Abstract 1: Abnormal Cardiac Differentiation Underlies Cardiac Hypertrophy in Noonan Syndrome With RAF1 Mutation

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Fabrice Jaffre ◽  
Gang Wang ◽  
Amy Roberts ◽  
William Pu ◽  
Andreas Hahn ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Noonan Syndrome ◽  
Germ Line ◽  
Mapk Pathway ◽  
Cardiac Differentiation ◽  
Subsequent Increase ◽  
Akt Activation ◽  
Stage Of Development ◽  
Hela Cell Lines ◽  
Induced Pluripotent

Noonan Syndrome (NS), an autosomal dominant RASopathy disorder, is caused by germ-line mutations that affect the canonical RAS-MAPK pathway. >95% of NS patients with an S257L mutation in Raf1 exhibit cardiac hypertrophy (CH) at a very young age. However, the molecular and developmental mechanisms that elicit CH in these patients remain unknown. Hence, we aimed at modeling CH by differentiating human NS Raf1 induced-pluripotent stem cells (iPSCs) towards the cardiomyocyte fate. We generated iPSCs from an NS pediatric patient with the S257L/+ mutation in the Raf1 gene and corrected the mutation using CRISPR-Cas9 double nickase system. Differentiation of the mutant NS line led to a significantly increased number of cardiomyocytes (CMs) as measured by FACS sorting at day 20, as compared to isogenic corrected cells (5.71 million ± 0.38 of cTNT+ cells vs 3.77±0.54, n=6, p<0.01). Differentiation of mutant iPSCs into mesodermal cells was normal; however, differentiation into cardiac progenitor cells was enhanced by the mutation, as demonstrated by the increased number of NKX2.5+ cells in the mutant culture at this stage of development. In addition, differentiated S257L/+ CMs had a significantly increased cell surface area compared to the control CMs (3,331 μm 2 ± 325 vs 1,638 μm 2 ± 97, n=6, p<0.01). To uncover the aberrant signaling pathways underlying these phenotypes, we generated RAF1 S257L/+ knockin and RAF1 null (KO) Hela cell lines using CRISPR-Cas9n technology. Interestingly, we found that ERK1/2 activity was enhanced in KO cells, both at baseline and in response to growth factor (GF) stimulation. At the opposite, ERK1/2 activity was reduced in mutant cells, suggesting that the RAF1 S257L/+ mutant actually suppresses ERK1/2 activation. Finally, we found that AKT activation was upregulated in S257L/+ cells under GF stimulation, due to decreased ERK1/2-dependent p70-S6 kinase activity and subsequent increase in mTORC2 activity (n=6). Similar experiments are underway in S257L/+, corrected and KO RAF1 iPSC-derived CMs. In conclusion, we propose that the S257L/+ mutation modulates development of CH in NS RAF1 patients through a reduction in ERK1/2 activity and overactivation of the AKT/mTOR signaling pathway, leading to both an increased number and size of CMs.

Abstract 397: Generation of Raf1 Mutant and Crispr-cas9 Corrected Isogenic iPSC-derived Cardiomyocytes to Model Hypertrophic Cardiomyopathy in Noonan Syndrome

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Fabrice Jaffré ◽  
Gang Wang ◽  
Amy Roberts ◽  
William Pu ◽  
Andreas Hahn ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Genome Editing ◽  
Noonan Syndrome ◽  
Molecular Mechanisms ◽  
Germ Line ◽  
Mapk Pathway ◽  
Heart Defects ◽  
Single Point ◽  
Cardiac Cells ◽  
Single Point Mutation

Background: Hypertrophic cardiomyopathy (HCM) is a major cause of death in infants and children. Noonan Syndrome (NS), an autosomal dominant RASopathy disorder, is characterized by multiple defects, including short stature, facial dysmorphia, and congenital heart defects that include HCM. RASopathies are caused by germ-line mutations that affect the canonical RAS-MAPK pathway. Indeed, 95% of NS patients with a mutation in Raf1 , a gene that plays an integral role in this signaling cascade, exhibit HCM. However, the molecular mechanisms that elicit HCM in these patients remain poorly understood. Objective: To generate human NS Raf1 induced-pluripotent stem cells (iPSCs), correct the mutation by genome editing and subsequently differentiate isogenic iPSC lines into cardiomyocytes to characterize the molecular and genetic basis of HCM in NS patients. Results: We generated iPSCs from skin fibroblasts obtained from a NS pediatric patient with a single point mutation in the Raf1 gene. Using electroporation of four episomal vectors containing the Yamanaka factors, we obtained several iPSC clones with normal karyotypes and strong expression of pluripotent markers (Nanog, Oct4, Lin28, Sox2) as detected by RT-qPCR and immunofluorescence. We next corrected the mutation in the NS Raf1 iPSCs using genome editing CRISPR-Cas9 nickase technology. Correction of the Raf1 mutation was determined at the clonal level by PCR followed by Restriction Fragment Length Polymorphism and confirmed by Sanger sequencing. In addition, by inducing a Cas9 nickase-dependent frame shift mutation we also generated an isogenic iPSC line where Raf1 gene was knockout (KO), as demonstrated at the RNA level by RT-qPCR and at the protein level by Western Blot. We next differentiated these multiple iPSC lines (mutant, corrected and KO) into isogenic beating cardiomyocytes, with more >98% of the cells positive for specific cardiomyocyte markers (α-actinin and cardiac TroponinT). Conclusion: We have successfully generated human NS Raf1 isogenic iPSC lines and corresponding cardiomyocytes. Currently, we are in progress of characterizing these cardiac cells to determine the molecular basis of NS-dependent HCM. Ultimately, our work should reveal new targets to treat HCM in NS patients.

scholarly journals Cues from human atrial extracellular matrix enrich the atrial differentiation of human induced pluripotent stem cell-derived cardiomyocytes

2021 ◽  
Author(s):  
Fernanda C. P. Mesquita ◽  
Jacquelynn Morrissey ◽  
Po-Feng Lee ◽  
Gustavo Monnerat ◽  
Yutao Xi ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cardiac Differentiation ◽  
Twofold Increase ◽  
Decellularized Extracellular Matrix ◽  
Induced Pluripotent

Decellularized extracellular matrix (dECM) from human atria preserves key native components that directed the cardiac differentiation of hiPSCs to an atrial-like phenotype, yielding a twofold increase of functional atrial-like cells.

In vitro propagation and cardiac differentiation of canine induced pluripotent stem cells on carbon nanotube substrates

2021 ◽  
pp. 101571
Author(s):  
Mahalakshmi Natarajan ◽  
Purnima Singh ◽  
Tanmay Mondal ◽  
Kuldeep Kumar ◽  
Kinsuk Das ◽  
...  
Keyword(s):  
Stem Cells ◽  
Carbon Nanotube ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
In Vitro Propagation ◽  
Cardiac Differentiation ◽  
Induced Pluripotent

Blockage of Angiotensin II type 2 receptor prevents thyroxine-mediated cardiac hypertrophy by blocking Akt activation

2010 ◽  
Vol 105 (3) ◽  
pp. 325-335 ◽  
Author(s):  
M. S. Carneiro-Ramos ◽  
G. P. Diniz ◽  
A. P. Nadu ◽  
J. Almeida ◽  
R. L. P. Vieira ◽  
...  
Keyword(s):  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Akt Activation

Abstract 837: Beta1 And Beta2 Adrenergic Receptor Transgenic Mice Display Distinct MicroRNA Expression Patterns That Converge On The Hypertrophic Signature

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Danish Sayed ◽  
Shweta Rane ◽  
Leng-Yi Chen ◽  
Minzhen He ◽  
Jacqueline Lypowy ◽  
...  
Keyword(s):  
Cardiac Hypertrophy ◽  
Adrenergic Receptor ◽  
Mapk Pathway ◽  
Expression Patterns ◽  
Pressure Overload ◽  
Mapk Signaling ◽  
Compensatory Hypertrophy ◽  
Over Expression ◽  
Mrna Targets ◽  
Dose Dependent

MicroRNA (miRNA) are ~22 ribonucleotides-long, with a potential to recognize multiple mRNA targets guided by sequence complimentarity. This class of molecules is functionally versatile, with the capacity to specifically inhibit translation, as well as, induce mRNA degradation, through targeting the 3′-untranslated regions. The levels of individual miRNA vary under different developmental, biological, or pathological conditions, thus, implicating them in normal and pathological cellular attributes. We have previously reported a miRNA signature that distinguishes pressure-overload compensatory hypertrophy by recapitulating the neonatal pattern. We hypothesized that this ’signature’ might aid in discriminating the underlying molecular differences in genetic models of cardiac hypertrophy, as seen in the beta1 and 2 adrenergic receptor (B1AR and B2AR) transgenic (Tg) mice. To address this, we used microarray analysis of RNA isolated from the hearts of 3 months old B1AR and B2AR mice. In general, while both mice exhibited an overlap with the hypertrophy signature including, upregulation of miR-21 and downregulation of miR-133a, miR-133b, and miR-185, the B2-AR Tg exhibited a more extensive overlap with the hypertrophy pattern, which further included upregulation of miR-199a*, miR-214, and miR-15b. To understand the functional significance of these miRNA in myocyte hypertrophy, we cloned them and their anti-sense sequences into adenoviral vectors. Significantly, over-expression miR-21 resulted in a, dose-dependent, branching (sprouting) of the cells. Computational predictions by ’TargetScanS’ identified sprouty as potential target. Subsequently, we confirmed down-regulation of sprouty by over-expression of miR-21 and vice versa. Sprouty is a known inhibitor of the Ras-MAPK signaling pathway and is, concordantly, downregulated in many forms of cancer. In the heart, sprouty has been suggested to control myocyte size and vascularization during cardiac hypertrophy. Thus, we propose that B1AR and B2AR Tg models exhibit distinct miRNA profiles that converge on that of pressure-overload cardiac hypertrophy. Moreover, the commonly over-expressed miR-21 plays a role in downregulating sprouty, an antagonist of the Ras-MAPK pathway.

scholarly journals The RASopathy Family: Consequences of Germline Activation of the RAS/MAPK Pathway

2018 ◽  
Vol 39 (5) ◽  
pp. 676-700 ◽  
Author(s):  
Mylène Tajan ◽  
Romain Paccoud ◽  
Sophie Branka ◽  
Thomas Edouard ◽  
Armelle Yart
Keyword(s):  
Noonan Syndrome ◽  
Mapk Pathway ◽  
Genetic Disorders ◽  
Mapk Signaling ◽  
Mendelian Inheritance ◽  
Disease Genes ◽  
Type I ◽  
Future Directions ◽  
Legius Syndrome ◽  
Anagen Hair

Abstract Noonan syndrome [NS; Mendelian Inheritance in Men (MIM) #163950] and related syndromes [Noonan syndrome with multiple lentigines (formerly called LEOPARD syndrome; MIM #151100), Noonan-like syndrome with loose anagen hair (MIM #607721), Costello syndrome (MIM #218040), cardio-facio-cutaneous syndrome (MIM #115150), type I neurofibromatosis (MIM #162200), and Legius syndrome (MIM #611431)] are a group of related genetic disorders associated with distinctive facial features, cardiopathies, growth and skeletal abnormalities, developmental delay/mental retardation, and tumor predisposition. NS was clinically described more than 50 years ago, and disease genes have been identified throughout the last 3 decades, providing a molecular basis to better understand their physiopathology and identify targets for therapeutic strategies. Most of these genes encode proteins belonging to or regulating the so-called RAS/MAPK signaling pathway, so these syndromes have been gathered under the name RASopathies. In this review, we provide a clinical overview of RASopathies and an update on their genetics. We then focus on the functional and pathophysiological effects of RASopathy-causing mutations and discuss therapeutic perspectives and future directions.

scholarly journals TBIO-27. RASOPATHIES AND BRAIN TUMOROGENESIS: ARE SOS1 MUTATIONS ARE CONCERNED?

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii471-iii471
Author(s):  
Nouha Bouayed Abdelmoula ◽  
Rim Louati ◽  
Balkiss Abdelmoula ◽  
Samir Aloulou
Keyword(s):  
Germ Line ◽  
Mapk Pathway ◽  
Heart Diseases ◽  
Genetic Defect ◽  
Nucleotide Exchange ◽  
Gene Encoding ◽  
Nucleotide Exchange Factor ◽  
Valvular Stenosis ◽  
Increased Risk ◽  
Linker Domain

Abstract Germ line gain-of-function mutations in several members of the RAS/MAPK pathway, including PTPN11 are associated with signalopathies named Rasopathies and known as Noonan syndrome and closely related conditions. Patients harboring Rasopathies are at increased risk of myeloproliferative diseases and solid tumors, such as neuroblastoma. Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, represent the second most frequent genetic defect in Rasopathies. However, SOS1 mutations are rare in human malignancies and patients with germline SOS1 mutations may not be at increased risk of developing cancer. Here, we report a SOS1 variant found to segregate in a Tunisian pedigree with many members affected by brain tumors as well as epileptic disorder. During our genetic counselling for congenital heart diseases, a 9-year-old female born at Sfax from a consanguineous couple and having pulmonic valvular stenosis, has been investigated at the molecular level. Screening of mutations in the entire coding sequence of PTPN11, Braf and SOS1, was conducted using HRM analysis and bidirectional sequencing. Heterozygous single nucleotide substitution of SOS1 gene: c.1655 G&gt;A was confirmed. This mutation affected the PH-REM linker domain with substitution of residue Arg552 to Lys: p.Arg552Lys. This mutation accounts for one-third of all mutations reported in SOS1 during Rasopathies. Although no other molecular exploration was done, family history revealed other affected children by neurodevelopmental and epileptic conditions as well as recurrent brain malignancies in the paternal family. Two aunts developed blindness and then died subsequently to tumor progression.

Leflunomide counterakts cardiac hypertrophy

2018 ◽  
Vol 132 (10) ◽  
pp. 1069-1073 ◽  
Author(s):  
Luciana A. Pescatore ◽  
Francisco R.M. Laurindo
Keyword(s):  
Heart Failure ◽  
Cardiac Hypertrophy ◽  
Independent Risk Factor ◽  
Cultured Cells ◽  
Protein Kinase B ◽  
Therapeutic Interventions ◽  
Recent Issue ◽  
Aortic Banding ◽  
Akt Activation ◽  
Anti Inflammatory

Cardiac hypertrophy (CH) is a major independent risk factor for heart failure and mortality. However, therapeutic interventions that target hypertrophy signaling in a load-independent way are unavailable. In a recent issue of Clinical Science (vol. 132, issue 6, 685-699), Ma et al. describe that the anti-inflammatory drug leflunomide markedly antagonized CH, dysfunction, and fibrosis induced by aortic banding or angiotensin-II in mice or by agonists in cultured cells. Unexpectedly, this occurred not via anti-inflammatory mechanisms but rather via inhibtion of Akt (protein kinase B, PKB) signaling. We further discuss the mechanisms underlying Akt activation and its effects on CH and review possible mechanisms of leflunomide effects. Despite some caveats, the availability of such a newly repurposed compound to treat CH can be a relevant advance.

scholarly journals Rabbit induced pluripotent stem cells retain capability of in vitro cardiac differentiation

2019 ◽  
Vol 68 (1) ◽  
pp. 35-47 ◽  
Author(s):  
Praopilas Phakdeedindan ◽  
Piyathip Setthawong ◽  
Narong Tiptanavattana ◽  
Sasitorn Rungarunlert ◽  
Praewphan Ingrungruanglert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cardiac Differentiation ◽  
Induced Pluripotent
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