Mutations in the ANKRD1 gene encoding CARP are responsible for human dilated cardiomyopathy

INSERM, U621, Paris F-75013, France.
European Heart Journal (Impact Factor: 15.2). 07/2009; 30(17):2128-36. DOI: 10.1093/eurheartj/ehp225
Source: PubMed


Dilated cardiomyopathy (DCM) is familial in approximately 30% of cases, and mutations have been identified in several genes. However, in a majority of familial cases, the responsible genes are still to be discovered. The ANKRD1 gene is over-expressed in heart failure in human and animal models. The encoded protein CARP interacts with partners such as myopalladin or titin, previously shown to be involved in DCM. We hypothesized that mutations in ANKRD1 could be responsible for DCM.
We sequenced the coding region of ANKRD1 from 231 independent DCM cases. We identified five missense mutations (three sporadic and two familial) absent from 400 controls and affecting highly conserved residues. Expression of the mutant CARP proteins after transfection in rat neonate cardiomyocytes indicated that most of them led to both significantly less repressor activity measured in a reporter gene assay and greater phenylephrin-induced hypertrophy, suggesting altered function of CARP mutant proteins.
On the basis of genetic and functional analysis of CARP mutations, we have identified ANKRD1 as a new gene associated with DCM, accounting for approximately 2% of cases.

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    • "Additionally, for the first time, we provide comparative analysis of MARP family proteins Ankrd2 and Ankrd1 in human cardiac tissue and NRCM. The role of Ankrd1 in heart and cardiac disorders is already well established (Kimura 2010; Miller et al. 2003; Zolk et al. 2002) and mutations in Ankrd1 are associated with HCM and DCM (Arimura et al. 2009; Duboscq-Bidot et al. 2009; Author's personal copy Fig. 6 Intracellular localization of endogenous Ankrd2 protein in nuclei and cytoplasm of NRCM. a Lower magnification (objective Fluotar 20×/0.50) and b higher magnification (objective Apo 63×/1.40 "
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    ABSTRACT: Muscle-specific mechanosensors Ankrd2/Arpp (ankyrin repeat protein 2) and Ankrd1/CARP (cardiac ankyrin repeat protein) have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. In skeletal muscle myofibrils, Ankrd2 has a structural role as a component of a titin associated stretch-sensing complex, while in the nucleus it exerts regulatory function as transcriptional co-factor. It is also involved in myogenic differentiation and coordination of myoblast proliferation. Although expressed in the heart, the role of Ankrd2 in the cardiac muscle is completely unknown. Recently, we have shown that hypertrophic and dilated cardiomyopathy pathways are altered upon Ankrd2 silencing suggesting the importance of this protein in cardiac tissue. Here we provide the underlying basis for the functional investigation of Ankrd2 in the heart. We confirmed reduced Ankrd2 expression levels in human heart in comparison with Ankrd1 using RNAseq and Western blot. For the first time we demonstrated that, apart from the sarcomere and nucleus, both proteins are localized to the intercalated disks of human cardiomyocytes. We further tested the expression and localization of endogenous Ankrd2 in rat neonatal cardiomyocytes, a well-established model for studying cardiac-specific proteins. Ankrd2 was found to be expressed in both the cytoplasm and nucleus, independently from maturation status of cardiomyocytes. In contrast to Ankrd1, it is not responsive to the cardiotoxic drug Doxorubicin, suggesting that different mechanisms govern their expression in cardiac cells.
    Histochemie 01/2015; 143(6). DOI:10.1007/s00418-015-1307-5 · 3.05 Impact Factor
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    • "Some studies indicated that CARP is a cardiac-restricted nuclear protein with a pivotal role in fetal development [23] or in failing and non-failing hearts of human [5], [24], but CARP was also detected in the cytoplasm rather than the nucleus of cardiomyocytes from HF patients [6], [25] or even in neonatal rat cardiomyocytes [9]. Generally, it is believed that CARP is both cytosolic and nuclear localization [8], [10] which functions as stretching-sense component in the cytoplasm and a co-factor of cardiac gene expression in the nuclei, and their subcellular localization depends on physiological and pathological stresses. The present study showed that CARP was mainly located in the nucleus of neonatal cardiomyocytes under physiological conditions and underwent translocation to the cytoplasm in response to stimulation by Ang II. "
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    ABSTRACT: The gene ankyrin repeat domain 1 (Ankrd1) is an enigmatic gene and may exert pleiotropic function dependent on its expression level, subcellular localization and even types of pathological stress, but it remains unclear how these factors influence the fate of cardiomyocytes. Here we attempted to investigate the role of CARP on cardiomyocyte hypertrophy. In neonatal rat ventricular cardiomyocytes (NRVCs), angiotensin II (Ang II) increased the expression of both calpain 1 and CARP, and also induced cytosolic translocation of CARP, which was abrogated by a calpain inhibitor. In the presence of Ang-II in NRVCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced myocyte hypertrophy, the upregulation of atrial natriuretic peptide and β-myosin heavy chain genes and calcineurin proteins as well as nuclear translocation of nuclear factor of activated T cells. Cyclosporin A attenuated Ad-Ankrd1-enhanced cardiomyocyte hypertrophy. Intra-myocardial injection of Ad-Ankrd1 in mice with transverse aortic constriction (TAC) markedly increased the cytosolic CARP level, the heart weight/body weight ratio, while short hairpin RNA targeting Ankrd1 inhibited TAC-induced hypertrophy. The expression of calcineurin was also significantly increased in Ad-Ankrd1-infected TAC mice. Olmesartan (an Ang II receptor antagonist) prevented the upregulation of CARP in both Ang II-stimulated NRVCs and hearts with pressure overload. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac remodeling through the enhancement of cytosolic translocation of CARP and upregulation of calcineurin.
    PLoS ONE 08/2014; 9(8):e104040. DOI:10.1371/journal.pone.0104040 · 3.23 Impact Factor
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    • "However, during postnatal hypertrophy induced by mechanical pressure overload or α1-and β-adrenergic agonist stimulation in animal models, CARP is markedly induced, suggesting that it is a member of the hypertrophic embryonic gene program [9], [10], [11], [12], [13], [14], [15]. Likewise, CARP is induced in patients with hypertrophic, dilated, ischemic, and arrhythmogenic right ventricular cardiomyopathy [16], [17], [18] and missense mutations in the Ankrd1 gene were recently shown to be causative for human dilated and hypertrophic cardiomyopathy [19], [20], [21]. Further evidence supporting an important role of CARP in regulating the cardiac hypertrophic response comes from a recent study of cardiac-specific CARP-overexpressing transgenic mice, which were found to show an attenuated cardiac hypertrophic response following mechanical pressure overload or isoproterenol infusion [22]. "
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    ABSTRACT: Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload.
    PLoS ONE 04/2014; 9(4):e93638. DOI:10.1371/journal.pone.0093638 · 3.23 Impact Factor
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