Induced pluripotent stem cell modelling of HLHS underlines the contribution of dysfunctional NOTCH signalling to impaired cardiogenesis

Abstract Hypoplastic left heart syndrome (HLHS) is among the most severe forms of congenital heart disease. Although the consensus view is that reduced flow through the left heart during development is a key factor in the development of the condition, the molecular mechanisms leading to hypoplasia of left heart structures are unknown. We have generated induced pluripotent stem cells (iPSC) from five HLHS patients and two unaffected controls, differentiated these to cardiomyocytes and identified reproducible in vitro cellular and functional correlates of the HLHS phenotype. Our data indicate that HLHS-iPSC have a reduced ability to give rise to mesodermal, cardiac progenitors and mature cardiomyocytes and an enhanced ability to differentiate to smooth muscle cells. HLHS-iPSC-derived cardiomyocytes are characterised by a lower beating rate, disorganised sarcomeres and sarcoplasmic reticulum and a blunted response to isoprenaline. Whole exome sequencing of HLHS fibroblasts identified deleterious variants in NOTCH receptors and other genes involved in the NOTCH signalling pathway. Our data indicate that the expression of NOTCH receptors was significantly downregulated in HLHS-iPSC-derived cardiomyocytes alongside NOTCH target genes confirming downregulation of NOTCH signalling activity. Activation of NOTCH signalling via addition of Jagged peptide ligand during the differentiation of HLHS-iPSC restored their cardiomyocyte differentiation capacity and beating rate and suppressed the smooth muscle cell formation. Together, our data provide firm evidence for involvement of NOTCH signalling in HLHS pathogenesis, reveal novel genetic insights important for HLHS pathology and shed new insights into the role of this pathway during human cardiac development.

[1]  E. Sernagor,et al.  An Induced Pluripotent Stem Cell Model of Hypoplastic Left Heart Syndrome (HLHS) Reveals Multiple Expression and Functional Differences in HLHS‐Derived Cardiac Myocytes , 2014, Stem cells translational medicine.

[2]  M. Mattson,et al.  Gamma secretase–mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke , 2006, Nature Medicine.

[3]  Yanmei Zhang,et al.  Conserved signal peptide of Notch3 inhibits interaction with proteasome. , 2007, Biochemical and biophysical research communications.

[4]  Vincent C. Chen,et al.  Notch signaling respecifies the hemangioblast to a cardiac fate , 2008, Nature Biotechnology.

[5]  E. Bove,et al.  Hypoplastic left heart syndrome. , 1997, BMJ.

[6]  R. Hinton,et al.  Hypoplastic left heart syndrome is associated with structural and vascular placental abnormalities and leptin dysregulation. , 2015, Placenta.

[7]  P. Grossfeld.  Hypoplastic left heart syndrome: new insights. , 2007, Circulation research.

[8]  Tsippi Iny Stein,et al.  The GeneCards Suite: From Gene Data Mining to Disease Genome Sequence Analyses , 2016, Current protocols in bioinformatics.

[9]  Gordon Keller,et al.  SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells , 2011, Nature Biotechnology.

[10]  Lior Pachter,et al.  Differential analysis of RNA-seq incorporating quantification uncertainty , 2016, Nature Methods.

[11]  W. Pearson,et al.  Current Protocols in Bioinformatics , 2002 .

[12]  E. J. Lee,et al.  Notch Inhibition Promotes Human Embryonic Stem Cell‐Derived Cardiac Mesoderm Differentiation , 2008, Stem cells.

[13]  Lisa J. Martin,et al.  Hypoplastic left heart syndrome links to chromosomes 10q and 6q and is genetically related to bicuspid aortic valve. , 2009, Journal of the American College of Cardiology.

[14]  M. B. Perryman,et al.  Hypoplastic left heart syndrome myocytes are differentiated but possess a unique phenotype. , 2003, Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology.

[15]  Qi-hua He,et al.  Timely Inhibition of Notch Signaling by DAPT Promotes Cardiac Differentiation of Murine Pluripotent Stem Cells , 2014, PloS one.

[16]  B. Arabin,et al.  Overweight and Obesity before, during and after Pregnancy: Part 2: Evidence-based Risk Factors and Interventions. , 2014, Geburtshilfe und Frauenheilkunde.

[17]  Godfrey L. Smith,et al.  The Use of Ratiometric Fluorescence Measurements of the Voltage Sensitive Dye Di-4-ANEPPS to Examine Action Potential Characteristics and Drug Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes , 2016, Toxicological sciences : an official journal of the Society of Toxicology.

[18]  L. Galletti,et al.  Identification of de novo mutations and rare variants in hypoplastic left heart syndrome , 2012, Clinical genetics.

[19]  R. Rizzi,et al.  Notch1 regulates the fate of cardiac progenitor cells , 2008, Proceedings of the National Academy of Sciences.

[20]  P. Thistlethwaite,et al.  Activation of Notch signaling by short-term treatment with Jagged-1 enhances store-operated Ca(2+) entry in human pulmonary arterial smooth muscle cells. , 2014, American journal of physiology. Cell physiology.

[21]  M. DePristo,et al.  The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. , 2010, Genome research.

[22]  Shilin Chen,et al.  FastUniq: A Fast De Novo Duplicates Removal Tool for Paired Short Reads , 2012, PloS one.

[23]  S. Nishikawa,et al.  Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development , 2006, Mechanisms of Development.

[24]  M H Paul,et al.  Experimental production of hypoplastic left heart syndrome in the chick embryo. , 1973, The American journal of cardiology.

[25]  M. de Andrade,et al.  Compound heterozygous NOTCH1 mutations underlie impaired cardiogenesis in a patient with hypoplastic left heart syndrome , 2015, Human Genetics.

[26]  Ramaswamy K. Iyer,et al.  Germline Variation in Cancer-Susceptibility Genes in a Healthy, Ancestrally Diverse Cohort: Implications for Individual Genome Sequencing , 2014, PloS one.

[27]  J. Borlak,et al.  A loss-of-function mutation in the binding domain of HAND1 predicts hypoplasia of the human hearts. , 2008, Human molecular genetics.

[28]  J. Cleveland,et al.  Cross-Talk Between the Toll-Like Receptor 4 and Notch1 Pathways Augments the Inflammatory Response in the Interstitial Cells of Stenotic Human Aortic Valves , 2012, Circulation.

[29]  K. Naruse,et al.  Directed Differentiation of Patient-Specific Induced Pluripotent Stem Cells Identifies the Transcriptional Repression and Epigenetic Modification of NKX2-5, HAND1, and NOTCH1 in Hypoplastic Left Heart Syndrome , 2014, PloS one.

[30]  K. Jones,et al.  The 11q terminal deletion disorder: A prospective study of 110 cases , 2004, American journal of medical genetics. Part A.

[31]  R. Ferrell,et al.  Truncating mutations in FOXC2 cause multiple lymphedema syndromes. , 2001, Human molecular genetics.

[32]  K. Stenmark,et al.  Gene expression and β-adrenergic signaling are altered in hypoplastic left heart syndrome. , 2014, The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation.

[33]  P. Simpson,et al.  Impact of MYH6 variants in hypoplastic left heart syndrome , 2016, Physiological genomics.

[34]  Kenny Q. Ye,et al.  An integrated map of genetic variation from 1,092 human genomes , 2012, Nature.

[35]  Lior Pachter,et al.  Near-optimal probabilistic RNA-seq quantification , 2016, Nature Biotechnology.

[36]  P. Shannon,et al.  Fetal reprogramming and senescence in hypoplastic left heart syndrome and in human pluripotent stem cells during cardiac differentiation. , 2013, The American journal of pathology.

[37]  S. Henikoff,et al.  Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm , 2009, Nature Protocols.

[38]  S. Sano,et al.  Transcoronary infusion of cardiac progenitor cells in hypoplastic left heart syndrome: Three-year follow-up of the Transcoronary Infusion of Cardiac Progenitor Cells in Patients With Single-Ventricle Physiology (TICAP) trial. , 2015, The Journal of thoracic and cardiovascular surgery.

[39]  David J. Miller,et al.  Gene Regulatory Networks in the Evolution and Development of the Heart , 2006 .

[40]  B. Lilly,et al.  Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease. , 2013, Journal of molecular and cellular cardiology.

[41]  Tsviya Olender,et al.  VarElect: the phenotype-based variation prioritizer of the GeneCards Suite , 2016, BMC Genomics.

[42]  Gordon Keller,et al.  Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. , 2011, Cell stem cell.

[43]  D. Elliott,et al.  Cardiac homeobox gene NKX2-5 mutations and congenital heart disease: associations with atrial septal defect and hypoplastic left heart syndrome. , 2003, Journal of the American College of Cardiology.

[44]  Shuhei Sato,et al.  Intracoronary Autologous Cardiac Progenitor Cell Transfer in Patients With Hypoplastic Left Heart Syndrome: The TICAP Prospective Phase 1 Controlled Trial , 2015, Circulation research.

[45]  B. Arabin,et al.  Overweight and Obesity before, during and after Pregnancy , 2014 .

[46]  Lior Pachter,et al.  Differential analysis of RNA-Seq incorporating quantification uncertainty , 2016 .

[47]  H. Kilbride,et al.  Haploinsufficiencies of FOXF1 and FOXC2 genes associated with lethal alveolar capillary dysplasia and congenital heart disease , 2010, American journal of medical genetics. Part A.

[48]  Lisa J. Martin,et al.  Hypoplastic left heart syndrome is heritable. , 2007, Journal of the American College of Cardiology.

[49]  David Sedmera,et al.  Increased Ventricular Preload Is Compensated by Myocyte Proliferation in Normal and Hypoplastic Fetal Chick Left Ventricle , 2007, Circulation research.

[50]  J. Barrett,et al.  Pooled Sequencing of 531 Genes in Inflammatory Bowel Disease Identifies an Associated Rare Variant in BTNL2 and Implicates Other Immune Related Genes , 2015, PLoS Genetics.

[51]  L. Garrett-Sinha,et al.  Deletion of ETS-1, a gene in the Jacobsen syndrome critical region, causes ventricular septal defects and abnormal ventricular morphology in mice. , 2010, Human molecular genetics.

[52]  L. Bailey,et al.  Identification of connexin43 (alpha1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE). , 2001, Mutation research.

[53]  Ross C. Brownson,et al.  Chronic disease epidemiology and control. , 2010 .

[54]  D. Schaffer,et al.  Transforming Growth Factor‐β and Notch Signaling Mediate Stem Cell Differentiation into Smooth Muscle Cells , 2010, Stem cells.

[55]  H. Burkhart,et al.  Regenerative therapy for hypoplastic left heart syndrome: first report of intraoperative intramyocardial injection of autologous umbilical-cord blood-derived cells. , 2015, The Journal of thoracic and cardiovascular surgery.

[56]  B. Black,et al.  Transcription factor pathways and congenital heart disease. , 2012, Current topics in developmental biology.

[57]  M. DePristo,et al.  A framework for variation discovery and genotyping using next-generation DNA sequencing data , 2011, Nature Genetics.

[58]  H. Garner,et al.  Inhibitory Role of Notch1 in Calcific Aortic Valve Disease , 2011, PloS one.

[59]  H. Hakonarson,et al.  ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data , 2010, Nucleic acids research.