Author + information
- Received August 21, 2008
- Revision received November 20, 2008
- Accepted December 3, 2008
- Published online July 21, 2009.
- Takuro Arimura, DVM, PhD⁎,
- J. Martijn Bos, MD†,
- Akinori Sato, MD⁎,
- Toru Kubo, MD, PhD‡,
- Hiroshi Okamoto, MD, PhD§,
- Hirofumi Nishi, MD, PhD∥,
- Haruhito Harada, MD, PhD¶,
- Yoshinori Koga, MD, PhD¶,
- Mousumi Moulik, MD#,
- Yoshinori L. Doi, MD, PhD‡,
- Jeffrey A. Towbin, MD⁎⁎,
- Michael J. Ackerman, MD, PhD† and
- Akinori Kimura, MD, PhD††,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Akinori Kimura, Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Bunkyo-Ku, Tokyo 113-8510, Japan
Objectives The purpose of this study was to explore a novel disease gene for hypertrophic cardiomyopathy (HCM) and to evaluate functional alterations caused by mutations.
Background Mutations in genes encoding myofilaments or Z-disc proteins of the cardiac sarcomere cause HCM, but the disease-causing mutations can be found in one-half of the patients, indicating that novel HCM-susceptibility genes await discovery. We studied a candidate gene, ankyrin repeat domain 1 (ANKRD1), encoding for the cardiac ankyrin repeat protein (CARP) that is a Z-disc component interacting with N2A domain of titin/connectin and N-terminal domain of myopalladin.
Methods We analyzed 384 HCM patients for mutations in ANKRD1and in the N2A domain of titin/connectin gene (TTN). Interaction of CARP with titin/connectin or myopalladin was investigated using coimmunoprecipitation assay to demonstrate the functional alteration caused by ANKRD1or TTNmutations. Functional abnormalities caused by the ANKRD1mutations were also examined at the cellular level in neonatal rat cardiomyocytes.
Results Three ANKRD1missense mutations, Pro52Ala, Thr123Met, and Ile280Val, were found in 3 patients. All mutations increased binding of CARP to both titin/connectin and myopalladin. In addition, TTNmutations, Arg8500His, and Arg8604Gln in the N2A domain were found in 2 patients, and these mutations increased binding of titin/connectin to CARP. Myc-tagged CARP showed that the mutations resulted in abnormal localization of CARP in cardiomyocytes.
Conclusions CARP abnormalities may be involved in the pathogenesis of HCM.
Cardiomyopathy is a primary heart muscle disorder caused by functional abnormalities of cardiomyocytes. There are several clinical subtypes of cardiomyopathy, and the most prevalent subtype is hypertrophic cardiomyopathy (HCM) (1,2). HCM is characterized by hypertrophy and diastolic dysfunction of cardiac ventricles accompanied by cardiomyocyte hypertrophy, fibrosis, and myofibrillar disarray (1). Although the etiologies of HCM have not been fully elucidated, 50% to 70% of the patients with HCM have apparent family histories consistent with autosomal dominant genetic trait (3), and recent genetic analyses have revealed that a significant percentage of HCM is caused by mutations in the genes encoding for myofilaments and Z-disc proteins of the cardiac sarcomere, with the majority of mutations identified in MYH7-encoded beta myosin heavy chain and MYBPC3-encoded myosin-binding protein C (3).
Ankyrin repeat domain 1 (ANKRD1)-encoded cardiac adriamycin responsive protein (4), or cardiac ankyrin repeat protein (CARP) (5), is a transcription cofactor and an early differentiation marker of cardiac myogenesis, expressed in the heart during embryonic and fetal development. CARP expression is up-regulated in the adult heart at end-stage heart failure (6). In addition, increased CARP expression was found in hypertrophied hearts from experimental murine models (7,8). These observations suggest a pivotal role for CARP in cardiac muscle function in both physiological and pathological conditions. Although CARP is known to be involved in the regulation of gene expression in the heart, Bang et al. (9) demonstrated that CARP located to both the sarcoplasm and nucleus, suggesting a shuttling of CARP in cellular components. Within the I-band region of sarcomere, CARP bound to both the N2A domain of titin/connectin encoded by titin/connectin gene (TTN) and the N-terminal domain of myopalladin encoded by MYPN. Hence, titin/connectin and myopalladin function in part as anchoring proteins of “sarcomeric CARP” (9,10).
Titin/connectin is the most giant protein expressed in the striated muscles, which is involved in sarcomere assembly, force transmission at the Z-disc, and maintenance of resting tension in the I-band region (11,12). In cardiac muscle, there are 2 titin isoforms, N2B and N2BA. The N2B isoform contains a cardiac specific N2B domain, and the N2BA isoform contains both N2B and N2A domains. Both N2A and N2B domains, within the extensible I-band region, function as a molecular spring that develops passive tension; the expression of N2B isoform results in a higher passive stiffness than that of N2AB isoform. We previously reported an HCM-associated mutation localizing to the N2B domain (13), and Gerull et al. (14) reported other TTNmutations in the Z/I transition domain. These observations suggest that the I-band region of titin/connectin contains elastic components extending with stretch to generate passive force, which plays an important role in the maintenance of cardiac function.
Another protein that anchors CARP at the Z/I band is myopalladin, a cytoskeletal protein containing 3 proline-rich motifs and 5 Ig domains. The proline-rich motifs in the central part is required for binding to nebulin/nebulette, and the Ig domains at the N-terminus and C-terminus are involved in the binding to CARP and sarcomeric a-actinin, respectively (9). It has been suggested that myopalladin played key roles in sarcomere/Z-disc assembly, myofibrillogenesis, recruitment of the other Z/I-band elements, and signaling in the Z/I-band (9).
In this study, we analyzed unrelated patients with heretofore genotype-negative HCM for mutations in ANKRD1and found 3 mutations that showed abnormal binding to myopalladin and titin/connectin. In addition, we searched for mutations in the reciprocal CARP-binding N2A domain of titin/connectin and identified 2 HCM-associated mutations in TTNcausing abnormal binding to CARP. We report here that abnormal CARP assembly in the cardiac muscles may be involved in the pathogenesis of HCM.
A total of 384 unrelated patients with HCM were included in this study. The patients were diagnosed based on medical history, physical examination, 12-lead electrocardiogram, echocardiography, and other special tests if necessary. The diagnostic criteria for HCM included left ventricular wall thickness >13 mm on echocardiography, in the absence of coronary artery disease, myocarditis, and hypertension. The patients had been analyzed previously for mutations in previously published myofilament- and Z-disc associated genes, and no mutation was found in any of the known HCM-susceptibility genes (15–18). Ethnically-matched healthy persons (400 from Japan, and 300 from the U.S.) were used as controls. Blood samples were obtained from the subjects after given informed consent. The protocol for research was approved by the Ethics Reviewing Committee of Medical Research Institute, Tokyo Medical and Dental University (Japan) and by the Mayo Foundation Institutional Review Board (U.S.).
Using intronic primers, each translated ANKRD1exon was amplified by polymerase chain reaction (PCR) from genomic DNA samples. TTNexons 99 to 104 corresponding to the N2A domain including binding domains to CARP and p94/calpain were amplified by PCR in exon-by-exon manner. Sequence of primers and PCR conditions used in this study are available upon request. PCR products were analyzed by direct sequencing or by denaturing high-performance liquid chromatography followed by sequencing analysis. Sequencing was performed using Big Dye Terminator chemistry (version 3.1, Applied Biosystems, Foster City, California) and ABI3100 DNA Analyzer (Applied Biosystems).
Coimmunoprecipitation (co-IP) assay
We obtained complementary deoxyribonucleic acid (cDNA) fragments of human ANKRD1and TTNby reverse-transcriptase PCR from adult heart messenger ribonucleic acid. A wild-type (WT) full-length CARP cDNA fragment spanned from bp249 to bp1208 of GenBank Accession No. NM_014391 (corresponding to aa1-aa319). Three equivalent mutant cDNA fragments containing C to G (Pro52Ala mutation), C to T (Thr123Met mutation), or A to G (Ile280Val mutation) substitutions were obtained by the primer-directed mutagenesis method. A WT TTNcDNA fragment encoding N2A domains (from bp25535 to bp26465 of NM_133378 corresponding to aa8437-aa8747) was obtained, and 3 TTNmutants carrying T to C (non–disease-associated Ile8474Thr polymorphism), G to A (HCM-associated Arg8500His mutation), or G to A (HCM-associated Arg8604Gln mutation) substitutions were created by the primer-mediated mutagenesis method. The cDNA fragments of ANKRD1were cloned into myc-tagged pCMV-Tag3 (Stratagene, La Jolla, California), and TTNand MYPNcDNA fragments were cloned into pEGFP-C1 (Clontech, Mountain View, California). These constructs were sequenced to ensure that no errors were introduced.
Cellular transfection and protein extractions were performed as described previously (19), and co-IP assays were performed using the Catch and Release version 2.0 Reversible Immunoprecipitation System according to the manufacturer's instructions (Millipore, Billerica, Massachusetts). Immunoprecipitates were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to a nitrocellulose membrane. After a pre-incubation with 3% skim milk in phosphate-buffered saline, the membrane was incubated with primary rabbit anti-myc polyclonal antibody (Ab) or mouse anti-GFP monoclonal antibody Ab (1:100, Santa Cruz Biotechnology, Santa Cruz, California), and with secondary goat anti-rabbit (for polyclonal Ab) or rabbit anti-mouse (for monoclonal Ab) IgG HRP-conjugated Ab (1:2,000, Dako A/S, Grostrup, Denmark). Signals were visualized by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and Luminescent Image Analyzer LAS-3000 mini (Fujifilm, Tokyo, Japan), and their densities were quantified by using Multi Gauge version 3.0 (Fujifilm, Tokyo, Japan). Numerical data were expressed as mean ± SEM. Statistical differences were analyzed using 1-way analysis of variance and the Student ttest for paired values. Means were compared by independent sample ttests without correction for multiple comparisons. A p value <0.05 was considered to be statistically significant.
Indirect immunofluorescence microscopy
All care and treatment of animals were in accordance with “Guidelines for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH Publication 85-23, revised 1985) and subjected to prior approval by the local animal protection authority. Neonatal rat cardiomyocytes were prepared as described previously (19). Eighteen hours and 48 h after the transfection, cardiomyocytes were washed with phosphate-buffered saline, fixed for 15 min in 100% ethanol at −20°C. Transfected cells were incubated in blocking solution, and stained by primary rabbit anti-myc polyclonal Ab (1:100, Santa Cruz Biotechnology) and mouse anti–α-actinin monoclonal Ab (1:800, Sigma-Aldrich, St. Louis, Missouri), followed by secondary sheep anti-rabbit IgG FITC-conjugated Ab (1:500, Chemicon, Boronia, Victoria, Australia) and Alexa fluor 568 goat anti-mouse IgG (1:500, Molecular Probes, Fugeue, Oregon). All cells were mounted on cover-glass using Mowiol 4-88 Reagent (Calbiochem, Darmstadt, Germany) with 4′6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich), and images from at least 200 transfected cells were analyzed with an LSM510 laser-scanning microscope (Carl Zeiss Microscopy, Jena, Germany).
Identification of ANKRD1(CARP) and TTNmutations in HCM
Eleven distinct sequence variations in ANKRD1were identified among the 384 patients with HCM (Fig. 1A).Four intronic variants, 2 nonsynonymous substitutions, and 1 synonymous variation were polymorphisms, because they were also found in the controls. A nonsense mutation (c.423C>T in exon 2 yielding Gln59ter) was found in 2 patients with familial HCM and was absent in the controls, but was not cosegregated with the disease in both families, suggesting that they were not associated with HCM. In contrast, 3 missense mutations, Pro52Ala (c.402C>G in exon 2), Thr123Met (c.616C>T in exon 4), and Ile280Val (c.1086A>G in exon 8), identified in 3 unrelated HCM patients, were not found in the controls.
Sequence variations in TTNat the N2A domain containing binding region to CARP and p94/calpain were searched for in the patients, and 8 variations were identified (Fig. 1B). An intronic variation and 3 synonymous variations were polymorphisms observed in the controls. Two nonsynonymous variations, Ile8474Thr (c.25645T>C in exon 99) and Asp8672Val (c.26239A>T in exon 102), were not associated with HCM, because Ile8474Thr was found in the controls and Asp8672Val did not cosegregate with the disease in a multiplex family. On the other hand, 2 missense mutations, Arg8500His (c.25723G>A in exon 99) and Arg8604Gln (c.26035G>A in exon 100), identified in familial HCM patients, were not found in the controls.
Clinical findings of the patients carrying the ANKRD1or TTNmutations are summarized in Table 1.All patients manifested with HCM except CM1288 II-2, who had mild cardiac hypertrophy. Her father had died suddenly of unknown etiology at the age of 30 years. Two unaffected brothers of the patient did not harbor the mutation (Fig. 1C). The proband patient with the TTNArg8606Gln mutation (CM1480) (Table 1) showed asymmetric septum hypertrophy. A family study revealed that his father had unexplained sudden cardiac death. His son (CM1481) (Table 1) was affected and carried the same mutation (Fig. 1D).
Altered interaction between titin/connectin and CARP caused by the TTN or CARP mutations
To investigate the functional alterations caused by the CARP mutations in the binding to titin/connectin N2A domain, WT-, Pro52Ala-, Thr123Met-, or Ile280Val-CARP construct was cotransfected with the WT TTN-N2A construct into COS-7 cells. Western blot analyses of immunoprecipitates from the transfected cells demonstrated that HCM-associated CARP mutations significantly increased binding to TTN-N2A (2.22 ± 0.76 arbitrary units [AU], p < 0.05; 1.98 ± 0.52 AU, p < 0.01; 2.16 ± 0.64 AU, p < 0.05, respectively) (Figs. 2Aand 2B). Reciprocally, the effect of titin/connectin mutations in binding to CARP was assessed. The TTN-N2A constructs, WT-, HCM-associated mutants (Arg8500His- and Arg8604Gln-TTN), or non–disease-related variant (Ile8474Thr) TTN-N2A were cotransfected with WT CARP. Western blot analyses showed that Arg8500His and Arg8604Gln significantly increased the binding to CARP (2.78 ± 0.40 AU or 3.16 ± 0.40 AU, respectively, p < 0.001 in each case) (Figs. 2A and 2B), whereas the non–disease-related variant (Ile8474Thr) did not alter the binding (1.18 ± 0.11 AU), despite equal expression of proteins.
Altered interaction between myopalladin and CARP caused by the CARP mutations
Because CARP bound also to myopalladin, we investigated the effects of CARP mutations in binding to myopalladin. The WT or mutant CARP construct was cotransfected with a MYPN construct. Western blot analysis revealed that binding of mutant CARPs, Pro52Ala, Thr123Met, or Ile280Val to myopalladin was significantly increased (3.60 ± 0.67 AU, p < 0.001; 1.87 ± 0.47 AU, p < 0.01; or 2.48 ± 0.45 AU, p < 0.001, respectively) (Figs. 2C and 2D).
Altered localization of CARP caused by the mutations
To further investigate the functional consequence of the CARP mutations, we examined cellular distribution of the mutant CARP proteins expressed in neonatal rat primary cardiomyocytes. Cells were transfected with myc-tagged WT or mutant CARP constructs, coimmunostained for myc (a marker for CARP) and α-actinin (a marker for Z-disc). The WT and mutant myc-CARP proteins were expressed at a similar level in the transfected cells as assessed by Western-blot analyses, suggesting that the mutations did not affect the expression level and stability of CARP proteins (data not shown). Control cells expressing myc-tag alone showed negative staining for myc-tag with striated staining pattern of sarcomeric α-actinin at the Z-disc (data not shown). In premature cardiomyocytes containing Z-bodies (Z-disc precursors), myc-tagged WT CARP was mainly targeted to nucleus and colocalization of CARP with α-actinin, which formed patchy dense bodies in the cytoplasm, was observed (Figs. 3Ato 3C). No apparent changes in localization of mutant CARP proteins were observed in the nascent and immature cardiomyocytes (Figs. 3D to 3L).
In the mature cardiomyocytes where Z-discs were well organized, myc-tagged WT CARP was assembled in the striated pattern at the Z-I bands and colocalized with α-actinin (Figs. 4Ato 4C). It was found that most (≈90%) of mature cardiomyocytes did not contain nuclear CARP (Figs. 4A to 4C). On the other hand, higher intensity of CARP-related fluorescence at the Z-I bands and diffused localization in cytoplasm was observed in most (≈80%) of the mature cardiomyocytes expressing myc-tagged mutant CARPs, albeit that the Z-disc assembly was not impaired (Figs. 4D to 4L). Quite interestingly, myc-tagged mutant CARP proteins displayed localization within the nuclear and/or at nuclear membrane in ≈60% of mature cardiomyocytes (Figs. 4D to 4L).
CARP encoded by ANKRD1is a nuclear transcription cofactor expressing in the embryonic hearts. Its expression progressively decreases in adult hearts (4,5) and reappears in the hypertrophied or failing adult heart (6,22), suggesting that CARP may be involved in the regulation of muscle gene expression. CARP also localizes in cardiac sarcomere although the roles of “sarcomeric CARP” are not fully elucidated. Several reports have demonstrated that CARP binds titin/connectin (10), myopalladin (9), and desmin (21) at the Z/I-region of sarcomere. In this study, we found that the HCM-associated ANKRD1mutations increased the binding of CARP to titin/connectin and myopalladin, and HCM-associated TTNmutations in its reciprocal CARP N2A-binding domain increased the binding of titin/connectin to CARP. These observations in association with HCM suggested that the assembly or binding of sarcomeric CARP with titin/connectin and/or myopalladin would be required for the maintenance of cardiac function.
In the nascent myofibrils, myc-tagged CARP proteins were detected within the nucleus irrespective of mutations. Because CARP is an early differentiation marker during heart development, recruitment of CARP into nuclei may be important in the embryonic gene expression. Interestingly, abnormal intranuclear accumulation of myc-tagged mutant CARP proteins was observed in mature myofibrils. It is well known that the embryonic and fetal gene program of cardiac cytoskeletal proteins is initiated during the cardiac remodeling (22,23). Hence, one could hypothesize that nuclear CARP may cause embryonic/fetal gene expression in mature myofibrils, and this abnormal gene expression is a possible mechanism leading to the pathogenesis of HCM. It was reported that CARP negatively regulated expression of cardiac genes including MYL2, TNNC1, and ANP(4,5). Conversely, another report suggested that different expression level of CARP did not correlate with the altered expression of cardiac genes such as MYL2, MYH7, ACTC, CACTN, TPM1, ACTN2, and DES(24). Thus, the role of CARP as a regulator of cardiac gene expression remains to be resolved. During the preparation of this paper, Cinquetti et al. (25) reported other CARP mutations, rearrangements, or Thr116Met, in association with the cyanotic congenital heart anomaly known as total anomalous pulmonary venous return. These mutations were demonstrated to be associated with increased expression or stability of CARP. It is not clear whether the mutations associated with HCM altered expression or stability of CARP, although our data suggested that HCM-associated CARP mutations did not alter the stability. The molecular mechanisms underlying the CARP-related pathogenesis should be different between total anomalous pulmonary venous return and HCM.
We identified 3 missense CARP mutations in <1% of unrelated patients with HCM, which not only increased the binding of sarcomeric CARP to I-band components but also resulted in the mislocalization of CARP to the nucleus. Although the molecular mechanisms of HCM due to the CARP mutations remain to be elucidated, our findings imply that HCM may be associated with the abnormal recruitment of CARP in cardiomyocytes, leading to pathological hypertrophy.
The authors thank Drs. Hironori Toshima, Chuichi Kawai, Keishiro Kawamura, Makoto Nagano, Tsuneaki Sugimoto, Satoshi Ogawa, Akira Matsumori, Shigetake Sasayama, Ryozo Nagai, and Yoshio Yazaki for their contributions in clinical evaluation and blood sampling from patients with cardiomyopathy, and Ms. Mieko Yanokura, Maki Emura, and Ayaka Nishimura for their technical assistance.
This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan; by a research grant from the Ministry of Health, Labour and Welfare, Japan, and the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation; by research grants from the Japan Heart Foundation and the Association Française contre les Myopathies (Grant No. 11737; Drs. Arimura and Kimura); and by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program (Dr. Ackerman). Dr. Moulik is supported by a Career Development grant from the National Institutes of Health (K08HL091176). Drs. Arimura and Bos contributed equally to this work.
- Abbreviations and Acronyms
- ankyrin repeat domain 1
- cardiac ankyrin repeat protein
- complementary deoxyribonucleic acid
- dilated cardiomyopathy
- hypertrophic cardiomyopathy
- polymerase chain reaction
- wild type
- Received August 21, 2008.
- Revision received November 20, 2008.
- Accepted December 3, 2008.
- American College of Cardiology Foundation
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