ANKRD1 Research Articles

Interleukin 11 therapy causes acute left ventricular dysfunction.

Interleukin 11 (IL11) was initially thought important for platelet production, which led to recombinant IL11 being developed as a drug to treat thrombocytopenia. IL11 was later found to be redundant for haematopoiesis and its use in patients is associated with unexplained and severe cardiac side effects. Here we aim to identify, for the first time, direct cardiomyocyte toxicities associated with IL11, which was previously believed cardioprotective. We injected recombinant mouse lL11 (rmIL11) into mice and studied its molecular effects in the heart using immunoblotting, qRT-PCR, bulk RNA-seq, single nuclei RNA-seq (snRNA-seq) and ATAC-seq. The physiological impact of IL11 was assessed by echocardiography in vivo and using cardiomyocyte contractility assays in vitro. To determine the activity of IL11 specifically in cardiomyocytes we made two cardiomyocyte-specific Il11ra1 knockout (CMKO) mouse models using either AAV9-mediated and Tnnt2-restricted (vCMKO) or Myh6 (m6CMKO) Cre expression and an Il11ra1 floxed mouse strain. In pharmacologic studies, we studied the effects of JAK/STAT inhibition on rmIL11-induced cardiac toxicities. Injection of rmIL11 caused acute and dose-dependent impairment of left ventricular ejection fraction (saline: 62.4% ± 1.9; rmIL11: 32.6% ± 2.9, p<0.001, n=5). Following rmIL11 injection, myocardial STAT3 and JNK phosphorylation were increased and bulk RNA-seq revealed upregulation of pro-inflammatory pathways (TNFα, NFκB and JAK/STAT) and perturbed calcium handling. snRNA-seq showed rmIL11-induced expression of stress factors (Ankrd1, Ankrd23, Xirp2), activator protein-1 (AP-1) transcription factor genes and Nppb in the cardiomyocyte compartment. Following rmIL11 injection, ATAC-seq identified the Ankrd1 and Nppb genes and loci enriched for stress-responsive, AP-1 transcription factor binding sites. Cardiomyocyte-specific effects were examined in vCMKO and m6CMKO mice, which were both protected from rmIL11-induced left ventricular impairment and molecular pathobiologies. In mechanistic studies, inhibition of JAK/STAT signalling with either ruxolitinib or tofacitinib prevented rmIL11-induced cardiac dysfunction. Injection of IL11 directly activates IL11RA/JAK/STAT3 in cardiomyocytes to cause acute heart failure. Our data overturn the earlier assumption that IL11 is cardioprotective and explain the serious cardiac side effects associated with IL11 therapy.

Integrating miRNA and full-length transcriptome profiling to elucidate the mechanism of muscle growth in Muscovy ducks reveals key roles for miR-301a-3p/ANKRD1

BackgroundThe popularity of Muscovy ducks is attributed not only to their conformation traits but also to their slightly higher content of breast and leg meat, as well as their stronger-tasting meat compared to that of typical domestic ducks. However, there is a lack of comprehensive systematic research on the development of breast muscle in Muscovy ducks. In addition, since the number of skeletal muscle myofibers is established during the embryonic period, this study conducted a full-length transcriptome sequencing and microRNA sequencing of the breast muscle. Muscovy ducks at four developmental stages, namely Embryonic Day 21 (E21), Embryonic Day 27 (E27), Hatching Day (D0), and Post-hatching Day 7 (D7), were used to isolate total RNA for analysis.ResultsA total of 68,161 genes and 472 mature microRNAs were identified. In order to uncover deeper insights into the regulation of mRNA by miRNAs, we conducted an integration of the differentially expressed miRNAs (known as DEMs) with the differentially expressed genes (referred to as DEGs) across various developmental stages. This integration allowed us to make predictions regarding the interactions between miRNAs and mRNA. Through this analysis, we identified a total of 274 DEGs that may serve as potential targets for the 68 DEMs. In the predicted miRNA‒mRNA interaction networks, let-7b, miR-133a-3p, miR-301a-3p, and miR-338-3p were the hub miRNAs. In addition, multiple DEMs also showed predicted target relationships with the DEGs associated with skeletal system development. These identified DEGs and DEMs as well as their predicted interaction networks involved in the regulation of energy homeostasis and muscle development were most likely to play critical roles in facilitating the embryo-to-hatchling transition. A candidate miRNA, miR-301a-3p, exhibited increased expression during the differentiation of satellite cells and was downregulated in the breast muscle tissues of Muscovy ducks at E21 compared to E27. A dual-luciferase reporter assay suggested that the ANKRD1 gene, which encodes a transcription factor, is a direct target of miR-301a-3p.ConclusionsmiR-301a-3p suppressed the posttranscriptional activity of ANKRD1, which is an activator of satellite cell proliferation, as determined with gain- and loss-of-function experiments. miR-301a-3p functions as an inducer of myogenesis by targeting the ANKRD1 gene in Muscovy ducks. These results provide novel insights into the early developmental process of black Muscovy breast muscles and will improve understanding of the underlying molecular mechanisms.

Unraveling the pathogenesis of novel ANKRD1 missense mutation causing familial dilated cardiomyopathy using human-induced pluripotent stem cells derived cardiomyocytes

Abstract Introduction Familial dilated cardiomyopathy(f-DCM) can be caused by mutations in 30 different genes encoding sarcomeric, cytoskeletal, mechanotransducers and transcription factors. One of the genes ANKRD1 encode Cardiac ankyrin repeat protein (CARP) which is involved in the mechanosensory unit located on the I-band of the sarcomere. Here we demonstrate a novel mutation in ANKRD1 gene causing f-DCM, and the usage of human-induced pluripotent(hiPSC) technology unraveling the pathogenesis of the disease. Purpose of the study: 1) Establishing patient specific hiPSC-derived cardiomyocytes from a family harboring a mutation in ANKRD1 gene causing f-DCM; 2) Phenotypical assessment of the morphological and ultrastructural properties of the diseased cells upon CARP activation; 3) Functional assessment of the 3 component of excitation-contraction coupling at the single-cell and tissue level. Methods and Results Patient-specific hiPSC were generated from a family harboring missense mutation in the ANKRD1 gene, with a substitution of the amino acid arginine to cysteine at position 182 of the Cardiac ankyrin repeat protein (CARP). ANKRD1-mut hiPSC were differentiated into cardiomyocytes and later matured using hormonal treatment. Morphological assessment using immunofluorescence(IF) techniques revealed that ANKRD1-mut hiPSC-CMs demonstrate an attenuated response to hypertrophy (upon Angiotensin-2 and Phenylephrine treatment). In addition ANKRD1-mut hiPSC-CMs displayed a higher cellular death rate upon increasing substrate stiffness and starvation. Electrophysiological assessment of the ANKRD1-mut cardiac tissues exhibit no alternations in action potential durations and conduction velocities while assessment of calcium transients demonstrated slower kinetics. In addition forces were quantified at the single-cell and tissue level , and demonstrated reduced active tension values and abnormal post-extrasystolic potentiation, while only single-cell experiments have demonstrated decreased passive tensions. These phenotypical alternations might be attributed to decreased nuclear translocation of CARP upon phenylephrine treatment as detected in IF experiments. Conclusion Novel mutation in ANKRD1 were found and validated to be causing f-DCM, by revealing phenotypical alternation using hiPSC-CM technology. Increased cellular death rate and impaired nuclear translocation of CARP might be the underlying causes of the morphological and contractile abnormalities detected in-vitro, leading to systolic dysfunction in the affected family.

Effects of N-Acetyl cysteine on gene expression of Protein kinase C-α and Ankyrin repeat domain-containing protein 1 in the cardiac tissue of rats exposed to lead

Objective: This study aimed to evaluate the effects of N-acetylcysteine (NAC) on the oxidative damage, inflammation and expression of PKC-α and ANKRD1 genes in the cardiac tissue of rats exposed to lead (Pb). Methods: Rats were randomly divided into five groups including G1 (control), G2 (single dose of Pb), G3 (continuous dose of Pb), G4 (single dose of Pb + NAC), and G5 (continuous dose of Pb + NAC). Levels of malondialdehyde (MDA), total antioxidant capacity (TAC), IL-10 and TNF-α were measured. Expression of PKC-α and ANKRD1 genes was considered using RT-PCR. Results: Continuous exposure to Pb caused a significant decrease in serum levels of TAC and IL-10, but increased MDA and TNF-α contents (p&lt;0.001). Continuous dose of Pb also dramatically increased the expression of PKC-α and ANKRD1 genes in the cardiac tissue by 4.27-fold and 3.07-fold, respectively (p&lt;0.001). NAC treatments not only improved morphological changes, oxidative stress and inflammatory biomarkers, but also compensated antioxidant capacity and expression of PKC-α and ANKRD1 genes in cardiac tissue. Conclusion: Pb exposure is strongly associated with cardiotoxicity through inducing oxidative stress, inflammation, and antioxidant depletion. NAC ameliorates Pb-induced cardiotoxicity by elevating antioxidants capacity, mitigating oxidative stress and down-regulating of PKC-α and ANKRD1 genes.

Identification of candidate genetic variants and altered protein expression in neural stem and mature neural cells support altered microtubule function to be an essential component in bipolar disorder

Identification of causative genetic variants leading to the development of bipolar disorder (BD) could result in genetic tests that would facilitate diagnosis. A better understanding of affected genes and pathways is also necessary for targeting of genes that may improve treatment strategies. To date several susceptibility genes have been reported from genome-wide association studies (GWAS), but little is known about specific variants that affect disease development. Here, we performed quantitative proteomics and whole-genome sequencing (WGS). Quantitative proteomics revealed NLRP2 as the most significantly up-regulated protein in neural stem cells and mature neural cells obtained from BD-patient cell samples. These results are in concordance with our previously published transcriptome analysis. Furthermore, the levels of FEZ2 and CADM2 proteins were also significantly differentially expressed in BD compared to control derived cells. The levels of FEZ2 were significantly downregulated in neural stem cells (NSC) while CADM2 was significantly up-regulated in mature neuronal cell culture. Promising novel candidate mutations were identified in the ANK3, NEK3, NEK7, TUBB, ANKRD1, and BRD2 genes. A literature search of candidate variants and deregulated proteins revealed that there are several connections to microtubule function for the molecules putatively involved. Microtubule function in neurons is critical for axon structure and axonal transport. A functional dynamic microtubule is also needed for an advocate response to cellular and environmental stress. If microtubule dynamics is compromised by mutations, it could be followed by deregulated expression forming a possible explanation for the inherited vulnerability to stressful life events that have been proposed to trigger mood episodes in BD patients.

P752Pericyte/mural cells of ischemic human hearts show impairment of mechanotransduction, attenuating YAP signaling

Abstract Background The Hippo pathway, that is upregulated in human heart failure, can restrain cardiac regeneration and impair arteriogenesis, preventing the nuclear translocation of the transcriptional coactivator YAP. Actin dynamics and mechanotransduction pathways are critical regulators of YAP subcellular localization. Aim To investigate if cardiac pathology alters mechanotransduction pathways, modulating the nucleocytoplasmic shuttling of YAP. Methods and results PDGFRb+ NG2+Tbx18+cardiac pericytes/mural cells (PM) cultured from normal atria (PMnorm, n=6) and from ischemic failing hearts (PMfail, n=6) were compared. From a transcriptomic standpoint, biological processes and molecular functions associated with cell adhesion and actin cytoskeleton were differentially expressed by PMfail vs PMnorm. The morphological analysis of the two cell types showed that PMfail were significantly larger (2,455±160μm2 vs 3518±334μm2, p=0.04) and less polarized (aspect ratio: 3±0.9 vs 2±0.1, p=0.002) with respect to PMnorm. Consistently, PMfail showed a significantly smaller number of Paxillin+ focal adhesions/cell area and impaired migratory properties. Furthermore, PMfail showed contact inhibition of cell proliferation at significantly lower cell density with respect to PMnorm, paralleled by a significantly lower fraction of cells expressing YAP in their nuclei at a density of 500 cells/mm2 (28.1±12.7% vs 87.0±23.6%, p=0.029). Given the link between mechanotransduction, response to substrate stiffness and extracellular matrix composition, we evaluated the responses of PM plated on a soft (16kPa), intermediate (231kPa) and very hard (in order to GPa) polyacrylamide gels coated with two different fibronectin (FN) concentrations (1 and 25μg/mL). Atomic force microscopy measure of cell stiffness, showed that, while PMnorm significantly increase their intracellular stiffness (804±311Pa vs 1,320±394Pa, p=0.014) as a function of extracellular stiffness (soft vs very hard), PMfail do not. These results were paralleled by YAP and Myocardin Related Transcription Factor-A (MRTF-A) shuttling, that are translocated in the nuclei of PMnorm in response to substrate stiffness and fibronectin concentration significantly more than in the nuclei of PMfail cells. Analysis of CTGF, CYR61 and ANKRD1 gene expression corroborated these data. Finally, we demonstrated that serum deprivation, inhibitors of both actin polymerization (latrunculin-A), and Rho-associated Kinase (ROCK, Y23763) significantly inhibited the nuclear translocation of both YAP and MRTF-A and cell proliferation. Conversely, the MEK1/2 inhibitor I-1040 exerted opposite effects. Conclusion PM isolated from ischemic hearts are characterized by altered mechanotransduction properties that impair a correct nuclear translocation of the cotranscriptional regulators YAP and MRTF-A in response to environmental cues. Pharmacologic modulation of regulators of this pathway may revert the pathologic phenotype.

Analyses of MicroRNA and mRNA Expression Profiles Reveal the Crucial Interaction Networks and Pathways for Regulation of Chicken Breast Muscle Development.

There is a lack of understanding surrounding the molecular mechanisms involved in the development of chicken skeletal muscle in the late postnatal stage, especially in the regulation of breast muscle development related genes, pathways, miRNAs and other factors. In this study, 12 cDNA libraries and 4 small RNA libraries were constructed from Gushi chicken breast muscle samples from 6, 14, 22, and 30 weeks. A total of 15,508 known transcripts, 25,718 novel transcripts, 388 known miRNAs and 31 novel miRNAs were identified by RNA-seq in breast muscle at the four developmental stages. Through correlation analysis of miRNA and mRNA expression profiles, it was found that 417, 370, 240, 1,418, 496, and 363 negatively correlated miRNA–mRNA pairs of W14 vs. W6, W22 vs. W6, W22 vs. W14, W30 vs. W6, W30 vs. W14, and W30 vs. W22 comparisons, respectively. Based on the annotation analysis of these miRNA–mRNA pairs, we constructed the miRNA–mRNA interaction network related to biological processes, such as muscle cell differentiation, striated muscle tissue development and skeletal muscle cell differentiation. The interaction networks for signaling pathways related to five KEGG pathways (the focal adhesion, ECM-receptor interaction, FoxO signaling, cell cycle, and p53 signaling pathways) and PPI networks were also constructed. We found that ANKRD1, EYA2, JSC, AGT, MYBPC3, MYH11, ACTC1, FHL2, RCAN1, FOS, EGR1, and FOXO3, PTEN, AKT1, GADD45, PLK1, CCNB2, CCNB3 and other genes were the key core nodes of these networks, most of which are targets of miRNAs. The FoxO signaling pathway was in the center of the five pathway-related networks. In the PPI network, there was a clear interaction among PLK1 and CDK1, CCNB2, CDK1, and GADD45B, and CDC45, ORC1 and MCM3 genes. These results increase the understanding for the molecular mechanisms of chicken breast muscle development, and also provide a basis for studying the interactions between genes and miRNAs, as well as the functions of the pathways involved in postnatal developmental regulation of chicken breast muscle.

Effects of Dietary Arginine Supplementation in Pregnant Mares on Maternal Metabolism and Foal Birthweight

Withers height is an important economic trait for the American Miniature Horses (AMHs) as the breeders' primary objective is to produce small and proportionate animals. Identification of Quantitative Trait Loci (QTLs) affecting height in this breed will result in a better understanding of the genetic architecture and biological pathways contributing to skeletal development. In earlier work, we genotyped 48 horses from 16 different breeds that represent extremes in body size, using the Equine SNP50 bead chip (Illumina Inc). In this study, we applied a dominant model Genome-wide association study (GWAS), a genome-wide fixation index (FST) estimation, as well as a cross-population composite likelihood ratio (XP-CLR) test between the eight large and eight small breeds from this data set. Our GWAS implicated the marker at ECA1:37,676,322 bp, positioned within an intron of the ANKRD1 gene, was significantly associated with height variation. An XP-CLR scan further supported a role in height for the ECA1:37,676,322 bp locus and the FST analysis supported correlations to IGF2BP2 at ECA19:23,815,750. To confirm our findings, we genotyped the candidate loci in an independent set of 89 AMHs. This set verified that the locus at ANKRD1 gene indeed correlates to height under a dominant mode of inheritance, but not the locus at the IGF2BP2 gene. ANKRD1, a transcription factor involved in myocyte growth and differentiation, may contribute to height by influencing the overall growth of the horses. This marker will be a valuable tool for the selection of breeding stock in breeds with height restrictions for registration.

Genome-Wide Scans Reveal a Quantitative Trait Locus for Withers Height in Horses Near the ANKRD1 Gene

Withers height is an important economic trait for the American Miniature Horses (AMHs) as the breeders' primary objective is to produce small and proportionate animals. Identification of Quantitative Trait Loci (QTLs) affecting height in this breed will result in a better understanding of the genetic architecture and biological pathways contributing to skeletal development. In earlier work, we genotyped 48 horses from 16 different breeds that represent extremes in body size, using the Equine SNP50 bead chip (Illumina Inc). In this study, we applied a dominant model Genome-wide association study (GWAS), a genome-wide fixation index (FST) estimation, as well as a cross-population composite likelihood ratio (XP-CLR) test between the eight large and eight small breeds from this data set. Our GWAS implicated the marker at ECA1:37,676,322 bp, positioned within an intron of the ANKRD1 gene, was significantly associated with height variation. An XP-CLR scan further supported a role in height for the ECA1:37,676,322 bp locus and the FST analysis supported correlations to IGF2BP2 at ECA19:23,815,750. To confirm our findings, we genotyped the candidate loci in an independent set of 89 AMHs. This set verified that the locus at ANKRD1 gene indeed correlates to height under a dominant mode of inheritance, but not the locus at the IGF2BP2 gene. ANKRD1, a transcription factor involved in myocyte growth and differentiation, may contribute to height by influencing the overall growth of the horses. This marker will be a valuable tool for the selection of breeding stock in breeds with height restrictions for registration.

The expression of gene ANKRD1 correlates with hypoxia status in muscle biopsies of treatment-näive adult dermatomyositis

OBJECTIVES. The ANKRD1 gene codes for the ankyrin repeat domain containing protein 1 and has an important role in myogenesis and possibly also in angiogenesis. Microvasculopathy is a cornerstone and an early pathological marker of change in dermatomyositis, leading to hypoxia and muscle perifascicular atrophy. These alterations could upregulate genes involved in myogenesis and angiogenesis such as ANKRD1. Therefore, we analyzed ANKRD1 expression in muscle biopsies of dermatomyositis and correlated with other hypoxia parameters and with histological changes. METHODS. Total RNA was extracted from frozen muscle biopsies samples of 29 dermatomyositis patients. A control group consisted of 20 muscle biopsies from adult patients with non-inflammatory myopathy diseases. The gene coding for hypoxia-inducible factor 1, alpha subunit (HIF1A), was analyzed to estimate the degree of hypoxia. ANKRD1 and HIF1A transcript expression levels were determined by quantitative real time PCR. RESULTS. Significantly higher ANKRD1 and HIF1A expression levels were observed in dermatomyositis relative to the control group (P<0.001, both genes). In addition, ANKRD1 and HIF1A were coexpressed (r=0.703, P=0.001) and their expression levels correlated positively to perifascicular atrophy (r=0.420, P=0.023 and r=0.404, P=0.030, respectively). CONCLUSIONS. Our results demonstrate ANKRD1 overexpression in dermatomyositis correlated to HIF1A expression and perifascicular atrophy. ANKRD1 involvement in myogenesis and angiogenesis mechanisms indicates that further investigation is worthwhile.

Distinct Skeletal Muscle Gene Regulation from Active Contraction, Passive Vibration, and Whole Body Heat Stress in Humans.

Skeletal muscle exercise regulates several important metabolic genes in humans. We know little about the effects of environmental stress (heat) and mechanical stress (vibration) on skeletal muscle. Passive mechanical stress or systemic heat stress are often used in combination with many active exercise programs. We designed a method to deliver a vibration stress and systemic heat stress to compare the effects with active skeletal muscle contraction. Purpose: The purpose of this study is to examine whether active mechanical stress (muscle contraction), passive mechanical stress (vibration), or systemic whole body heat stress regulates key gene signatures associated with muscle metabolism, hypertrophy/atrophy, and inflammation/repair. Methods: Eleven subjects, six able-bodied and five with chronic spinal cord injury (SCI) participated in the study. The six able-bodied subjects sat in a heat stress chamber for 30 minutes. Five subjects with SCI received a single dose of limb-segment vibration or a dose of repetitive electrically induced muscle contractions. Three hours after the completion of each stress, we performed a muscle biopsy (vastus lateralis or soleus) to analyze mRNA gene expression. Results: We discovered repetitive active muscle contractions up regulated metabolic transcription factors NR4A3 (12.45 fold), PGC-1α (5.46 fold), and ABRA (5.98 fold); and repressed MSTN (0.56 fold). Heat stress repressed PGC-1α (0.74 fold change; p < 0.05); while vibration induced FOXK2 (2.36 fold change; p < 0.05). Vibration similarly caused a down regulation of MSTN (0.74 fold change; p < 0.05), but to a lesser extent than active muscle contraction. Vibration induced FOXK2 (p < 0.05) while heat stress repressed PGC-1α (0.74 fold) and ANKRD1 genes (0.51 fold; p < 0.05). Conclusion: These findings support a distinct gene regulation in response to heat stress, vibration, and muscle contractions. Understanding these responses may assist in developing regenerative rehabilitation interventions to improve muscle cell development, growth, and repair.

Overexpression of ankyrin repeat domain 1 enhances cardiomyocyte apoptosis by promoting p53 activation and mitochondrial dysfunction in rodents.

The Ankrd1 (ankyrin repeat domain 1) gene is known to be up-regulated in heart failure and acts as a co-activator of p53, modulating its transcriptional activity, but it remains inconclusive whether this gene promotes or inhibits cell apoptosis. In the present study, we attempted to investigate the role of Ankrd1 on AngII (angiotensin II)- or pressure-overload-induced cardiomyocyte apoptosis. In the failing hearts of mice with pressure overload, the protein expression of Ankrd1-encoded CARP (cardiac ankyrin repeat protein) was significantly increased. In NRCs (neonatal rat cardiomyocytes), AngII increased the expression of Ankrd1 and CARP. In the presence of AngII in NRCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced the mitochondrial translocation of Bax and phosphorylated p53, increased mitochondrial permeability and cardiomyocyte apoptosis, and reduced cell viability, whereas these effects were antagonized by silencing of Ankrd1. Intra-myocardial injection of Ad-Ankrd1 in mice with TAC (transverse aortic constriction) markedly exacerbated cardiac dysfunction with an increase in the lung weight/body weight ratio and a decrease in left ventricular fractional shortening. Cardiomyocyte apoptosis and the expression of phosphorylated p53 were also significantly increased in Ad-Ankrd1-infected TAC mice, whereas knockdown of Ankrd1 significantly inhibited the apoptotic signal pathway as well as cardiomyocyte apoptosis in pressure-overload mice. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac dysfunction through enhancement of cardiomyocyte apoptosis mediated by the up-regulation of p53.

The Muscle Ankyrin Repeat Proteins CARP, Ankrd2, and DARP Are Not Essential for Normal Cardiac Development and Function at Basal Conditions and in Response to Pressure Overload

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.

Novel mutations in the sarcomeric protein myopalladin in patients with dilated cardiomyopathy

Recently, missense mutations in titin-associated proteins have been linked to the pathogenesis of dilated cardiomyopathy (DCM). The objective of this study was to search for novel disease-associated mutations in the two human titin-binding proteins myopalladin and its amino-terminal-interacting partner cardiac ankyrin-repeat protein (CARP). In a cohort of 255 cases with familial and sporadic DCM, we analyzed the coding regions and all corresponding intron flanks located in the MYPN and CARP-encoding ANKRD1 gene. Two heterozygous missense mutations were detected in the MYPN gene (p.R955W and p.P961L), but neither of these mutations was found in 300 healthy controls. Both mutations were located in the α-actinin-binding region of myopalladin. Endomyocardial biopsies from the p.R955W carrier showed normal subcellular localization of myopalladin and α-actinin in cardiac myocytes, while their regular sarcomeric staining pattern was significantly disrupted in the p.P961L carrier, indicating that disturbed myofibrillogenesis and altered sarcomere assembly are the cause of the disease. In the ANKRD1 gene, we identified synonymous base exchanges (c.108T>C and c.-79C>T, respectively), but no non-synonymous mutations. In summary, we have identified novel missense mutations in the third immunoglobulin-like domain of myopalladin, which have either no or profound effects on the molecular composition of the sarcomere. According to our epidemiological data, the prevalence of ANKRD1 mutations seems to be lower than that of its binding partner myopalladin, indicating the clinical significance of myopalladin for the functional integrity of the sarcomeric apparatus and the protection against DCM.

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

Dilated Cardiomyopathy (DCM) is familial in about 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 overexpressed 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 hypothesised that mutations in ANKRD1 could be responsible for DCM. We sequenced the coding region of ANKRD1 from 231 independent DCM cases. We identified 5 missense mutations (3 sporadic and 2 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 significant less repressor activity measured in a reporter gene assay and greater phenylephrin induced hypertrophy suggesting altered function of CARP mutant proteins. Based on genetic and functional analysis of CARP mutations, we have identified ANKRD1 as a new gene associated with DCM, accounting for about 2% of cases.

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

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.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium

The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.

H006 Mutations in the ANKRD1 gene encoding carp are responsible for human dilated cardiomyopathy

Dilated Cardiomyopathy (DCM) is familial in about 30 % of cases, and to date, 15 responsible genes have been identified in isolated forms and up to 25 associated with additional phenotypes including myopathy, arrythmias or more complex syndromes. No major gene for the disease has been identified, demonstrating the genetic heterogeneity of DCM. However, in a majority of families the responsible genes are still to be discovered. The ANKRD1 gene is overexpressed in heart failure in human or animal models. The encoded protein CARP is interacting with partners such as Myopalladin or Titin, previously involved in DCM. We hypothesised that mutations in ANKRD1 could be responsible for DCM. We have screened a DCM affected population consisting on 231 caucasian independent familial (158) and sporadic (73) cases by direct sequencing of PCR-amplified coding exons. We identified 5 missense mutations: 3 sporadic (mutations p.Glu57Gln, p.Arg66Gln and p.Leu199Arg) and 2 familial (mutations p.Thr116Met and p.Ala276Val) absent from 400 controls and affecting highly conserved residues. Expression of the mutant CARP proteins in rat neonate cardiomyocytes indicated that at least 3 of the mutations identified (p.Glu57Gln, p.Leu199Arg, p.Ala276Val) led both to significant less repressor activity and to greater phenylephrin induced hypertrophy suggesting altered function of CARP mutant proteins. Based on genetic and functional analysis of CARP mutations, we have identified ANKRD1 as a new gene associated with DCM, accounting for about 4 % of cases.

Transcriptional deregulation and a missense mutation define ANKRD1 as a candidate gene for total anomalous pulmonary venous return

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect in which the pulmonary veins fail to enter the left atrium and drain instead into the right atrium or one of its venous tributaries. Although a genetic basis for TAPVR has long been recognized, no single gene involved in the pathogenesis of this disease has been identified to date. We previously reported a TAPVR patient bearing a de novo 10;21 balanced translocation. In this work, we cloned both translocation breakpoints from this patient and mapped the ANKRD1 gene, encoding a cardiac transcriptional regulator, 130 kb proximally to the breakpoint on chromosome 10. In situ hybridization analysis performed on murine embryos showed ANKRD1 expression in the developing pulmonary veins, suggesting a possible role for this gene in TAPVR pathogenesis. Moreover, ANKRD1 expression levels were found to be highly increased in lymphoblastoid cell lines derived from both the translocation-bearing proband and a second independent sporadic TAPVR patient, suggesting that disruption of the normal ANKRD1 expression pattern is associated with TAPVR. Finally, a nonconservative missense mutation in the ANKRD1 gene was found in a third sporadic TAPVR patient. In vitro calpain-mediated degradation assays, coupled to reporter gene analysis in transfected HeLa cells, strongly suggested that this mutation enhances both the stability of the ANKRD1/CARP protein and its transcriptional repression activity upon the cardiac-specific atrial natriuretic factor (ANF) promoter. Taken together, these results define ANKRD1 as a possible candidate gene for TAPVR pathogenesis.

The enigmatic role of the ankyrin repeat domain 1 gene in heart development and disease

It has been proposed that the ankyrin repeat domain 1 (ANKRD1) factor (also known as CARP) plays a critical role in transcriptional regulation, myofibrillar assembly and stretch sensing during heart development and cardiac insults. ANKRD1/CARP has also been reported to negatively regulate cardiac gene expression in cell-based promoter-reporter assays. Consequently, rapid up-regulation of the ankrd1 gene in myocardium in response to developmental stimuli or pathological insults has tended to be interpreted in the context of the inhibitory effects of ANKRD1 on cardiomyocyte gene expression. Surprisingly, a total ankrd1 knockout resulted in a complete lack of phenotype, suggesting that ANKRD1/CARP is not crucial for regulation of cardiac gene expression in vivo. In this essay, we summarize (1) the accumulated evidence for the apparent multifunctional properties of this enigmatic protein, (2) the distinct chamber-dependent regulation of ankrd1 expression patterns in the heart, both during development and cardiac injury, and (3) ANKRD1 involvement in networks regulating adaptation of the myocardium to stress. Whenever feasible, we present the results obtained in patients together with those obtained in the relevant animal and cellular models. A close examination of the findings still fails to define ANKRD1 as a negative regulator of cardiac gene expression in vivo, but rather indicates that its augmented expression can represent an adaptive response of the myocardium to stress both during development and various heart insults.