NOTCH Activation Promotes Valve Formation by Regulating the Endocardial Secretome.

The endocardium is a specialized endothelium that lines the inner surface of the heart. Functional studies in mice and zebrafish have established that the endocardium is a source of instructive signals for the development of cardiac structures, including the heart valves and chambers. Here, we characterized the NOTCH-dependent endocardial secretome by manipulating NOTCH activity in mouse embryonic endocardial cells (MEEC) followed by mass spectrometry-based proteomics. We profiled different sets of soluble factors whose secretion not only responds to NOTCH activation but also shows differential ligand specificity, suggesting that ligand-specific inputs may regulate the expression of secreted proteins involved in different cardiac development processes. NOTCH signaling activation correlates with a transforming growth factor-β2 (TGFβ2)-rich secretome and the delivery of paracrine signals involved in focal adhesion and extracellular matrix (ECM) deposition and remodeling. In contrast, NOTCH inhibition is accompanied by the up-regulation of specific semaphorins that may modulate cell migration. The secretome protein expression data showed a good correlation with gene profiling of RNA expression in embryonic endocardial cells. Additional characterization by in situ hybridization in mouse embryos revealed expression of various NOTCH candidate effector genes (Tgfβ2, Loxl2, Ptx3, Timp3, Fbln2, and Dcn) in heart valve endocardium and/or mesenchyme. Validating these results, mice with conditional Dll4 or Jag1 loss-of-function mutations showed gene expression alterations similar to those observed at the protein level in vitro These results provide the first description of the NOTCH-dependent endocardial secretome and validate MEEC as a tool for assaying the endocardial secretome response to a variety of stimuli and the potential use of this system for drug screening.We thank C. Martí Gómez-Aldaraví for help with graphic representation and critical reading of the manuscript, and S. Bartlett for English editing. RTC is supported by a Foundation La Caixa PhD fellowship (Ref LCF/BQ/ES15/10360023). LLZ is supported by a Ramón y Cajal postdoctoral contract (Ref: RYC-2016-20917). JLdlP is funded by grants SAF2016-78370-R, CB16/11/00399 (CIBER CV), and RD16/0011/0021 (TERCEL) from the Ministerio de Ciencia, Innovación y Universidades, and grants from the Fundación BBVA (Ref.: BIO14_298) and Fundación La Marató TV3 (Ref.: 20153431). JV is supported by grants BIO2015-67580-P and CB16/11/00277 (CIBER CV) from the Ministerio de Ciencia, Innovación y Universidades, and Carlos III Institute of Health-Fondo de Investigación Sanitaria (Grant ProteoRed-PRB3-IPT17/0019-ISCIII-SGEFI/ERDF), the Fundación La Marató TV3 (Ref. 122/C/2015) and “La Caixa” Banking Foundation (project code HR17-00247). The cost of this publication was supported in part with funds from the ERDF. The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve.

The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.This study was supported by grants PID2019-104776RB-I00 and PID2020-120326RB-I00, CB16/11/00399 (CIBER CV) financed by MCIN/AEI/10.13039/501100011033, a grant from the Fundación BBVA (Ref. BIO14_298), and a grant from Fundació La Marató de TV3 (Ref. 20153431) to J.L.d.l.P. M.S.-A. was supported by a PhD contract from the Severo Ochoa Predoctor-al Program (SVP-2014-068723) of the MCIN/AEI/10.13039/501100011033. J.R.G.-B. was supported by SEC/FEC-INV-BAS 21/021. A.R. was funded by grants from MCIN (PID2021123925OB-I00), TerCel (RD16/0011/0024), AGAUR (2017-SGR-899), and Fundació La Marató de TV3 (201534-30). J.M.P.-P. was supported by RTI2018-095410-B-I00 (MCIN) and PY2000443 (Junta de Andalucía). B.I. was supported by the European Commission (H2020-HEALTH grant No. 945118) and by MCIN (PID2019-107332RB-I00). DO’R was sup-ported by the Medical Research Council (MC-A658-5QEB0) and KAMcG by the British Heart Foundation (RG/19/6/34387, RE/18/4/34215). The cost of this publication was supported in part with funds from the European Regional Devel-opment Fund. The Centro Nacional de Investigaciones Cardiovasculares is sup-ported by the ISCIII, the MCIN, and the Pro Centro Nacional de Investigaciones Cardiovasculares Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020001041-S) financed by MCIN/AEI/10.13039/501100011033. For the purpose of open access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising.S

Novel Association of the NOTCH Pathway Regulator MIB1 Gene With the Development of Bicuspid Aortic Valve.

IMPORTANCE Nonsyndromic bicuspid aortic valve (nsBAV) is the most common congenital heart valve malformation. BAV has a heritable component, yet only a few causative genes have been identified; understanding BAV genetics is a key point in developing personalized medicine. OBJECTIVE To identify a new gene for nsBAV. DESIGN, SETTING, AND PARTICIPANTS This was a comprehensive, multicenter, genetic association study based on candidate gene prioritization in a familial cohort followed by rare and common association studies in replication cohorts. Further validation was done using in vivo mice models. Study data were analyzed from October 2019 to October 2022. Three cohorts of patients with BAV were included in the study: (1) the discovery cohort was a large cohort of inherited cases from 29 pedigrees of French and Israeli origin; (2) the replication cohort 1 for rare variants included unrelated sporadic cases from various European ancestries; and (3) replication cohort 2 was a second validation cohort for common variants in unrelated sporadic cases from Europe and the US. MAIN OUTCOMES AND MEASURES To identify a candidate gene for nsBAV through analysis of familial cases exome sequencing and gene prioritization tools. Replication cohort 1 was searched for rare and predicted deleterious variants and genetic association. Replication cohort 2 was used to investigate the association of common variants with BAV. RESULTS A total of 938 patients with BAV were included in this study: 69 (7.4%) in the discovery cohort, 417 (44.5%) in replication cohort 1, and 452 (48.2%) in replication cohort 2. A novel human nsBAV gene, MINDBOMB1 homologue MIB1, was identified. MINDBOMB1 homologue (MIB1) is an E3-ubiquitin ligase essential for NOTCH-signal activation during heart development. In approximately 2% of nsBAV index cases from the discovery and replication 1 cohorts, rare MIB1 variants were detected, predicted to be damaging, and were significantly enriched compared with population-based controls (2% cases vs 0.9% controls; P = .03). In replication cohort 2, MIB1 risk haplotypes significantly associated with nsBAV were identified (permutation test, 1000 repeats; P = .02). Two genetically modified mice models carrying Mib1 variants identified in our cohort showed BAV on a NOTCH1-sensitized genetic background. CONCLUSIONS AND RELEVANCE This genetic association study identified the MIB1 gene as associated with nsBAV. This underscores the crucial role of the NOTCH pathway in the pathophysiology of BAV and its potential as a target for future diagnostic and therapeutic intervention.This study was supported in part by grants PID2019-104776RB-I00 and CB16/ 11/00399 (Dr de la Pompa) from the Spanish Ministerio de Ciencia e Innovación (MCIN/ AEI/ 10.13039/501100011033/); a grant from Hadassah France Association (Drs Gilon and Tessler); a grant from the Center for Interdisciplinary Data Science Research of the Hebrew University of Jerusalem (Dr Tessler); grant R35 CA220340 from the National Institutes of Health (Dr Blacklow), and grants R21HL150373, R01HL114823 (Dr Body); BSF grants 2013269 and 2017245 (Drs. Sprinzak and Blacklow); a consolidator grant from the European Research Council (Genomia – ERC-COG-2017-771945; Dr Loeys); the European Reference Network on rare multisystemic vascular disorders (VASCERN - project ID: 769036 partly cofunded by the European Union Third Health Programme (Drs Loeys and Verstraeten); funding from the Outreach project (Dutch Heart Foundation; Dr Luyckx); funding from Heart and Stroke Foundation of Canada/Robert M Freedom Chair of Cardiovascular Science (Dr Mital); sample biobanking and sequencing from Canada were supported by grants from the Leducq Foundation Transatlantic Networks of Excellence grant, and the Ted Rogers Centre for Heart Research; ISF grant 1053/12 (Dr Durst); and grant R01HL150401 from National Heart, Lung, and Blood Institute (Dr Muehlschlegel).S

Estudio del papel de Gata6 en el desarrollo valvular y enfermedad y su potencial interacción con la vía de señalización de NOTCH

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 13-04-2023Esta tesis tiene embargado el acceso al texto completo hasta el 13-10-2024Bicuspid aortic valve (BAV) is the most common congenital heart defect, affecting up to 0.5 to 2% of the population. It consists in the presence of two asymmetrical leaflets instead of three symmetrical ones. This malformation is more prevalent in males than in females, shows dominant inheritance, and incomplete penetrance. Individuals with BAV are more at risk of having valve stenosis, and calcific aortic valve disease (CAVD) and usually require valve replacement. Previous genome wide association studies have shown an association with chromosome region 18q with BAV. This region harbors the Gata6 and Mib1 genes. More recent studies have shown that Gata6 loss of function mutants display RL-type BAV, the most common morphology found in humans. NOTCH1 mutations have been associated with familial BAV and studies from our laboratory have shown that conditional deletion of Mib1 or Jag1 in endocardium and myocardium results in dysmorphic aortic valves and BAV. So, we wanted to elucidate the role of Gata6 in the aortic valve development and disease. Moreover, we studied the relationship between Gata6 and Mib1/NOTCH in this pathology. First, we generated Gata6STOP/+ mouse line by CRISPR-Cas9 technology, with 70% BAV and 43% ventricular septal defect (VSD). Defective outflow tract (OFT) development is one of the most important causes for BAV, therefore we characterized Gata6STOP/+ OFT and they show a defect on OFT development, displaying a smaller caliber and shorter length at E12.5. Since OFT is originated from second heart field (SHF), we observed a SHF differentiation defect and a proliferation defect at E9.5, suggesting those as a potential causes for the smaller size of the OFT. We also studied cardiac neural crest cells (cNCCs), necessary for the correct septation of the OFT, and its contribution to the OFT is reduced, leading to OFT septation defects. To understand which cell type is responsible for the BAV phenotype, we obtained a previously generated conditional Gata6flox allele and inactivated this gene in the endocardium and endothelium using theTie2-Cre driver, in the myocardium and endocardium with the Nkx2.5-Cre driver, and in the second heart field (SHF) with the Mef2c-AHF-Cre driver. The Gata6flox; Mef2cAHF-Cre line recapitulates the BAV and VSD phenotypes of the Gata6 standard knockout, suggesting the important role of Gata6 in SHF at early stages of OFT development. Moreover, to elucidate the molecular mechanisms underlying the BAV phenotype, we performed bulk RNA-Seq of the OFT at E11.5. We found that an altered transcriptional landscape in Gata6STOP/+ mutant involving cellular migratory processes. We focused on Ackr3, which encodes the CXCR7 receptor. Cxcr7-/- mice recapitulate Gata6STOP/+ phenotype of BAV and VSD. Ex vivo assays with explanted E11.5 Gata6STOP/+ OFT tissue demonstrated that cellular migration and invasion were decreased relative to controls. Supplementing WT OFT explants with CXCR7 agonists or with the CXCL12 ligand decreases cell migration. Taken together the data suggest that CXCR7 could mediate Gata6 function in OFT morphogenesis. We also generated double heterozygous mice with Mib1 missense and non-sense mutations in a Gata6 haploinssuficient background, and examined their BAV phenotype. To study if NOTCH pathway interacts with Gata6, we generated double heterozygous Gata6STOP/+; Notch1KO/+ mice and we observed BAV phenotype in Gata6 mutants is NOTCH-independent, besides, VSD penetrance was increased, suggesting a common regulation between Gata6 and NOTCH in ventricular septal formation. This doctoral thesis contributes to increase our mechanistic understanding of BAV formation and may contribute to the design of therapies to prevent this pathologyEste proyecto ha sido realizado en el laboratorio de Señalización intercelular durante el desarrollo y la enfermedad cardiovascular, liderado por el Dr. José Luis de la Pompa Mínguez en el Centro Nacional de Investigaciones Cardiovasculares (CNIC) en Madrid. Para su realización, Rebeca Piñeiro Sabarís percibió una Ayuda del programa de Formación de Personal Investigador, concedida en resolución del Ministerio de Ciencia e Innovación el día 1 de junio de 2018 y con referencia BES-2017-079720. La estancia predoctoral en el laboratorio del Prof. Jonathan Butcher en el Departamento de Ingeniería Biomñedica en Cornell University (Ithaca, Nueva York, EEUU) fue financiada por una beca short-term de la European Molecular Biology Organization (EMBO) (1/9/2022 – 1/12/2022). La financiación del proyecto provino de los siguientes proyectos adjudicados al Dr. José Luis de la Pompa: SAF2016-79502-R, PID2019-104776RB-I00 y CB16/11/00399 por parte del Ministerio de Ciencia e Innovación (MCIN), la Agencia Estatal de Investigación (AEI) 10.13039/501100011033 y el Instituto de Salud Carlos II

A Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve

Background:The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. Methods:We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. Results:Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. Conclusions:These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers