an increase of interstitial fibrosis without evident myocardial disarray in the heterozygous mice when compared to WT (Fig. 5d). To confirm the asymmetric septal hypertrophy observed by echo in the cMyBP-C^+/– mice, IVS and PW thicknesses were measured in cardiac sections in five different mice. This showed an increase of the IVS/PW ratio when compared to four WT mice (1.36±0.04 vs. 1.02±0.01, P<0.01; Fig. 5e). This validated the echo data obtained at the same age (Table 2).

View larger version (69K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Ventricular morphology and histology in cMyBP-C+/–, cMyBP-C–/– and WT mice. (a) Gross ventricular morphology at 9 months of age. KO mice exhibited ventricular enlargement when compared to WT or HET mice. (b) Light micrographs (40 x) of 6-µm ventricular sections at 9 months of age stained with Hematoxylin & Eosin (HE), modified Gomori's Trichrome (GT) and collagen-specific Sirius red F3B (SR) in WT and KO mice. KO mice exhibited myocardial disarray and increased interstitial fibrosis when compared to WT. Note the presence of calcification in the fibrotic areas in the KO mice (right panels, 20 x). (c) Determination of the ventricular weight/body ratio in HET (n=9) and WT (n=7) at 10–11 months of age. (d) Light micrographs of 6-µm transversal ventricular sections in WT and HET at 11 months of age stained with Hematoxylin & Eosin showing all the myocardium (upper panels, 1 x) or the septum specifically (lower panels 20 x). Note that the IVS thickness increased substantially in HET when compared to WT. This is associated with a significant increase of interstitial fibrosis. (e) Determination of the interventricular septum/posterior wall thickness ratio (IVS/PW) in HET (n=5) and WT (n=4) at 10–11 months of age. Abbreviations used are: KO, cMyBP-C–/– mice; HET, cMyBP-C+/– mice; and WT, wild type mice. **Significantly different vs. WT; P<0.01.

 
The absence of cMyBP-C in the cMyBP-C^–/– mice was confirmed by immunohistochemistry with a specific antibody (Fig. 6a). In contrast, cMyBP-C^+/– mice exhibited normal cMyBP-C striations. Immunohistochemistry performed with antibodies directed against the S1–S2 region of the MyHC (Fig. 6b), the Z1 domain (Fig. 6c) or the M8 domain (Fig. 6d) of the titin showed regular sarcomere striations in the three groups of mice. This indicates that there is no myofibrillar disarray in both cMyBP-C^–/– and cMyBP-C^+/– mice hearts.

View larger version (172K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Cardiac immunohistochemistry in cMyBP-C+/–, cMyBP-C–/– and WT mice at 9 months of age. Ventricular sections were stained with antibodies directed against cMyBP-C (C0–C1 domains; a) MyHC (S1–S2 region; b), titin (Z1 domain; c), and titin (M8 domain; d). Inserts correspond to a magnification of the pictures. Absence of cMyBP-C is clearly shown in KO. No alteration of the MyHC and titin striation patterns was found in both KO and HET when compared to WT. Abbreviations used are: WT, wild type mice; HET, cMyBP-C+/– mice; KO, cMyBP-C–/– mice. Magnification x 100.

 

	4. Discussion

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

 
In the present study, we developed cMyBP-C null mice and we show that homozygous and heterozygous mice exhibit different cardiac disorders. The major finding of this study is the asymmetric septal hypertrophy in the heterozygous cMyBP-C null mice. Additional disruption of the second allele of the mouse cMyBP-C gene results in eccentric LVH and impaired relaxation.

The heterozygous cMyBP-C^+/– mice developed significant asymmetric septal hypertrophy at 10–11 months of age without impairment of LV function as described in human HCM [12,13]. This is different from another targeted mice model that expressed a truncated cMyBP-C at the heterozygous state, and which developed concentric LVH at 30 months of age [23]. The most likely explanation for the phenotype discrepancy between the two models is that the molecular mechanisms are different. Particularly, the present heterozygotes do not express any mutant in the ventricles whereas truncated cMyBP-C is expressed in the other model and may therefore act as a "poison peptide" [23]. In addition, the phenotype of the present heterozygous null mice is associated with a slight but significant decrease of the amount of cMyBP-C protein. We hypothesize that it may impair a molecular pathway through a deficit of interaction with a septum-specific protein, not yet identified, and result in the development of asymmetric septal hypertrophy in the heterozygous cMyBP-C null mice. In humans, a decrease of protein amount was not observed in myocardial tissue of two patients carrying a frameshift cMyBP-C gene mutation [18,19]. However, more recently, we precisely quantified the amount of both mRNA and protein in myocardial tissue of two patients carrying a frameshift cMyBP-C mutation and demonstrated cMyBP-C deficiency either at the mRNA level or at the protein level (unpublished observation). Therefore, the present heterozygous cMyBP-C null mice are a good model to further analyze the pathogenesis and the molecular mechanisms of human FHC related to cMyBP-C mutations.

The homozygous cMyBP-C null mice developed eccentric LVH together with depressed FS at 3–4 months of age. This does not represent a typical form of human dilated cardiomyopathy (DCM) associated with wall thinning [29] but this was found in some cases of human DCM and it is suggested that it plays a protective or beneficial role against further cavity dilation [12]. The phenotype of the present homozygous mice is identical to what was observed in a similar deficient cMyBP-C mouse model [24] and in homozygous cMyBP-C^t/t and -MyHC^403/403 mutant mice [30,31]. Moreover, the present homozygous cMyBP-C^–/– null mice showed a striking impairment of LV relaxation (–dP/dt_min and Exp. Tau) both at rest and after β-adrenergic stimulation after 9 months of age, which is consistent with hypertrophy and/or possible impairment of Ca²⁺ uptake by the sarcoplasmic reticulum. In contrast, no decrease of contractility (dP/dt_max) was observed. This finding in the presence of significantly reduced FS on echocardiography was unexpected, but may relate in part to the sizeable variability observed in the relatively small numbers of animals studied and to increased LV end-systolic meridional wall stress in the cMyBP-C^–/– group when compared to WT mice (51.2 vs. 29.8 dyn/cm², p<0.05), which itself can cause depressed FS [32,33].

All the different heterozygous mice created so far (cMyBP-C^+/–, cMyBP-C^t/+ and -MyHC^403/+) develop HCM, whereas the homozygous mice develop eccentric LVH or DCM [23,24,30,34]. This is similar to what is found in human FHC, in which homozygotes, double heterozygotes and compound heterozygotes, which carry two mutated alleles, develop a more severe cardiac phenotype (large degree of LVH, LV and left atrial dilations) than the corresponding single heterozygote individuals [14,35,36]. In addition, the homozygous -MyHC^403/403 mice develop profound cardiac dilation and die by day 10 after birth [31], whereas the homozygous cMyBP-C^t/t and cMyBP-C^–/– mice can both live at least 1 year [24,30]. Thus, the severity of the disease in homozygous mice bearing sarcomeric protein mutations parallels the severity of HCM in heterozygous mice bearing the same mutation. This suggests that the signal determining the severity of the cardiac disease is the same in homozygous and heterozygous animals.

In conclusion, we show that inactivation of one or two alleles of mouse cMyBP-C gene results in asymmetric LVH and eccentric LVH, respectively. To our knowledge, the heterozygous cMyBP-C null mice represent the first mouse model with the major feature of human HCM, which is usually characterized by asymmetric septal hypertrophy. The elucidation of the cellular events involved in the homozygous and heterozygous cMyBP-C null mice should provide insights into the development of LVH in many cardiomyopathies.

	Acknowledgements

 
We thank Julie Anderson for expert animal assistance. This study was supported by INSERM and AFM grants (Paris) and Leducq Foundation (Paris and UCSD). Dr. R. Knöll was supported by DFG Kn 448/6-1, Dr. D.I. Keller by the ADUMED-donation and Swiss National Foundation grants, and Dr. P. Bausero by the Leducq Foundation.

	Notes

 
¹ Contributed equally to the work.

Time for primary review 27 days

	References

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

 

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