Filamin A heart valve disease as a genetic cause of inherited bicuspid and tricuspid aortic valve disease.

OBJECTIVE: Variants in the FLNA gene have been associated with mitral valve dystrophy (MVD), and even polyvalvular disease has been reported. This study aimed to analyse the aortic valve and root involvement in FLNA-MVD families and its impact on outcomes. METHODS: 262 subjects (37 (18-53) years, 140 male, 79 carriers: FLNA+) from 4 FLNA-MVD families were included. Echocardiography was performed in 185 patients and histological analysis in 3 explanted aortic valves. The outcomes were defined as aortic valve surgery or all-cause mortality. RESULTS: Aortic valve alterations were found in 58% of FLNA+ compared with 6% of FLNA- (p<0.001). 9 (13.4%) FLNA+ had bicuspid aortic valve compared with 4 (3.4%) FLNA- (p=0.03). Overall, the transvalvular mean gradient was slightly increased in FLNA+ (4.8 (4.1-6.1) vs 4.0 (2.9-4.9) mm Hg, p=0.02). The sinuses of Valsalva and sinotubular junction diameters were enlarged in FLNA+ subjects (all p<0.05). 8 FLNA+ patients underwent aortic valve surgery (0 in relatives; p<0.001). Myxomatous remodelling with an infiltration of immune cells was observed. Overall survival was similar between FLNA+ versus FLNA- subjects (86±5% vs 85±6%, p=0.36). There was no statistical evidence for an interaction between genetic status and sex (p=0.15), but the survival tended to be impaired in FLNA+ men (p=0.06) whereas not in women (p=0.71). CONCLUSION: The patients with FLNA variants present frequent aortic valve disease and worse outcomes. Bicuspid aortic valve is more frequent in patients carrying the FLNA-MVD variants. These unique features should be factored into the management of patients with dystrophic and/or bicuspid aortic valve.

Genetics and pathophysiology of mitral valve prolapse.

Mitral valve prolapse (MVP) is a common condition affecting 2-3% of the general population, and the most complex form of valve pathology, with a complication rate up to 10-15% per year in advanced stages. Complications include mitral regurgitation which can lead to heart failure and atrial fibrillation, but also life-threatening ventricular arrhythmia and cardiovascular death. Sudden death has been recently brought to the forefront of MVP disease, increasing the complexity of management and suggesting that MVP condition is not properly understood. MVP can occur as part of syndromic conditions such as Marfan syndrome, but the most common form is non-syndromic, isolated or familial. Although a specific X-linked form of MVP was initially identified, autosomal dominant inheritance appears to be the primary mode of transmission. MVP can be stratified into myxomatous degeneration (Barlow), fibroelastic deficiency, and Filamin A-related MVP. While FED is still considered a degenerative disease associated with aging, myxomatous MVP and FlnA-MVP are recognized as familial pathologies. Deciphering genetic defects associated to MVP is still a work in progress; although FLNA, DCHS1, and DZIP1 have been identified as causative genes in myxomatous forms of MVP thanks to familial approaches, they explain only a small proportion of MVP. In addition, genome-wide association studies have revealed the important role of common variants in the development of MVP, in agreement with the high prevalence of this condition in the population. Furthermore, a potential genetic link between MVP and ventricular arrhythmia or a specific type of cardiomyopathy is considered. Animal models that allow to advance in the genetic and pathophysiological knowledge of MVP, and in particular those that can be easily manipulated to express a genetic defect identified in humans are detailed. Corroborated by genetic data and animal models, the main pathophysiological pathways of MVP are briefly addressed. Finally, genetic counseling is considered in the context of MVP.

Multimodality imaging and transcriptomics to phenotype mitral valve dystrophy in a unique knock-in Filamin-A rat model.

AIMS: Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD. METHODS AND RESULTS: Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-ß and inflammation in the disease. CONCLUSION: The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.

Highly Ordered Boron Nitride/Epigraphene Epitaxial Films on Silicon Carbide by Lateral Epitaxial Deposition.

The realization of high-performance nanoelectronics requires control of materials at the nanoscale. Methods to produce high quality epitaxial graphene (EG) nanostructures on silicon carbide are known. The next step is to grow van der Waals semiconductors on top of EG nanostructures. Hexagonal boron nitride (h-BN) is a wide bandgap semiconductor with a honeycomb lattice structure that matches that of graphene, making it ideally suited for graphene-based nanoelectronics. Here, we describe the preparation and characterization of multilayer h-BN grown epitaxially on EG using a migration-enhanced metalorganic vapor phase epitaxy process. As a result of the lateral epitaxial deposition (LED) mechanism, the grown h-BN/EG heterostructures have highly ordered epitaxial interfaces, as desired in order to preserve the transport properties of pristine graphene. Atomic scale structural and energetic details of the observed row-by-row growth mechanism of the two-dimensional (2D) epitaxial h-BN film are analyzed through first-principles simulations, demonstrating one-dimensional nucleation-free-energy-barrierless growth. This industrially relevant LED process can be applied to a wide variety of van der Waals materials.

Obinutuzumab plus fludarabine and cyclophosphamide in previously untreated, fit patients with chronic lymphocytic leukemia: a subgroup analysis of the GREEN study.

GREEN (NCT01905943) is a nonrandomized, open-label, single-arm, phase 3b study investigating the safety and efficacy of obinutuzumab alone or in combination with chemotherapy in chronic lymphocytic leukemia (CLL). We report the preplanned subgroup analysis of 140 previously untreated, fit CLL patients who received obinutuzumab plus fludarabine and cyclophosphamide (G-FC). The primary endpoint was safety and tolerability. Efficacy was the secondary endpoint. Obinutuzumab 1000 mg was administered intravenously on Day (D)1 (dose split D1‒2), D8 and D15 of Cycle (C)1, and D1 of C2-6 (28-day cycles). Standard intravenous/oral doses of fludarabine and cyclophosphamide were administered on D1-3 of C1-6. Overall, 87.1% of patients experienced grade ≥ 3 adverse events (AEs), including neutropenia (67.1%) and thrombocytopenia (17.1%). Serious AEs were experienced by 42.1% of patients. Rates of grade ≥ 3 infusion-related reactions and infections were 19.3% and 15.7%, respectively. Overall response rate was observed in 90.0%, with 46.4% of patients achieving complete response (CR; including CR with incomplete marrow recovery). Minimal residual disease negativity rates were 64.3% in peripheral blood and 35.7% in bone marrow (intent-to-treat analysis). After a median observation time of 25.6 months, 2 year progression-free survival was 91%. Frontline G-FC represents a promising treatment option for fit patients with CLL.

Critical Structural Defects Explain Filamin A Mutations Causing Mitral Valve Dysplasia.

Mitral valve diseases affect ∼3% of the population and are the most common reasons for valvular surgery because no drug-based treatments exist. Inheritable genetic mutations have now been established as the cause of mitral valve insufficiency, and four different missense mutations in the filamin A gene (FLNA) have been found in patients suffering from nonsyndromic mitral valve dysplasia (MVD). The filamin A (FLNA) protein is expressed, in particular, in endocardial endothelia during fetal valve morphogenesis and is key in cardiac development. The FLNA-MVD-causing mutations are clustered in the N-terminal region of FLNA. How the mutations in FLNA modify its structure and function has mostly remained elusive. In this study, using NMR spectroscopy and interaction assays, we investigated FLNA-MVD-causing V711D and H743P mutations. Our results clearly indicated that both mutations almost completely destroyed the folding of the FLNA5 domain, where the mutation is located, and also affect the folding of the neighboring FLNA4 domain. The structure of the neighboring FLNA6 domain was not affected by the mutations. These mutations also completely abolish FLNA's interactions with protein tyrosine phosphatase nonreceptor type 12, which has been suggested to contribute to the pathogenesis of FLNA-MVD. Taken together, our results provide an essential structural and molecular framework for understanding the molecular bases of FLNA-MVD, which is crucial for the development of new therapies to replace surgery.

Non-syndromic Mitral Valve Dysplasia Mutation Changes the Force Resilience and Interaction of Human Filamin A.

Filamin A (FLNa), expressed in endocardial endothelia during fetal valve morphogenesis, is key in cardiac development. Missense mutations in FLNa cause non-syndromic mitral valve dysplasia (FLNA-MVD). Here, we aimed to reveal the currently unknown underlying molecular mechanism behind FLNA-MVD caused by the FLNa P637Q mutation. The solved crystal structure of the FLNa3-5 P637Q revealed that this mutation causes only minor structural changes close to mutation site. These changes were observed to significantly affect FLNa's ability to transmit cellular force and to interact with its binding partner. The performed steered molecular dynamics simulations showed that significantly lower forces are needed to split domains 4 and 5 in FLNA-MVD than with wild-type FLNa. The P637Q mutation was also observed to interfere with FLNa's interactions with the protein tyrosine phosphatase PTPN12. Our results provide a crucial step toward understanding the molecular bases behind FLNA-MVD, which is critical for the development of drug-based therapeutics.

Safety of obinutuzumab alone or combined with chemotherapy for previously untreated or relapsed/refractory chronic lymphocytic leukemia in the phase IIIb GREEN study.

The safety of obinutuzumab, alone or with chemotherapy, was studied in a non-randomized, open-label, non-comparative, phase IIIb study (GREEN) in previously untreated or relapsed/refractory chronic lymphocytic leukemia. Patients received obinutuzumab 1000 mg alone or with chemotherapy (investigator's choice of fludarabine-cyclophosphamide for fit patients, chlorambucil for unfit patients, or bendamustine for any patient) on days 1, 8 and 15 of cycle 1, and day 1 of cycles 2-6 (28-day cycles), with the cycle 1/day 1 dose administered over two days. The primary end point was safety/tolerability. Between October 2013 and March 2016, 972 patients were enrolled and 971 treated (126 with obinutuzumab monotherapy, 193 with obinutuzumab-fludarabine-cyclophosphamide, 114 with obinutuzumab-chlorambucil, and 538 with obinutuzumab-bendamustine). Grade ≥3 adverse events occurred in 80.3% of patients, and included neutropenia (49.9%), thrombocytopenia (16.4%), anemia (9.6%), and pneumonia (9.0%); rates were similar in first-line and relapsed/refractory patients, and in first-line fit and unfit patients. Using expanded definitions, infusion-related reactions were observed in 65.4% of patients (grade ≥3, 19.9%; mainly seen during the first obinutuzumab infusion), tumor lysis syndrome in 6.4% [clinical and laboratory; highest incidence with obinutuzumab-bendamustine (9.3%)], and infections in 53.7% (grade ≥3, 20.1%). Serious and fatal adverse events were seen in 53.1% and 7.3% of patients, respectively. In first-line patients, overall response rates at three months post treatment exceeded 80% for all obinutuzumab-chemotherapy combinations. In the largest trial of obinutuzumab to date, toxicities were generally manageable in this broad patient population. Safety data were consistent with previous reports, and response rates were high. (clinicaltrials.gov identifier: 01905943).

Genetics of syndromic and non-syndromic mitral valve prolapse.

Mitral valve prolapse (MVP) is a common condition that affects 2%-3% of the general population. MVP is thought to include syndromic forms such as Marfan syndrome and non-syndromic MVP, which is the most frequent form. Myxomatous degeneration and fibroelastic deficiency (FED) are regarded as two different forms of non-syndromic MVP. While FED is still considered a degenerative disease associated with ageing, frequent familial clustering has been demonstrated for myxomatous MVP. Familial and genetic studies led to the recognition of reduced penetrance and large phenotypic variability, and to the identification of prodromal or atypical forms as a part of the complex spectrum of the disease. Whereas autosomal dominant mode is the common inheritance pattern, an X linked form of non-syndromic MVP was recognised initially, related to Filamin-A gene, encoding for a cytoskeleton protein involved in mechanotransduction. This identification allowed a comprehensive description of a new subtype of MVP with a unique association of leaflet prolapse and paradoxical restricted motion in diastole. In autosomal dominant forms, three loci have been mapped to chromosomes 16p11-p12, 11p15.4 and 13q31-32. Although deciphering the underlying genetic defects is still a work in progress, DCHS1 mutations have been identified (11p15.4) in typical myxomatous disease, highlighting new molecular pathways and pathophysiological mechanisms leading to the development of MVP. Finally, a large international genome-wide association study demonstrated the implication of frequent variants in MVP development and opened new directions for future research. Hence, this review focuses on phenotypic, genetic and pathophysiological aspects of MVP.

Loss of vascular expression of nucleoside triphosphate diphosphohydrolase-1/CD39 in hypertension.

Ectonucleoside triphosphate diphosphohydrolase-1, the major vascular/immune ectonucleotidase, exerts anti-thrombotic and immunomodulatory actions by hydrolyzing extracellular nucleotides (danger signals). Hypertension is characterized by vascular wall remodeling, endothelial dysfunction, and immune infiltration. Here our aim was to investigate the impact of arterial hypertension on CD39 expression and activity in mice. Arterial expression of CD39 was determined by reverse transcription quantitative real-time PCR in experimental models of hypertension, including angiotensin II (AngII)-treated mice (1 mg/kg/day, 21 days), deoxycorticosterone acetate-salt mice (1% salt and uninephrectomy, 21 days), and spontaneously hypertensive rats. A decrease in CD39 expression occurred in the resistance and conductance arteries of hypertensive animals with no effect on lymphoid organs. In AngII-treated mice, a decrease in CD39 protein levels (Western blot) was corroborated by reduced arterial nucleotidase activity, as evaluated by fluorescent (etheno)-ADP hydrolysis. Moreover, serum-soluble ADPase activity, supported by CD39, was significantly decreased in AngII-treated mice. Experiments were conducted in vitro on vascular cells to determine the elements underlying this downregulation. We found that CD39 transcription was reduced by proinflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor alpha on vascular smooth muscle cells and by IL-6 and anti-inflammatory and profibrotic cytokine transforming growth factor beta 1 on endothelial cells. In addition, CD39 expression was downregulated by mechanical stretch on vascular cells. Arterial expression and activity of CD39 were decreased in hypertension as a result of both a proinflammatory environment and mechanical strain exerted on vascular cells. Reduced ectonucleotidase activity may alter the vascular condition, thus enhancing arterial damage, remodeling, or thrombotic events.

New insights into mitral valve dystrophy: a Filamin-A genotype-phenotype and outcome study.

Aims: Filamin-A (FLNA) was identified as the first gene of non-syndromic mitral valve dystrophy (FLNA-MVD). We aimed to assess the phenotype of FLNA-MVD and its impact on prognosis. Methods and results: We investigated the disease in 246 subjects (72 mutated) from four FLNA-MVD families harbouring three different FLNA mutations. Phenotype was characterized by a comprehensive echocardiography focusing on mitral valve apparatus in comparison with control relatives. In this X-linked disease valves lesions were severe in men and moderate in women. Most men had classical features of mitral valve prolapse (MVP), but without chordal rupture. By contrast to regular MVP, mitral leaflet motion was clearly restricted in diastole and papillary muscles position was closer to mitral annulus. Valvular abnormalities were similar in the four families, in adults and young patients from early childhood suggestive of a developmental disease. In addition, mitral valve lesions worsened over time as encountered in degenerative conditions. Polyvalvular involvement was frequent in males and non-diagnostic forms frequent in females. Overall survival was moderately impaired in men (P = 0.011). Cardiac surgery rate (mainly valvular) was increased (33.3 ± 9.8 vs. 5.0 ± 4.9%, P < 0.0001; hazard ratio 10.5 [95% confidence interval: 2.9-37.9]) owing mainly to a lifetime increased risk in men (76.8 ± 14.1 vs. 9.1 ± 8.7%, P < 0.0001). Conclusion: FLNA-MVD is a developmental and degenerative disease with complex phenotypic expression which can influence patient management. FLNA-MVD has unique features with both MVP and paradoxical restricted motion in diastole, sub-valvular mitral apparatus impairment and polyvalvular lesions in males. FLNA-MVD conveys a substantial lifetime risk of valve surgery in men.

Generation of gene-edited rats by delivery of CRISPR/Cas9 protein and donor DNA into intact zygotes using electroporation.

The generation of gene-edited animals using the CRISPRs/Cas9 system is based on microinjection into zygotes which is inefficient, time consuming and demands high technical skills. We report the optimization of an electroporation method for intact rat zygotes using sgRNAs and Cas9 protein in combination or not with ssODNs (~100 nt). This resulted in high frequency of knockouts, between 15 and 50% of analyzed animals. Importantly, using ssODNs as donor template resulted in precise knock-in mutations in 25-100% of analyzed animals, comparable to microinjection. Electroporation of long ssDNA or dsDNA donors successfully used in microinjection in the past did not allow generation of genome-edited animals despite dsDNA visualization within zygotes. Thus, simultaneous electroporation of a large number of intact rat zygotes is a rapid, simple, and efficient method for the generation of a variety of genome-edited rats.

The alternatively spliced LRRFIP1 Isoform-1 is a key regulator of the Wnt/ß-catenin transcription pathway.

The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the ß-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.

MVP-Associated Filamin A Mutations Affect FlnA-PTPN12 (PTP-PEST) Interactions.

Although the genetic basis of mitral valve prolapse (MVP) has now been clearly established, the molecular and cellular mechanisms involved in the pathological processes associated to a specific mutation often remain to be determined. The FLNA gene (encoding Filamin A; FlnA) was the first gene associated to non-syndromic X-linked myxomatous valvular dystrophy, but the impacts of the mutations on its function remain un-elucidated. Here, using the first repeats (1-8) of FlnA as a bait in a yeast two-hybrid screen, we identified the tyrosine phosphatase PTPN12 (PTP-PEST) as a specific binding partner of this region of FlnA protein. In addition, using yeast two-hybrid trap assay pull down and co-immunoprecipitation experiments, we showed that the MVP-associated FlnA mutations (G288R, P637Q, H743P) abolished FlnA/PTPN12 interactions. PTPN12 is a key regulator of signaling pathways involved in cell-extracellular matrix (ECM) crosstalk, cellular responses to mechanical stress that involve integrins, focal adhesion transduction pathways, and actin cytoskeleton dynamics. Interestingly, we showed that the FlnA mutations impair the activation status of two PTPN12 substrates, the focal adhesion associated kinase Src, and the RhoA specific activating protein p190RhoGAP. Together, these data point to PTPN12/FlnA interaction and its weakening by FlnA mutations as a mechanism potentially involved in the physiopathology of FlnA-associated MVP.

Mitral valve disease--morphology and mechanisms.

Mitral valve disease is a frequent cause of heart failure and death. Emerging evidence indicates that the mitral valve is not a passive structure, but--even in adult life--remains dynamic and accessible for treatment. This concept motivates efforts to reduce the clinical progression of mitral valve disease through early detection and modification of underlying mechanisms. Discoveries of genetic mutations causing mitral valve elongation and prolapse have revealed that growth factor signalling and cell migration pathways are regulated by structural molecules in ways that can be modified to limit progression from developmental defects to valve degeneration with clinical complications. Mitral valve enlargement can determine left ventricular outflow tract obstruction in hypertrophic cardiomyopathy, and might be stimulated by potentially modifiable biological valvular-ventricular interactions. Mitral valve plasticity also allows adaptive growth in response to ventricular remodelling. However, adverse cellular and mechanobiological processes create relative leaflet deficiency in the ischaemic setting, leading to mitral regurgitation with increased heart failure and mortality. Our approach, which bridges clinicians and basic scientists, enables the correlation of observed disease with cellular and molecular mechanisms, leading to the discovery of new opportunities for improving the natural history of mitral valve disease.

A long QT mutation substitutes cholesterol for phosphatidylinositol-4,5-bisphosphate in KCNQ1 channel regulation.

INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. METHODS AND RESULTS: COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model. CONCLUSIONS: We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.

Selective involvement of serum response factor in pressure-induced myogenic tone in resistance arteries.

OBJECTIVE: In resistance arteries, diameter adjustment in response to pressure changes depends on the vascular cytoskeleton integrity. Serum response factor (SRF) is a dispensable transcription factor for cellular growth, but its role remains unknown in resistance arteries. We hypothesized that SRF is required for appropriate microvascular contraction. METHODS AND RESULTS: We used mice in which SRF was specifically deleted in smooth muscle or endothelial cells, and their control. Myogenic tone and pharmacological contraction was determined in resistance arteries. mRNA and protein expression were assessed by quantitative real-time PCR (qRT-PCR) and Western blot. Actin polymerization was determined by confocal microscopy. Stress-activated channel activity was measured by patch clamp. Myogenic tone developing in response to pressure was dramatically decreased by SRF deletion (5.9±2.3%) compared with control (16.3±3.2%). This defect was accompanied by decreases in actin polymerization, filamin A, myosin light chain kinase and myosin light chain expression level, and stress-activated channel activity and sensitivity in response to pressure. Contractions induced by phenylephrine or U46619 were not modified, despite a higher sensitivity to p38 blockade; this highlights a compensatory pathway, allowing normal receptor-dependent contraction. CONCLUSIONS: This study shows for the first time that SRF has a major part to play in the control of local blood flow via its central role in pressure-induced myogenic tone in resistance arteries.

Mass spectrometry-based identification of native cardiac Nav1.5 channel α subunit phosphorylation sites.

Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified in situ phosphorylation sites. Taken together, the results presented provide the first in situ map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit.

Multifocal ectopic Purkinje-related premature contractions: a new SCN5A-related cardiac channelopathy.

OBJECTIVES: The aim of this study was to describe a new familial cardiac phenotype and to elucidate the electrophysiological mechanism responsible for the disease. BACKGROUND: Mutations in several genes encoding ion channels, especially SCN5A, have emerged as the basis for a variety of inherited cardiac arrhythmias. METHODS: Three unrelated families comprising 21 individuals affected by multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by narrow junctional and rare sinus beats competing with numerous premature ventricular contractions with right and/or left bundle branch block patterns were identified. RESULTS: Dilated cardiomyopathy was identified in 6 patients, atrial arrhythmias were detected in 9 patients, and sudden death was reported in 5 individuals. Invasive electrophysiological studies demonstrated that premature ventricular complexes originated from the Purkinje tissue. Hydroquinidine treatment dramatically decreased the number of premature ventricular complexes. It normalized the contractile function in 2 patients. All the affected subjects carried the c.665G>A transition in the SCN5A gene. Patch-clamp studies of resulting p.Arg222Gln (R222Q) Nav1.5 revealed a net gain of function of the sodium channel, leading, in silico, to incomplete repolarization in Purkinje cells responsible for premature ventricular action potentials. In vitro and in silico studies recapitulated the normalization of the ventricular action potentials in the presence of quinidine. CONCLUSIONS: A new SCN5A-related cardiac syndrome, MEPPC, was identified. The SCN5A mutation leads to a gain of function of the sodium channel responsible for hyperexcitability of the fascicular-Purkinje system. The MEPPC syndrome is responsive to hydroquinidine.

Developmental basis for filamin-A-associated myxomatous mitral valve disease.

AIMS: We hypothesized that the structure and function of the mature valves is largely dependent upon how these tissues are built during development, and defects in how the valves are built can lead to the pathological progression of a disease phenotype. Thus, we sought to uncover potential developmental origins and mechanistic underpinnings causal to myxomatous mitral valve disease. We focus on how filamin-A, a cytoskeletal binding protein with strong links to human myxomatous valve disease, can function as a regulatory interface to control proper mitral valve development. METHODS AND RESULTS: Filamin-A-deficient mice exhibit abnormally enlarged mitral valves during foetal life, which progresses to a myxomatous phenotype by 2 months of age. Through expression studies, in silico modelling, 3D morphometry, biochemical studies, and 3D matrix assays, we demonstrate that the inception of the valve disease occurs during foetal life and can be attributed, in part, to a deficiency of interstitial cells to efficiently organize the extracellular matrix (ECM). This ECM organization during foetal valve gestation is due, in part, to molecular interactions between filamin-A, serotonin, and the cross-linking enzyme, transglutaminase-2 (TG2). Pharmacological and genetic perturbations that inhibit serotonin-TG2-filamin-A interactions lead to impaired ECM remodelling and engender progression to a myxomatous valve phenotype. CONCLUSIONS: These findings illustrate a molecular mechanism by which valve interstitial cells, through a serotonin, TG, and filamin-A pathway, regulate matrix organization during foetal valve development. Additionally, these data indicate that disrupting key regulatory interactions during valve development can set the stage for the generation of postnatal myxomatous valve disease.