Don't Break the Axis: Placental Inflammation Leads to Congenital Heart Disease.

This study was supported by grants PID2019-104776RB-I00 and CB16/11/00399 (CIBER CV [Centro de Investigación Biomedica en Red]) financed by MCIN/AEI/10.13039/501100011033 (Ministerio de Ciencia e Innovación/La Agencia Estatal de Investigación). The Centro Nacional de Investigaciones Cardiovasculares is supported by the Instituto de Salud Carlos III, the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020001041-S), financed by MCIN/ AEI/10.13039/501100011033.S

Cooperative Response to Endocardial Notch Reveals Interaction With Hippo Pathway.

BACKGROUND The endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve formation and calcific aortic valve disease, but knowledge is very limited about the role played in valve development and disease by noncoding endocardial regulatory regions and upstream factors. METHODS We manipulated Notch signaling in mouse embryonic endocardial cells by short-term and long-term coculture with OP9 stromal cells expressing Notch ligands and inhibition of Notch activity. We examined the transcriptional profile and chromatin accessibility landscape for each condition, integrated transcriptomic, transcription factor occupancy, chromatin accessibility, and proteomic datasets. We generated in vitro and in vivo models with CRISPR-Cas9-edited deletions of various noncoding regulatory elements and validated their regulatory potential. RESULTS We identified primary and secondary transcriptional responses to Notch ligands in the mouse embryonic endocardium, and a NOTCH-dependent transcriptional signature in valve development and disease. By defining the changes in the chromatin accessibility landscape and integrating with the landscape in developing mouse endocardium and adult human valves, we identify potential noncoding regulatory elements, validated selected candidates, propose interacting cofactors, and define the timeframe of their regulatory activity. Additionally, we found cooperative transcriptional repression with Hippo pathway by inhibiting nuclear Yap (Yes-associated protein) activity in the endocardium during cardiac valve development. CONCLUSIONS Sequential Notch-dependent transcriptional regulation in the embryonic endocardium involves multiple factors. Notch activates certain noncoding elements through these factors and simultaneously suppresses elements that could hinder cardiac valve development and homeostasis. Biorxviv: https://www.biorxiv.org/content/10.1101/2023.03.23.533882v1.full.S

Mesenchymal Stem Cell Migration and Proliferation Are Mediated by Hypoxia-Inducible Factor-1 alpha Upstream of Notch and SUMO Pathways

Mesenchymal stem cells (MSCs) are effective in treating several pathologies. We and others have demonstrated that hypoxia or hypoxia-inducible factor 1 alpha (HIF-1 alpha) stabilization improves several MSC functions, including cell adhesion, migration, and proliferation, thereby increasing their therapeutic potential. To further explore the mechanisms induced by HIF-1 alpha in MSCs, we studied its relationship with Notch signaling and observed that overexpression of HIF-1 alpha in MSCs increased protein levels of the Notch ligands Jagged 1-2 and Delta-like (Dll) 1, Dll3, and Dll4 and potentiated Notch signaling only when this pathway was activated. Crosstalk between HIF and Notch resulted in Notch-dependent migration and spreading of MSCs, which was abolished by gamma-secretase inhibition. However, the HIF-1-induced increase in MSC proliferation was independent of Notch signaling. The ubiquitin family member, small ubiquitin-like modifier (SUMO), has important functions in many cellular processes and increased SUMO1 protein levels have been reported in hypoxia. To investigate the potential involvement of SUMOylation in HIF/Notch crosstalk, we measured general SUMOylation levels and observed increased SUMOylation in HIF-1-expressing MSCs. Moreover, proliferation and migration of MSCs were reduced in the presence of a SUMOylation inhibitor, and this effect was particularly robust in HIF-MSCs. Immunoprecipitation studies demonstrated SUMOylation of the intracellular domain of Notch1 (N1ICD) in HIF-1-expressing MSCs, which contributed to Notch pathway activation and resulted in increased levels of N1ICD nuclear translocation as assessed by subcellular fractionation. SUMOylation of N1ICD was also observed in HEK293T cells with stabilized HIF-1 alpha expression, suggesting that this is a common mechanism in eukaryotic cells. In summary, we describe, for the first time, SUMOylation of N1ICD, which is potentiated by HIF signaling. These phenomena could be relevant for the therapeutic effects of MSCs in hypoxia or under conditions of HIF stabilization.This work was supported, in part, by grants from the Instituto de Salud Carlos III PI13/00414, PI16/0107, RE-TICS RD12/0019/0025 to P.S. and RETICS RD12/0019/0003 (TERCEL) to J.L.D.L.P cofunded by FEDER ``una manera de hacer Europa. It was also supported by the Regenerative Medicine Program of Instituto de Salud Carlos III and Valencian Community to Centro de Investigacion Principe Felipe. The authors are grateful to Dr. A. Dorronsoro for critical review of the work and Dr. K McCreath for manuscript editing.S

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

Morphogenesis of myocardial trabeculae in the mouse embryo

Formation of trabeculae in the embryonic heart and the remodelling that occurs prior to birth is a conspicuous, but poorly understood, feature of vertebrate cardiogenesis. Mutations disrupting trabecular development in the mouse are frequently embryonic lethal, testifying to the importance of the trabeculae, and aberrant trabecular structure is associated with several human cardiac pathologies. Here, trabecular architecture in the developing mouse embryo has been analysed using high-resolution episcopic microscopy (HREM) and three-dimensional (3D) modelling. This study shows that at all stages from mid-gestation to birth, the ventricular trabeculae comprise a complex meshwork of myocardial strands. Such an arrangement defies conventional methods of measurement, and an approach based upon fractal algorithms has been used to provide an objective measure of trabecular complexity. The extent of trabeculation as it changes along the length of left and right ventricles has been quantified, and the changes that occur from formation of the four-chambered heart until shortly before birth have been mapped. This approach not only measures qualitative features evident from visual inspection of 3D models, but also detects subtle, consistent and regionally localised differences that distinguish each ventricle and its developmental stage. Finally, the combination of HREM imaging and fractal analysis has been applied to analyse changes in embryonic heart structure in a genetic mouse model in which trabeculation is deranged. It is shown that myocardial deletion of the Notch pathway component Mib1 (Mib1(flox/flox); cTnT-cre) results in a complex array of abnormalities affecting trabeculae and other parts of the heart.This work was supported by funding to TJM from the Medical Research Council (U117562103); to JCM by the Higher Education Funding Council for England; to JLdlP and GL by grants SAF2010-17555 and SAF2013-45543-R from the Spanish Ministry of Economy and Competitiveness (MINECO); and to GC by a research fellowship at the University College London Biomedical Research Centre from the UK National Institutes of Health Research Cardiometabolic Programme.S

A novel source of arterial valve cell linked to bicuspid aortic valve without rephe in mice

Abnormalities of the arterial valve leaflets, predominantly bicuspid aortic valve, are the commonest congenital malformations. Although many studies have investigated the development of the arterial valves, it has been assumed that, as with the atrioventricular valves, endocardial to mesenchymal transition (EndMT) is the predominant mechanism. We show that arterial is distinctly different from atrioventricular valve formation. Whilst the four septal valve leaflets are dominated by NCC and EndMT-derived cells, the intercalated leaflets differentiate directly from Tnnt2-Cre+/Isl1+ progenitors in the outflow wall, via a Notch-Jag dependent mechanism. Further, when this novel group of progenitors are disrupted, development of the intercalated leaflets is disrupted, resulting in leaflet dysplasia and bicuspid valves without raphe, most commonly affecting the aortic valve. This study thus overturns the dogma that heart valves are formed principally by EndMT, identifies a new source of valve interstitial cells, and provides a novel mechanism for causation of bicuspid aortic valves without raphe.British Heart Foundation RG/12/15/29935 Lorriane Eley Rachel V Richardson Lindsay Murphy Bill Chaudhry Deborah J Henderson; British Heart Foundation PG/15/46/31589 Lorriane Eley Bill Chaudhry Deborah J Henderson; Ministerio de Ciencia, Innovacion y Universidades of Spain CB16/11/00399 (Ciber Cardiovascular) Donal MacGrogan Alejandro Salguero-Jimenez Jose Luis de La Pompa; Ministerio de Ciencia, Innovacion y Universidades of Spain SAF2016-78370-R Donal MacGrogan Alejandro Salguero-Jimenez Jose Luis de La Pompa; Ministerio de Ciencia, Innovacion y Universidades of Spain RD16/0011/0021 (Red de Terapia Celular, TERCEL) Donal MacGrogan Alejandro Salguero-Jimenez Jose Luis de La Pompa; The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.S

iPSC-Based Modeling of Variable Clinical Presentation in Hypertrophic Cardiomyopathy.

BACKGROUND Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and a frequent cause of heart failure and sudden cardiac death. Our understanding of the genetic bases and pathogenic mechanisms underlying HCM has improved significantly in the recent past, but the combined effect of various pathogenic gene variants and the influence of genetic modifiers in disease manifestation are very poorly understood. Here, we set out to investigate genotype-phenotype relationships in 2 siblings with an extensive family history of HCM, both carrying a pathogenic truncating variant in the MYBPC3 gene (p.Lys600Asnfs*2), but who exhibited highly divergent clinical manifestations. METHODS We used a combination of induced pluripotent stem cell (iPSC)-based disease modeling and CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing to generate patient-specific cardiomyocytes (iPSC-CMs) and isogenic controls lacking the pathogenic MYBPC3 variant. RESULTS Mutant iPSC-CMs developed impaired mitochondrial bioenergetics, which was dependent on the presence of the mutation. Moreover, we could detect altered excitation-contraction coupling in iPSC-CMs from the severely affected individual. The pathogenic MYBPC3 variant was found to be necessary, but not sufficient, to induce iPSC-CM hyperexcitability, suggesting the presence of additional genetic modifiers. Whole-exome sequencing of the mutant carriers identified a variant of unknown significance in the MYH7 gene (p.Ile1927Phe) uniquely present in the individual with severe HCM. We finally assessed the pathogenicity of this variant of unknown significance by functionally evaluating iPSC-CMs after editing the variant. CONCLUSIONS Our results indicate that the p.Ile1927Phe variant of unknown significance in MYH7 can be considered as a modifier of HCM expressivity when found in combination with truncating variants in MYBPC3. Overall, our studies show that iPSC-based modeling of clinically discordant subjects provides a unique platform to functionally assess the effect of genetic modifiers.The funding for this research was provided by the Spanish Ministry of Science and Innovation-MCIN (grants PID2021-123925OB-I00, PID2019-104776RB-I00, CB06/01/1056, and CB16/11/00399 financed by MCIN/AEI/10.13039/501100011033), AGAUR (2021-SGR-974), Fundació La Marató de TV3 (201534-30), Fundación BBVA (BIO14_298), Fundació Obra Social la Caixa, and CERCA Program/ Generalitat de Catalunya. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MCIN, and the Pro CNIC Foundation. I. Lazis was partially supported by a predoctoral fellowship from MCIN (PRE2019-087901).S

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.We thank the Leducq Fondation for supporting Tui Neri, and funding this research under the framework of the MITRAL network and for generously awarding us for the equipment of our cell imaging facility in the frame of their program “Equipement de Recherche et Plateformes Technologiques” (ERPT to M.P.), the Genopole at Evry and the Fondation de la recherche Medicale (grant DEQ20100318280) for supporting the laboratory of Michel Puceat. Part of this work in South Carolina University was conducted in a facility constructed with support from the National Institutes of Health, Grant Number C06 RR018823 from the Extramural Research Facilities Program of the National Center for Research Resources. Other funding sources: National Heart Lung and Blood Institute: RO1-HL33756 (R.R.M.), COBRE P20RR016434–07 (R.R.M., R.A. N.), P20RR016434–09S1 (R.R.M. and R.A.N.); American Heart Association: 11SDG5270006 (R.A.N.); National Science Foundation: EPS-0902795 (R.R.M. and R.A. N.); American Heart Association: 10SDG2630130 (A.C.Z.), NIH: P01HD032573 (A.C. Z.), NIH: U54 HL108460 (A.C.Z), NCATS: UL1TR000100 (A.C.Z.); EH was supported by a fellowship of the Ministere de la recherche et de l’éducation in France.TM-M was supported by a fellowship from the Fondation Foulon Delalande and the Leducq Foundation. P.v.V. was sponsored by a UC San Diego Cardiovascular Scholarship Award and a Postdoctoral Fellowship from the California Institute for Regenerative Medicine (CIRM) Interdisciplinary Stem Cell Training Program II. S.M.E. was funded by a grant from the National Heart, Lung, and Blood Institute (HL-117649). A.T. is supported by the National Heart, Lung, and Blood Institute (R01-HL134664).S

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Notch and interacting signalling pathways in cardiac development, disease, and regeneration.

Cardiogenesis is a complex developmental process involving multiple overlapping stages of cell fate specification, proliferation, differentiation, and morphogenesis. Precise spatiotemporal coordination between the different cardiogenic processes is ensured by intercellular signalling crosstalk and tissue-tissue interactions. Notch is an intercellular signalling pathway crucial for cell fate decisions during multicellular organismal development and is aptly positioned to coordinate the complex signalling crosstalk required for progressive cell lineage restriction during cardiogenesis. In this Review, we describe the role of Notch signalling and the crosstalk with other signalling pathways during the differentiation and patterning of the different cardiac tissues and in cardiac valve and ventricular chamber development. We examine how perturbation of Notch signalling activity is linked to congenital heart diseases affecting the neonate and adult, and discuss studies that shed light on the role of Notch signalling in heart regeneration and repair after injury.The authors thank present and past members of the laboratory for their contribution to this Review and apologize for the omission of studies not discussed or cited because of space limitations. J.L.d.l.P. is funded by grants SAF2016-78370-R, CB16/11/00399 (CIBER CV), and RD16/0011/0021 (TERCEL) from the Ministerio de Ciencia, Innovacion y Universidades and grants from the Fundacion BBVA (Ref. BIO14_298) and Fundacion La Marato (Ref. 20153431). J.L.d.l.P.'s work was supported in part with funds from the ERDF. CNIC is supported by the Ministerio de Ciencia, Innovacion y Universidades and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S