Desmoglein-2 mutations in propeptide cleavage-site causes arrhythmogenic right ventricular cardiomyopathy/dysplasia by impairing extracellular 1-dependent desmosomal interactions upon cellular stress.

AIMS: Desmoglein-2 (DSG2) mutations, which encode a heart-specific cadherin crucial for desmosomal adhesion, are frequent in arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). DSG2 mutations have been associated with higher risk of biventricular involvement. Among DSG2 mutations, mutations of the inhibitory propeptide consensus cleavage-site (Arg-X-Arg/Lys-Arg), are particularly frequent. In the present work, we explored the functional consequences of DSG2 propeptide cleavage site mutations p.Arg49His, p.Arg46Trp, and p.Arg46Gln on localization, adhesive properties, and desmosome incorporation of DSG2. METHODS AND RESULTS: We studied the expression of mutant-DSG2 in human heart and in epithelial and cardiac cellular models expressing wild-type or mutant (p.Arg49His, p.Arg46Trp, and p.Arg46Gln) proDSG2-GFP fusion proteins. The consequences of the p.Arg46Trp mutation on DSG2 adhesiveness were studied by surface plasmon resonance. Incorporation of mutant p.Arg46Trp DSG2 into desmosomes was studied under low-calcium culture conditions and cyclic mechanical stress. We demonstrated in human heart and cellular models that all three mutations prevented N-terminal propeptide cleavage, but did not modify intercellular junction targeting. Surface plasmon resonance experiments showed a propeptide-dependent loss of interaction between the cadherin N-terminal extracellular 1 (EC1) domains. Additionally, proDSG2 mutant proteins were abnormally incorporated into desmosomes under low-calcium culture conditions or following mechanical stress. This was accompanied by an epidermal growth factor receptor-dependent internalization of proDSG2, suggesting increased turnover of unprocessed proDSG2. CONCLUSION: Our results strongly suggest weakened desmosomal adhesiveness due to abnormal incorporation of uncleaved mutant proDSG2 in cellular stress conditions. These results provide new insights into desmosomal cadherin regulation and ARVC/D pathophysiology, in particular, the potential role of mechanical stress on desmosomal dysfunction.

Shaping of Drosophila Neural Cell Lineages Through Coordination of Cell Proliferation and Cell Fate by the BTB-ZF Transcription Factor Tramtrack-69.

Cell diversity in multicellular organisms relies on coordination between cell proliferation and the acquisition of cell identity. The equilibrium between these two processes is essential to assure the correct number of determined cells at a given time at a given place. Using genetic approaches and correlative microscopy, we show that Tramtrack-69 (Ttk69, a Broad-complex, Tramtrack and Bric-à-brac - Zinc Finger (BTB-ZF) transcription factor ortholog of the human promyelocytic leukemia zinc finger factor) plays an essential role in controlling this balance. In the Drosophila bristle cell lineage, which produces the external sensory organs composed by a neuron and accessory cells, we show that ttk69 loss-of-function leads to supplementary neural-type cells at the expense of accessory cells. Our data indicate that Ttk69 (1) promotes cell cycle exit of newborn terminal cells by downregulating CycE, the principal cyclin involved in S-phase entry, and (2) regulates cell-fate acquisition and terminal differentiation, by downregulating the expression of hamlet and upregulating that of Suppressor of Hairless, two transcription factors involved in neural-fate acquisition and accessory cell differentiation, respectively. Thus, Ttk69 plays a central role in shaping neural cell lineages by integrating molecular mechanisms that regulate progenitor cell cycle exit and cell-fate commitment.

Light-induced formation of NO in endothelial cells by photoactivatable NADPH analogues targeting nitric-oxide synthase.

BACKGROUND: Nitric-oxide synthases (NOS) catalyze the formation of NO using NADPH as electron donor. We have recently designed and synthesized a new series of two-photon absorbing and photoactivatable NADPH analogues (NT). These compounds bear one or two carboxymethyl group(s) on the 2'- or/and 3'-position(s) of the ribose in the adenosine moiety, instead of a 2'-phosphate group, and differ by the nature of the electron donor in their photoactivatable chromophore (replacing the nicotinamide moiety). Here, we addressed the ability of NTs to photoinduce eNOS-dependent NO production in endothelial cells. METHODS: The cellular fate of NTs and their photoinduced effects were studied using multiphoton fluorescence imaging, cell viability assays and a BODIPY-derived NO probe for NO measurements. The eNOS dependence of photoinduced NO production was addressed using two NOS inhibitors (NS1 and L-NAME) targeting the reductase and the oxygenase domains, respectively. RESULTS: We found that, two compounds, those bearing a single carboxymethyl group on the 3'-position of the ribose, colocalize with the Golgi apparatus (the main intracellular location of eNOS) and display high intracellular two-photon brightness. Furthermore, a eNOS-dependent photooxidation was observed for these two compounds only, which is accompanied by a substantial intracellular NO production accounting for specific photocytotoxic effects. CONCLUSIONS: We show for the first time that NT photoactivation efficiently triggers electron flow at the eNOS level and increases the basal production of NO by endothelial cells. GENERAL SIGNIFICANCE: Efficient photoactivatable NADPH analogues targeting NOS could have important implications for generating apoptosis in tumor cells or modulating NO-dependent physiological processes.

Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides.

Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family.

A role for the serine/arginine-rich (SR) protein B52/SRSF6 in cell growth and myc expression in Drosophila.

Serine-/arginine-rich (SR) proteins are RNA-binding proteins that are primarily involved in alternative splicing. Expression of some SR proteins is frequently upregulated in tumors, and previous reports have demonstrated that these proteins can directly participate in cell transformation. Identifying factors that can rescue the effects of SR overexpression in vivo is, therefore, of potential therapeutic interest. Here, we analyzed phenotypes induced by overexpression of the SR protein B52 during Drosophila development and identified several proteins that can rescue these phenotypes. Using the mechanosensory bristle lineage as a developmental model, we show that B52 expression level influences cell growth, but not differentiation, in this lineage. In particular, B52 overexpression increases cell growth, upregulates myc transcription, and gives rise to flies lacking thoracic bristles. Using a genetic screen, we identified several suppressors of the phenotypes induced by overexpression of B52 in vivo in two different organs. We show that upregulation of brain tumor (brat), a tumor suppressor and post-transcriptional repressor of myc, and downregulation of lilliputian (lilli), a subunit of the superelongation complex involved in transcription elongation, efficiently rescue the phenotypes induced by B52 overexpression. Our results demonstrate a role of this SR protein in cell growth and identify candidate proteins that may overcome the effects of SR protein overexpression in mammals.

Screening of genes encoding junctional candidates in arrhythmogenic right ventricular cardiomyopathy/dysplasia.

AIMS: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited cardiomyopathy characterized by fibro-fatty replacement of the right ventricle and ventricular arrhythmias. The major disease-causing genes encode cardiac desmosomal components but are involved in only ∼50% of patients. To identify the missing genetic determinants, we used a candidate gene approach, focusing on the 3'-untranslated region (UTR) of the main ARVC/D gene PKP2 and on additional genes involved in desmosomal structure or function. METHODS AND RESULTS: We screened a population of 64 ARVC/D probands with no identified mutations in any of the five known desmosomal genes (PKP2, DSG2, DSP, DSC2, and JUP). No putative mutation was identified in the 3'-UTR of PKP2 or in PNN, CTNNA3, CAV1, or PLN coding sequences. In a single proband, we identified two rare heterozygous missense variants affecting evolutionary conserved residues: c.175G>A (p.Gly59Arg) in PERP and c.1811A>G (p.Asp604Gly) in PKP4 (minor allele frequency <0.5% in control population). CONCLUSION: Our study suggests that mutations in the candidate genes studied and regulation of PKP2 mRNA via 3'-UTR dependent mechanisms are unlikely to be major causes of ARVC/D in the studied population. Additional studies are needed to investigate the putative effects of rare PKP4 and PERP variants in this disease.

De novo heterozygous desmoplakin mutations leading to Naxos-Carvajal disease.

STUDY/PRINCIPLES: Arrythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an autosomal-dominantly inherited disease caused by mutations in genes encoding desmosomal proteins and is characterised by fibrofatty replacement occurring predominantly in the right ventricle and can result in sudden cardiac death. Naxos and Carvajal syndrome, autosomal recessive forms of ARVC/D, are characterised by involvement of the right and/or left ventricle in association with palmoplantar keratoderma and woolly hair. The aim of the present study has been to screen for mutations in the desmosomal protein genes of two unrelated patients with Naxos-Carvajal syndrome. METHODS AND RESULTS: Desmosomal protein genes were screened for mutations by polymerase chain reaction as well as direct sequencing approach. In each patient we identified a single heterozygous de novo mutation in the desmoplakin gene DSP, p.Leu583Pro and p.Thr564Ile, leading to severe combined cardiac/dermatological and cardiac/dermatological/dental phenotypes. The DSP missense mutations are localised in the N terminal domain of desmoplakin. CONCLUSION: The identified variations in DSP involve highly conserved residues. Moreover, the variations are de novo mutations and they are localised in critical protein domains that appear to be mutation hot spots. We assume that these heterozygous variations are causal for the mixed Naxos-Carvajal syndrome phenotype in the screened patients.

Brugada ECG pattern: a physiopathological prospective study based on clinical, electrophysiological, angiographic, and genetic findings.

INTRODUCTION: Brugada syndrome (BrS) is considered a primary electrical disease. However, morphological abnormalities have been reported and localized arrhythmogenic right ventricular (RV) dysplasia/cardiomyopathy (ARVD/C) may mimic its phenotype, raising the question of an overlap between these two conditions and making difficult the therapeutic management of patients with borderline forms. The main objective of this study was to assess prospectively the prevalence of BrS and ARVD/C on the basis of international criteria, in patients with BrS-ECG and normal echocardiography, looking for a potential overlap between the two pathologies. The secondary objectives were to describe and quantify angiographic structural alterations, hemodynamics, electrophysiology, and genetics in the setting of BrS-ECG. MATERIALS AND METHODS: Hundred and fourteen consecutive patients matched in age underwent prospectively cardiac catheterization and quantitative biventricular contrast angiography to rule out a structural heart disease. Fifty-one patients with a BrS-ECG (BrS group, 7 F, 44 M, 43 ± 11 y) had a spontaneous or ajmaline-induced BrS coved type ECG. For angiographic comparison, 49 patients with localized ARVD/C but without ST segment elevation in the right precordial leads (14 F, 35 M, 39 ± 13 y) were also studied. They fulfilled international ESC/WHF 2000 criteria and presented angiographic localized forms, mainly confined to hypokinetic anteroapical zone (characterized by trabecular dysarray and hypertrophy), and/or diaphragmatic wall, thus resulting in RV normal volumes and preserved systolic function. These two populations were also compared with 14 control patients (7 F, 7 M, 38 ± 16 y). Among BrS group, we identified three main angiographic phenotypes: BrS group I = patients with normal RV (n = 15, 29%); BrS group II = patients with segmental RV wall motion abnormalities but no structural arguments for ARVD/C (n = 26, 51%); BrS group III = patients with localized abnormalities suggestive of focal ARVD/C (n = 10, 20%). RESULTS: Among BrS group, 34/51 patients (67%) fulfilled BrS HRS/EHRA 2005 criteria. Nineteen (37%) were symptomatic for aborted sudden death, agonal nocturnal respiration or syncope. Ventricular stimulation was positive in 14 patients (28%). Angiography showed RV abnormalities in 36/51 patients (71%) of BrS group (BrS groups II and III). Late potentials were present in 73% (100% sensitivity and NPV for an angiographic ARVD/C, but poor specificity and PPV, both 37%). In BrS group III, 8/10 patients (16% of BrS patients) finally fulfilled international ESC/WHF 2000 ARVD/C criteria and 5/10 (10% of BrS patients) fulfilled BrS diagnostic criteria. An overlap was observed in 4 patients (8% of BrS patients) who fulfilled both ARVD/C and BrS criteria. Among the 45 genotyped patients, only one presented a SCN5A mutation, whereas a TRPM4 mutation was found in another patient. Both belonged to BrS group II. MOG1 gene analysis was negative for all patients, as were PKP2, DSP, DSG2, and DSC2 analyzes performed in BrS group III. CONCLUSIONS: Seventy-one percent of patients with a BrS-ECG had abnormal RV wall motion and 16 had structural alterations corresponding to localized (anteroapical and/or diaphragmatic) ARVD/C. Moreover, 8% of BrS-ECG patients fulfilled both BrS and ARVD/C criteria. Our results support the hypothesis of an overlap between BrS and localized forms of ARVD/C. Conversely, genetic screening was poorly contributive for both diseases in the present series.

Desmosomal gene analysis in arrhythmogenic right ventricular dysplasia/cardiomyopathy: spectrum of mutations and clinical impact in practice.

AIMS: Five desmosomal genes have been recently implicated in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) but the clinical impact of genetics remains poorly understood. We wanted to address the potential impact of genotyping. METHODS AND RESULTS: Direct sequencing of the five genes (JUP, DSP, PKP2, DSG2, and DSC2) was performed in 135 unrelated patients with ARVD/C. We identified 41 different disease-causing mutations, including 28 novel ones, in 62 patients (46%). In addition, a genetic variant of unknown significance was identified in nine additional patients (7%). Distribution of genes was 31% (PKP2), 10% (DSG2), 4.5% (DSP), 1.5% (DSC2), and 0% (JUP). The presence of desmosomal mutations was not associated with familial context but was associated with young age, symptoms, electrical substrate, and extensive structural damage. When compared with other genes, DSG2 mutations were associated with more frequent left ventricular involvement (P = 0.006). Finally, complex genetic status with multiple mutations was identified in 4% of patients and was associated with more frequent sudden death (P = 0.047). CONCLUSION: This study supports the use of genetic testing as a new diagnostic tool in ARVC/D and also suggests a prognostic impact, as the severity of the disease appears different according to the underlying gene or the presence of multiple mutations.

Sporadic arrhythmogenic right ventricular cardiomyopathy/dysplasia due to a de novo mutation.

We report the case of a 41-year-old man with a diagnosis of sporadic arrhythmogenic right ventricular cardiomyopathy (ARVC). Genetic screening identified the heterozygous missense mutation R49H in the desmoglein-2 gene. The mutation was absent in both parents, and we demonstrated that it was a de novo mutation. To the best of our knowledge, this is the first description of a de novo mutation in ARVC. This has important implications, including for clinical practice, since individuals with sporadic ARVC caused by a de novo mutation can transmit the disease gene to 50% of their offspring. This suggests that the benefit of molecular genetics can be extended to sporadic ARVC and may improve genetic counselling.

Long QT syndrome in neonates: conduction disorders associated with HERG mutations and sinus bradycardia with KCNQ1 mutations.

OBJECTIVES: We hypothesized that neonatal long QT syndrome (LQTS) with 2:1 atrioventricular block (AVB) could be related to HERG mutations. BACKGROUND: Early onset of LQTS is rare but carries a high risk of life-threatening events such as ventricular arrhythmias and conduction disorders. There are no data on possible gene specificity. METHODS: We analyzed the characteristics and outcomes of 23 neonate probands from our LQTS population. Samples of DNA were available in 18 cases. RESULTS: Long QT syndrome was diagnosed because of corrected QT interval (QTc) prolongation (mean QTc of 558 +/- 62 ms) and neonatal bradycardia attributable to sinus bradycardia (n = 8) or 2:1 AVB (n = 15). Symptoms included syncope (n = 2), torsades de pointes (n = 7), and hemodynamic failure (n = 6). Three infants with 2:1 AVB died during the first month of life. During the neonatal period, all living patients received beta-blockers (BB) and 13 had a combination of BB and permanent cardiac pacing. Under treatment, patients remained asymptomatic, with a mean follow-up of seven years. Mutations were identified in HERG (n = 8) and KCNQ1 (n = 8), and one child had three mutations (HERG, KCNQ1, and SCN5A). Conduction disorders were associated with LQT2, whereas sinus bradycardia was associated with LQT1. CONCLUSIONS: Two-to-one AVB seems preferentially associated with HERG mutations, either isolated or combined. Long QT syndrome with relative bradycardia attributable to 2:1 AVB has a poor prognosis during the first month of life. In contrast, sinus bradycardia seems to be associated with KCNQ1 mutations, with a good short-term prognosis under BB therapy.

Immunolocalization of a nuclear protein bound to the sphere organelle during oogenesis and embryogenesis inPleurodeles waltl.

The distribution of a nuclear antigen ofPleurodeles waltl oocytes, recognized by the monoclonal antibody B24/1, has been studied during oogenesis and early embryonic development. In stage I oocytes the antigen was localized in the nucleoplasm and on two atypical structures of lampbrush chromosomes, the spheres (S) and the mass (M). The immunostaining increased as the oocyte developed. In stage VI oocytes, the nucleoplasm and spheres showed intense staining. At this stage, the nucleoplasm often contained free spheres which were also labelled. The staining of M diminished during oogenesis, as did its size. Immunoblots of nuclear proteins of oocytes at different stages confirmed that there was an accumulation of this protein during oogenesis. During embryonic development, the nuclei of all the cells of blastula and gastrula were labelled by this antibody: there was no embryonic regionalization. Starting from the neurula stage, the staining progressively disappeared from the nuclei of ectodermal and mesodermal cells. In the tailbud stage, only the endodermal cell nuclei showed faint staining. Immunoblots of proteins from embryos of different stages showed that the quantity of this protein was constant until the young gastrula stage and then decreased progressively; in the young tailbud stage, this protein was practically absent. B24/1 is the first described protein of the sphere. This protein is accumulated in the oocyte nucleus and behaves like a maternal polypeptide, shifting early in the nuclei during embryonic development. Thus, B24/1 probably has a function required from the early developmental stages, perhaps in relation with small nuclear ribonucleoproteins.