Dr. Mercola is Professor of Medicine and Professor in the Stanford Cardiovascular Institute. He completed postdoctoral training at the Dana-Farber Cancer Institute and Harvard Medical School, was on the faculty in the Department of Cell Biology at Harvard Medical School for 12 years, and later at the Sanford-Burnham-Prebys Institute and Department of Bioengineering at the University of California, San Diego before relocating to Stanford in 2015.
Prof. Mercola is known for identifying many of the factors that are responsible for inducing and forming the heart, including the discovery that Wnt inhibition is a critical step in cardiogenesis that provided the conceptual basis and reagents for the large-scale production of cardiovascular tissues from pluripotent stem cells. He has collaborated with medicinal chemists, optical engineers and software developers to pioneer the use of patient iPSC-cardiomyocytes for disease modeling, safety pharmacology and drug development. His academic research is focused on developing and using quantitative assays of patient-specific cardiomyocyte function to discover druggable targets for preserving contractile function in heart failure and promoting regeneration following ischemic injury. He co-established drug screening and assay development at the Conrad Prebys Drug Discovery Center (San Diego), which operated as one of 4 large screening centers of the US National Institutes of Health (NIH) Molecular Libraries screening initiative and continues as one of the largest academic drug screening centers.
Prof. Mercola received an NIH MERIT award for his work on heart formation, and authored over 130 papers. He holds numerous patents, including describing the invention of the first engineered dominant negative protein and small molecules for stem cell and cancer applications. He serves on multiple editorial and advisory boards, including Vala Sciences,, Regencor, The Ted Rogers Centre for Heart Research and the Human Biomolecular Research Institute. His laboratory is funded by the NIH, California Institute for Regenerative Medicine and the Fondation Leducq.
Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.
Rationale: Diabetes mellitus (DM) is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII activation may occur in DM and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive-oxygen species (ROS) mediated oxidation of CaMKII, that can increase post-infarction mortality. Objective: To test whether high extracellular [glucose] (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. Methods and Results: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using H2DCFDA and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation, an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac specific CaMKIIdelta knockout mice. CaMKII knockout or inhibition also prevented Hi-Glu induced sarcoplasmic reticulum (SR) Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu induced ROS generation and SR Ca leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (Thm-G), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1) prevented ROS induction in response to either Hi-Glu or Thm-G. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIdelta was ablated (S280A) neither Hi-Glu nor Thm-G induced myocyte ROS generation. So CaMKIIdelta-S280 is required for the Hi-Glu-induced (and GlcNAc-dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (Mito-Tempo) and NOS pathway inhibitors (L-NAME, L-NIO and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/Thm-G induced ROS generation. Conclusions: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIdelta at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.
View details for DOI 10.1161/CIRCRESAHA.119.316288
View details for PubMedID 32134364
Small-Molecule Modulation of TDP-43 Recruitment to Stress Granules Prevents Persistent TDP-43 Accumulation in ALS/FTD
2019; 103 (5): 802-+
Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.
View details for DOI 10.1016/j.neuron.2019.05.048
View details for Web of Science ID 000484400200009
View details for PubMedID 31272829
View details for PubMedCentralID PMC6728177
Identification of a potent inhibitor of notch signaling
AMER CHEMICAL SOC. 2019
View details for Web of Science ID 000525061502335
Integrated analysis of transcriptional regulation in PLN R14del cardiomyopathy
NATURE PUBLISHING GROUP. 2019: 790–91
View details for Web of Science ID 000489313106094
- Stars in the Night Sky: iPSC-Cardiomyocytes Return the Patient Context to Drug Screening CELL STEM CELL 2019; 24 (4): 506–7
Stars in the Night Sky: iPSC-Cardiomyocytes Return the Patient Context to Drug Screening.
Cell stem cell
2019; 24 (4): 506–7
iPSC-derived cardiomyocytes have the potential to revolutionize the discovery of new medicines for serious heart conditions; however, heart failure remains a major cause of mortality worldwide. In this issue of Cell Stem Cell, Fiedler etal. (2019) describe using iPSC-derived cardiomyocytes to screen new chemical entities, discovering a small molecule for ischemic injury.
View details for PubMedID 30951657
- A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay CIRCULATION 2019; 139 (6): 799–811
AlleleProfileR: A versatile tool to identify and profile sequence variants in edited genomes.
2019; 14 (12): e0226694
Gene editing strategies, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9), are revolutionizing biology. However, quantitative and sensitive detection of targeted mutations are required to evaluate and quantify the genome editing outcomes. Here we present AlleleProfileR, a new analysis tool, written in a combination of R and C++, with the ability to batch process the sequence analysis of large and complex genome editing experiments, including the recently developed base editing technologies.
View details for DOI 10.1371/journal.pone.0226694
View details for PubMedID 31877162
Disruption of NOTCH signaling by a small molecule inhibitor of the transcription factor RBPJ.
2019; 9 (1): 10811
NOTCH plays a pivotal role during normal development and in congenital disorders and cancer. γ-secretase inhibitors are commonly used to probe NOTCH function, but also block processing of numerous other proteins. We discovered a new class of small molecule inhibitor that disrupts the interaction between NOTCH and RBPJ, which is the main transcriptional effector of NOTCH signaling. RBPJ Inhibitor-1 (RIN1) also blocked the functional interaction of RBPJ with SHARP, a scaffold protein that forms a transcriptional repressor complex with RBPJ in the absence of NOTCH signaling. RIN1 induced changes in gene expression that resembled siRNA silencing of RBPJ rather than inhibition at the level of NOTCH itself. Consistent with disruption of NOTCH signaling, RIN1 inhibited the proliferation of hematologic cancer cell lines and promoted skeletal muscle differentiation from C2C12 myoblasts. Thus, RIN1 inhibits RBPJ in its repressing and activating contexts, and can be exploited for chemical biology and therapeutic applications.
View details for DOI 10.1038/s41598-019-46948-5
View details for PubMedID 31346210
Crataegus Extract WS1442 Stimulates Cardiomyogenesis and Angiogenesis From Stem Cells: A Possible New Pharmacology for Hawthorn?
Frontiers in pharmacology
2019; 10: 1357
Extracts from the leaves and flowers of Crataegus spp. (i.e., hawthorn species) have been traditionally used with documented preclinical and clinical activities in cardiovascular medicine. Based on reported positive effects on heart muscle after ischemic injury and the overall cardioprotective profile, the present study addressed potential contributions of Crataegus extracts to cardiopoietic differentiation from stem cells. The quantified Crataegus extract WS1442 stimulated cardiomyogenesis from murine and human embryonic stem cells (ESCs). Mechanistically, this effect was found to be induced by promoting differentiation of cardiovascular progenitor cell populations but not by proliferation. Bioassay-guided fractionation, phytochemical and analytical profiling suggested high-molecular weight ingredients as the active principle with at least part of the activity due to oligomeric procyanidines (OPCs) with a degree of polymerization between 3 and 6 (DP3-6). Transcriptome profiling in mESCs suggested two main, plausible mechanisms: These were early, stress-associated cellular events along with the modulation of distinct developmental pathways, including the upregulation of brain-derived neurotrophic factor (BDNF) and retinoic acid as well as the inhibition of transforming growth factor beta/bone morphogenetic protein (TGFbeta/BMP) and fibroblast growth factor (FGF) signaling. In addition, WS1442 stimulated angiogenesis ex vivo in Sca-1+ progenitor cells from adult mice hearts. These in vitro data provide evidence for a differentiation promoting activity of WS1442 on distinct cardiovascular stem/progenitor cells that could be valuable for therapeutic heart regeneration after myocardial infarction. However, the in vivo relevance of this new pharmacological activity of Crataegus spp. remains to be investigated and active ingredients from bioactive fractions will have to be further characterized.
View details for DOI 10.3389/fphar.2019.01357
View details for PubMedID 31849643
High-Throughput Phenotypic Screening Using Induced Pluripotent Stem Cell Derived Cardiomyocytes Identifies Compounds That Rescue Genetic Dilated Cardiomyopathy
LIPPINCOTT WILLIAMS & WILKINS. 2018: E72
View details for Web of Science ID 000454198400026
Mechanosensitive miR-376c Modulates Arrhythmia Susceptibility Via Regulation Of KCNJ2 In hiPSC-derived Cardiomyocytes
LIPPINCOTT WILLIAMS & WILKINS. 2018: E79
View details for Web of Science ID 000454198400051
- Use of human induced pluripotent stem cell-derived cardiomyocytes to assess drug cardiotoxicity NATURE PROTOCOLS 2018; 13 (12): 3018–41
Use of human induced pluripotent stem cell-derived cardiomyocytes to assess drug cardiotoxicity.
Cardiotoxicity has historically been a major cause of drug removal from the pharmaceutical market. Several chemotherapeutic compounds have been noted for their propensities to induce dangerous cardiac-specific side effects such as arrhythmias or cardiomyocyte apoptosis. However, improved preclinical screening methodologies have enabled cardiotoxic compounds to be identified earlier in the drug development pipeline. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can be used to screen for drug-induced alterations in cardiac cellular contractility, electrophysiology, and viability. We previously established a novel 'cardiac safety index' (CSI) as a metric that can evaluate potential cardiotoxic drugs via high-throughput screening of hiPSC-CMs. This metric quantitatively examines drug-induced alterations in CM function, using several in vitro readouts, and normalizes the resulting toxicity values to the in vivo maximum drug blood plasma concentration seen in preclinical or clinical pharmacokinetic models. In this ~1-month-long protocol, we describe how to differentiate hiPSCs into hiPSC-CMs and subsequently implement contractility and cytotoxicity assays that can evaluate drug-induced cardiotoxicity in hiPSC-CMs. We also describe how to carry out the calculations needed to generate the CSI metric from these quantitative toxicity measurements.
View details for PubMedID 30413796
b-Annulated 1,4-dihydropyridines as Notch inhibitors
BIOORGANIC & MEDICINAL CHEMISTRY LETTERS
2018; 28 (20): 3363–67
The Notch signaling pathway is involved in cell proliferation and differentiation, and has been recognized as an active pathway in regenerating tissue and cancerous cells. Notch signaling inhibition is considered a viable approach to the treatment of a variety of conditions including colorectal cancer, pancreatic cancer, breast cancer and metastatic melanoma. The discovery that the b-annulated dihydropyridine FLI-06 (1) is an inhibitor of the Notch pathway with an EC50 ≈ 2.5 μM prompted us to screen a library of related analogs. After structure activity studies were conducted, racemic compound 7 was identified with an EC50 = 0.36 μM. Synthesis of individual enantiomers provided (+)-7 enantiomer with an EC50 = 0.13 μM, or about 20-fold the potency of 1.
View details for PubMedID 30201292
A Premature Termination Codon Mutation in MYBPC3 Causes Hypertrophic Cardiomyopathy via Chronic Activation of Nonsense-Mediated Decay.
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in myosin binding protein C3 ( MYBPC3) resulting in a premature termination codon (PTC). The underlying mechanisms of how PTC mutations in MYBPC3 lead to the onset and progression of HCM are poorly understood. This study's aim was to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with MYBPC3 PTC mutations by utilizing human isogenic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs).METHODS: Isogenic iPSC lines were generated from patients harboring MYBPC3 PTC mutations (p.R943x; p.R1073P_Fsx4) using genome editing and then differentiated into cardiomyocytes. Comprehensive phenotypical and transcriptome analyses were performed.RESULTS: We observed aberrant calcium handling properties with prolonged decay kinetics and elevated diastolic calcium levels in HCM iPSC-CMs compared to isogenic controls without structural abnormalities or contractile dysfunction. The mRNA expression levels of MYBPC3 were significantly reduced in mutant iPSC-CMs, but the protein levels were comparable among isogenic iPSC-CMs, suggesting that haploinsufficiency of MYBPC3 does not contribute to the pathogenesis of HCM in vitro. Furthermore, truncated MYBPC3 peptides were not detected. At the molecular level, the nonsense-mediated decay (NMD) pathway was activated, and a set of genes involved in major cardiac signaling pathways was dysregulated in HCM iPSC-CMs, indicating an HCM gene signature in vitro. Specific inhibition of the NMD pathway in mutant iPSC-CMs resulted in reversal of the molecular phenotype and normalization of calcium handling abnormalities.CONCLUSIONS: iPSC-CMs carrying MYBPC3 PTC mutations displayed aberrant calcium signaling and molecular dysregulations in the absence of significant haploinsufficiency of MYBPC3 protein. Here we provided the first evidence of the direct connection between the chronically activated NMD pathway and HCM disease development.
View details for PubMedID 30586709
- Will iPSC-cardiomyocytes revolutionize the discovery of drugs for heart disease? CURRENT OPINION IN PHARMACOLOGY 2018; 42: 55–61
A Novel Inhibitor Targets Both Wnt Signaling and ATM/p53 in Colorectal Cancer
2018; 78 (17): 5072–83
For 2017, the estimated lifetime risk of developing colorectal cancer was 1 in 22. Even though preventative colonoscopy screening and standard-of-care surgery, radiation, and chemotherapy have decreased the death rate from colorectal cancer, new therapies are needed for metastatic colorectal cancer. Here, we developed a novel small molecule, compound 2, that inhibited proliferation and viability of human colorectal cancer cells (HCT-116, DLD-1, SW480, and 10.1). Compound 2 inhibited cell migration, invasion, and epithelial-mesenchymal transition processes and potently increased cell apoptosis in human colorectal cancer cells. Compound 2 also modulated mitotic stress signaling, leading to both inhibition of Wnt responsiveness and stabilization and activation of p53 to cause cell-cycle arrest. In mouse xenografts, treatment with compound 2 (20 mg/kg/day, i.p.) induced cell death and inhibited tumor growth more than four-fold compared with vehicle at day 34. Neither acute cytotoxicity nor toxicity in animals (up to 1,000 mg/kg, i.p.) were observed for compound 2 To our knowledge, compound 2 is the first reported potent small molecule that inhibits Wnt/β-catenin signaling, activates p53 signaling regardless of p53 mutation status, and binds microtubules without detectable toxicity. Thus, compound 2 offers a novel mechanism of action and a new strategy to treat colorectal cancer.Significance: These findings identify a potent small molecule that may be therapeutically useful for colon cancer that works by inhibiting Wnt/β-catenin signaling, activating p53, and binding microtubules without detectable toxicity. Cancer Res; 78(17); 5072-83. ©2018 AACR.
View details for PubMedID 30032112
Novel tertiary sulfonamides as potent anti-cancer agents
BIOORGANIC & MEDICINAL CHEMISTRY
2018; 26 (15): 4441–51
For adult women in the United States, breast cancer is the most prevalent form of cancer. Compounds that target dysregulated signal transduction can be efficacious anti-cancer therapies. A prominent signaling pathway frequently dysregulated in breast cancer cells is the Wingless-related integration site (Wnt) pathway. The purpose of the work was to optimize a "hit" from a screening campaign. 76,000 compounds were tested in a Wnt transcription assay and revealed potent and reproducible "hit," compound 1. Medicinal chemistry optimization of 1 led to more potent and drug-like molecules, 19, 24 and 25 (i.e., Wnt pathway IC50 values = 11, 18 and 7 nM, respectively). The principal results showed compounds 19, 24 and 25 were potent anti-proliferative agents in breast cancer cell lines, MCF-7 (i.e., IC50 values = 10, 7 and 4 nM, respectively) and MDA-MB 231 (i.e., IC50 values = 13, 13 and 16 nM, respectively). Compound 19 synergized anti-proliferation with chemotherapeutic Doxorubicin in vitro. A major conclusion was that compound 19 enhanced anti-proliferation of Doxorubicin in vitro and in a xenograft animal model of breast cancer.
View details for PubMedID 30075999
Will iPSC-cardiomyocytes revolutionize the discovery of drugs for heart disease?
Current opinion in pharmacology
2018; 42: 55–61
Cardiovascular disease remains the largest single cause of mortality in the Western world, despite significant advances in clinical management over the years. Unfortunately, the development of new cardiovascular medicines is stagnating and can in part be attributed to the difficulty of screening for novel therapeutic strategies due to a lack of suitable models. The advent of human induced pluripotent stem cells and the ability to make limitless numbers of cardiomyocytes could revolutionize heart disease modeling and drug discovery. This review summarizes the state of the art in the field, describes the strengths and weaknesses of the technology, and applications where the model system would be most appropriate.
View details for PubMedID 30081259
Using iPSC Models to Probe Regulation of Cardiac Ion Channel Function
CURRENT CARDIOLOGY REPORTS
2018; 20 (7): 57
Cardiovascular disease is the leading contributor to mortality and morbidity. Many deaths of heart failure patients can be attributed to sudden cardiac death due primarily to ventricular arrhythmia. Currently, most anti-arrhythmics modulate ion channel conductivity or β-adrenergic signaling, but these drugs have limited efficacy for some indications, and can potentially be proarrhythmic.Recent studies have shown that mutations in proteins other than cardiac ion channels may confer susceptibility to congenital as well as acquired arrhythmias. Additionally, ion channels themselves are subject to regulation at the levels of channel expression, trafficking and post-translational modification; thus, research into the regulation of ion channels may elucidate disease mechanisms and potential therapeutic targets for future drug development. This review summarizes the current knowledge of the molecular mechanisms of arrhythmia susceptibility and discusses technological advances such as induced pluripotent stem cell-derived cardiomyocytes, gene editing, functional genomics, and physiological screening platforms that provide a new paradigm for discovery of new therapeutic targets to treat congenital and acquired diseases of the heart rhythm.
View details for PubMedID 29802473
- EXOSOMAL MIR-106A-363 CLUSTER FROM THE HYPOXIC HUMAN IPSC-DERIVED CARDIOMYOCYTES RESTORE THE ISCHEMIC MYOCARDIUM ELSEVIER SCIENCE INC. 2018: 14
High-Throughput Functional Screening Assay of Force and Stiffness in IPSC Derived Cardiomyocytes
CELL PRESS. 2018: 312A
View details for Web of Science ID 000430450000068
Id genes are essential for early heart formation
GENES & DEVELOPMENT
2017; 31 (13): 1325–38
Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.
View details for PubMedID 28794185
View details for PubMedCentralID PMC5580654
High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.
Science translational medicine
2017; 9 (377)
Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.
View details for DOI 10.1126/scitranslmed.aaf2584
View details for PubMedID 28202772
Bringing new dimensions to drug discovery screening: impact of cellular stimulation technologies.
Drug discovery today
The current mandate for the drug discovery industry is to develop more efficient drugs faster while reducing the costs associated with their development. Incorporation of cell stimulation technologies during screening assays is expected to revolutionize the discovery of novel drugs as well as safety pharmacology. In this review, we highlight 'classical' and emerging cell stimulation technologies that provide the ability to evaluate the effects of drug candidates on cells in different functional states to assess clinically relevant phenotypes.
View details for DOI 10.1016/j.drudis.2017.01.015
View details for PubMedID 28179145
miR-25 Tough Decoy Enhances Cardiac Function in Heart Failure.
Molecular therapy : the journal of the American Society of Gene Therapy
MicroRNAs are promising therapeutic targets, because their inhibition has the potential to normalize gene expression in diseased states. Recently, our group found that miR-25 is a key SERCA2a regulating microRNA, and we showed that multiple injections of antagomirs against miR-25 enhance cardiac contractility and function through SERCA2a restoration in a murine heart failure model. However, for clinical application, a more stable suppressor of miR-25 would be desirable. Tough Decoy (TuD) inhibitors are emerging as a highly effective method for microRNA inhibition due to their resistance to endonucleolytic degradation, high miRNA binding affinity, and efficient delivery. We generated a miR-25 TuD inhibitor and subcloned it into a cardiotropic AAV9 vector to evaluate its efficacy. The AAV9 TuD showed selective inhibition of miR-25 in vitro cardiomyoblast culture. In vivo, AAV9-miR-25 TuD delivered to the murine pressure-overload heart failure model selectively decreased expression of miR-25, increased levels of SERCA2a protein, and ameliorated cardiac dysfunction and fibrosis. Our data indicate that miR-25 TuD is an effective long-term suppressor of miR-25 and a promising therapeutic candidate to treat heart failure.
View details for PubMedID 29273502
Id genes are essential for early heart formation
Genes & Dev.
2017; 31: 1325-1338
View details for DOI 10.1101/gad.300400.117v1
An Automated Platform for Assessment of Congenital and Drug-Induced Arrhythmia with hiPSC-Derived Cardiomyocytes.
Frontiers in physiology
2017; 8: 766
The ability to produce unlimited numbers of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) harboring disease and patient-specific gene variants creates a new paradigm for modeling congenital heart diseases (CHDs) and predicting proarrhythmic liabilities of drug candidates. However, a major roadblock to implementing hiPSC-CM technology in drug discovery is that conventional methods for monitoring action potential (AP) kinetics and arrhythmia phenotypes in vitro have been too costly or technically challenging to execute in high throughput. Herein, we describe the first large-scale, fully automated and statistically robust analysis of AP kinetics and drug-induced proarrhythmia in hiPSC-CMs. The platform combines the optical recording of a small molecule fluorescent voltage sensing probe (VoltageFluor2.1.Cl), an automated high throughput microscope and automated image analysis to rapidly generate physiological measurements of cardiomyocytes (CMs). The technique can be readily adapted on any high content imager to study hiPSC-CM physiology and predict the proarrhythmic effects of drug candidates.
View details for PubMedID 29075196
View details for PubMedCentralID PMC5641590
miR-322/-503 cluster is expressed in the earliest cardiac progenitor cells and drives cardiomyocyte specification
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2016; 113 (34): 9551-9556
Understanding the mechanisms of early cardiac fate determination may lead to better approaches in promoting heart regeneration. We used a mesoderm posterior 1 (Mesp1)-Cre/Rosa26-EYFP reporter system to identify microRNAs (miRNAs) enriched in early cardiac progenitor cells. Most of these miRNA genes bear MESP1-binding sites and active histone signatures. In a calcium transient-based screening assay, we identified miRNAs that may promote the cardiomyocyte program. An X-chromosome miRNA cluster, miR-322/-503, is the most enriched in the Mesp1 lineage and is the most potent in the screening assay. It is specifically expressed in the looping heart. Ectopic miR-322/-503 mimicking the endogenous temporal patterns specifically drives a cardiomyocyte program while inhibiting neural lineages, likely by targeting the RNA-binding protein CUG-binding protein Elav-like family member 1 (Celf1). Thus, early miRNAs in lineage-committed cells may play powerful roles in cell-fate determination by cross-suppressing other lineages. miRNAs identified in this study, especially miR-322/-503, are potent regulators of early cardiac fate.
View details for DOI 10.1073/pnas.1608256113
View details for PubMedID 27512039
- The All-Chemical Approach: A Solution for Converting Fibroblasts Into Myocytes. Circulation research 2016; 119 (4): 505-507
High throughput physiological screening of iPSC-derived cardiomyocytes for drug development
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH
2016; 1863 (7): 1717-1727
Cardiac drug discovery is hampered by the reliance on non-human animal and cellular models with inadequate throughput and physiological fidelity to accurately identify new targets and test novel therapeutic strategies. Similarly, adverse drug effects on the heart are challenging to model, contributing to costly failure of drugs during development and even after market launch. Human induced pluripotent stem cell derived cardiac tissue represents a potentially powerful means to model aspects of heart physiology relevant to disease and adverse drug effects, providing both the human context and throughput needed to improve the efficiency of drug development. Here we review emerging technologies for high throughput measurements of cardiomyocyte physiology, and comment on the promises and challenges of using iPSC-derived cardiomyocytes to model disease and introduce the human context into early stages of drug discovery. This article is part of a Special Issue entitled: Cardiomyocyte biology: Integration of Developmental and Environmental Cues in the Heart edited by Marcus Schaub and Hughes Abriel.
View details for DOI 10.1016/j.bbamcr.2016.03.003
View details for Web of Science ID 000378360400004
View details for PubMedID 26952934
View details for PubMedCentralID PMC4885786
Identification of miRNAs promoting human cardiomyocyte proliferation by regulating Hippo pathway
OXFORD UNIV PRESS. 2016: S17
View details for Web of Science ID 000379812500045
Notch-independent RBPJ controls angiogenesis in the adult heart
Increasing angiogenesis has long been considered a therapeutic target for improving heart function after injury such as acute myocardial infarction. However, gene, protein and cell therapies to increase microvascularization have not been successful, most likely because the studies failed to achieve regulated and concerted expression of pro-angiogenic and angiostatic factors needed to produce functional microvasculature. Here, we report that the transcription factor RBPJ is a homoeostatic repressor of multiple pro-angiogenic and angiostatic factor genes in cardiomyocytes. RBPJ controls angiogenic factor gene expression independently of Notch by antagonizing the activity of hypoxia-inducible factors (HIFs). In contrast to previous strategies, the cardiomyocyte-specific deletion of Rbpj increased microvascularization of the heart without adversely affecting cardiac structure or function even into old age. Furthermore, the loss of RBPJ in cardiomyocytes increased hypoxia tolerance, improved heart function and decreased pathological remodelling after myocardial infarction, suggesting that inhibiting RBPJ might be therapeutic for ischaemic injury.
View details for DOI 10.1038/ncomms12088
View details for Web of Science ID 000379113200001
View details for PubMedID 27357444
View details for PubMedCentralID PMC4931341
Metallic Nanoislands on Graphene as Highly Sensitive Transducers of Mechanical, Biological, and Optical Signals
2016; 16 (2): 1375-1380
This article describes an effect based on the wetting transparency of graphene; the morphology of a metallic film (≤20 nm) when deposited on graphene by evaporation depends strongly on the identity of the substrate supporting the graphene. This control permits the formation of a range of geometries, such as tightly packed nanospheres, nanocrystals, and island-like formations with controllable gaps down to 3 nm. These graphene-supported structures can be transferred to any surface and function as ultrasensitive mechanical signal transducers with high sensitivity and range (at least 4 orders of magnitude of strain) for applications in structural health monitoring, electronic skin, measurement of the contractions of cardiomyocytes, and substrates for surface-enhanced Raman scattering (SERS, including on the tips of optical fibers). These composite films can thus be treated as a platform technology for multimodal sensing. Moreover, they are low profile, mechanically robust, semitransparent and have the potential for reproducible manufacturing over large areas.
View details for DOI 10.1021/acs.nanolett.5b04821
View details for Web of Science ID 000370215200081
View details for PubMedID 26765039
View details for PubMedCentralID PMC4751512
Epicardial FSTL1 reconstitution regenerates the adult mammalian heart.
2015; 525 (7570): 479-485
The elucidation of factors that activate the regeneration of the adult mammalian heart is of major scientific and therapeutic importance. Here we found that epicardial cells contain a potent cardiogenic activity identified as follistatin-like 1 (Fstl1). Epicardial Fstl1 declines following myocardial infarction and is replaced by myocardial expression. Myocardial Fstl1 does not promote regeneration, either basally or upon transgenic overexpression. Application of the human Fstl1 protein (FSTL1) via an epicardial patch stimulates cell cycle entry and division of pre-existing cardiomyocytes, improving cardiac function and survival in mouse and swine models of myocardial infarction. The data suggest that the loss of epicardial FSTL1 is a maladaptive response to injury, and that its restoration would be an effective way to reverse myocardial death and remodelling following myocardial infarction in humans.
View details for DOI 10.1038/nature15372
View details for PubMedID 26375005
Retinoic Acid Activity in Undifferentiated Neural Progenitors Is Sufficient to Fulfill Its Role in Restricting Fgf8 Expression for Somitogenesis
2015; 10 (9)
Bipotent axial stem cells residing in the caudal epiblast during late gastrulation generate neuroectodermal and presomitic mesodermal progeny that coordinate somitogenesis with neural tube formation, but the mechanism that controls these two fates is not fully understood. Retinoic acid (RA) restricts the anterior extent of caudal fibroblast growth factor 8 (Fgf8) expression in both mesoderm and neural plate to control somitogenesis and neurogenesis, however it remains unclear where RA acts to control the spatial expression of caudal Fgf8. Here, we found that mouse Raldh2-/- embryos, lacking RA synthesis and displaying a consistent small somite defect, exhibited abnormal expression of key markers of axial stem cell progeny, with decreased Sox2+ and Sox1+ neuroectodermal progeny and increased Tbx6+ presomitic mesodermal progeny. The Raldh2-/- small somite defect was rescued by treatment with an FGF receptor antagonist. Rdh10 mutants, with a less severe RA synthesis defect, were found to exhibit a small somite defect and anterior expansion of caudal Fgf8 expression only for somites 1-6, with normal somite size and Fgf8 expression thereafter. Rdh10 mutants were found to lack RA activity during the early phase when somites are small, but at the 6-somite stage RA activity was detected in neural plate although not in presomitic mesoderm. Expression of a dominant-negative RA receptor in mesoderm eliminated RA activity in presomitic mesoderm but did not affect somitogenesis. Thus, RA activity in the neural plate is sufficient to prevent anterior expansion of caudal Fgf8 expression associated with a small somite defect. Our studies provide evidence that RA restriction of Fgf8 expression in undifferentiated neural progenitors stimulates neurogenesis while also restricting the anterior extent of the mesodermal Fgf8 mRNA gradient that controls somite size, providing new insight into the mechanism that coordinates somitogenesis with neurogenesis.
View details for DOI 10.1371/journal.pone.0137894
View details for Web of Science ID 000361601100172
View details for PubMedID 26368825
View details for PubMedCentralID PMC4569375
1,5-Disubstituted benzimidazoles that direct cardiomyocyte differentiation from mouse embryonic stem cells
BIOORGANIC & MEDICINAL CHEMISTRY
2015; 23 (17): 5282-5292
Cardiomyopathy is the leading cause of death worldwide. Despite progress in medical treatments, heart transplantation is one of the only current options for those with infarcted heart muscle. Stem cell differentiation technology may afford cell-based therapeutics that may lead to the generation of new, healthy heart muscle cells from undifferentiated stem cells. Our approach is to use small molecules to stimulate stem cell differentiation. Herein, we describe a novel class of 1,5-disubstituted benzimidazoles that induce differentiation of stem cells into cardiac cells. We report on the evaluation in vitro for cardiomyocyte differentiation and describe structure-activity relationship results that led to molecules with drug-like properties. The results of this study show the promise of small molecules to direct stem cell lineage commitment, to probe signaling pathways and to develop compounds for the stimulation of stem cells to repair damaged heart tissue.
View details for DOI 10.1016/j.bmc.2015.07.073
View details for Web of Science ID 000360349900004
View details for PubMedID 26278027
View details for PubMedCentralID PMC4766108
Developmental origin of age-related coronary artery disease.
2015; 107 (2): 287-294
Age and injury cause structural and functional changes in coronary artery smooth muscle cells (caSMCs) that influence the pathogenesis of coronary artery disease. Although paracrine signalling is widely believed to drive phenotypic changes in caSMCs, here we show that developmental origin within the fetal epicardium can have a profound effect as well.Fluorescent dye and transgene pulse-labelling techniques in mice revealed that the majority of caSMCs are derived from Wt1(+), Gata5-Cre(+) cells that migrate before E12.5, whereas a minority of cells are derived from a later-emigrating, Wt1(+), Gata5-Cre(-) population. We functionally evaluated the influence of early emigrating cells on coronary artery development and disease by Gata5-Cre excision of Rbpj, which prevents their contribution to coronary artery smooth muscle cells. Ablation of the Gata5-Cre(+) population resulted in coronary arteries consisting solely of Gata5-Cre(-) caSMCs. These coronary arteries appeared normal into early adulthood; however, by 5-8 months of age, they became progressively fibrotic, lost the adventitial outer elastin layer, were dysfunctional and leaky, and animals showed early mortality.Taken together, these data reveal heterogeneity in the fetal epicardium that is linked to coronary artery integrity, and that distortion of the coronaries epicardial origin predisposes to adult onset disease.
View details for DOI 10.1093/cvr/cvv167
View details for PubMedID 26054850
Cholesterol-derived glucocorticoids control early fate specification in embryonic stem cells
STEM CELL RESEARCH
2015; 15 (1): 88-95
Aside from its role in cell membrane integrity, cholesterol is a key component in steroid hormone production. The vital functions of steroid hormones such as estrogen, testosterone, glucocorticoids (Gcrts) and mineralocorticoids (Mnrts) in perinatal and adult life are well understood; however, their role during early embryonic development remains largely unexplored. Here we show that siRNA-mediated perturbation of steroid hormone production during mesoderm formation has important consequences on cardiac differentiation in mouse embryonic stem cells (mESC). Both Gcrts and Mnrts are capable of driving cardiac differentiation in mESC. Interestingly, the Gcrt receptor is widely expressed during gastrulation in the mouse, and is exclusively localized in the nuclei-and thus active-in visceral endoderm cells, suggesting that it functions much earlier than previously anticipated. We therefore studied Gcrt signaling in mESC as a model of the gastrulating embryo, and found that Gcrt signaling regulates expression of the transcription factor Hnf4a and the secreted Nodal and BMP inhibitor Cer1 in the early visceral endoderm. RNAi-mediated knockdown of Gcrt function blocked cardiomyocyte differentiation, with limited effects on other cardiovascular cell types including vascular endothelial cells and smooth muscle. Furthermore, the cardiogenic effect of Gcrts required Hnf4a and paracrine Cer1. These results establish a novel function for cholesterol-derived steroid hormones and identify Gcrt signaling in visceral endoderm cells as a regulator of Cer1 and cardiac fate.
View details for DOI 10.1016/j.scr.2015.04.010
View details for Web of Science ID 000359994400009
View details for PubMedID 26024790
View details for PubMedCentralID PMC4516691
Cyclic stretch of embryonic cardiomyocytes increases proliferation, growth, and expression while repressing Tgf-beta signaling
JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY
2015; 79: 133-144
Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.
View details for DOI 10.1016/j.yjmcc.2014.11.003
View details for Web of Science ID 000353525800014
View details for PubMedID 25446186
View details for PubMedCentralID PMC4302020
High content screening for modulators of cardiac differentiation in human pluripotent stem cells.
Methods in molecular biology (Clifton, N.J.)
2015; 1263: 43-61
Chemical genomics has the unique potential to expose novel mechanisms of complex cellular biology through screening of small molecules in in vitro assays of a biological phenotype of interest, followed by target identification. In the case of disease-specific assays, the cellular proteins identified might constitute novel drug targets, and the small molecules themselves might be developed as drug leads. In cardiovascular biology, a chemical genomics approach to study the formation of cardiomyocyte, vascular endothelial, and smooth muscle lineages might contribute to therapeutic regeneration. Here, we describe methods used to develop high content screening assays implementing multipotent cardiovascular progenitors derived from human pluripotent stem cells and have identified novel compounds that direct cardiac differentiation.
View details for DOI 10.1007/978-1-4939-2269-7_4
View details for PubMedID 25618335
View details for PubMedCentralID PMC4766105
Inhibition of miR-25 improves cardiac contractility in the failing heart
2014; 508 (7497): 531-?
Heart failure is characterized by a debilitating decline in cardiac function, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy. MicroRNAs (miRNAs) are dysregulated in heart failure but whether they control contractility or constitute therapeutic targets remains speculative. Using high-throughput functional screening of the human microRNAome, here we identify miRNAs that suppress intracellular calcium handling in heart muscle by interacting with messenger RNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a (also known as ATP2A2). Of 875 miRNAs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas adeno-associated virus 9 (AAV9)-mediated overexpression of miR-25 in vivo resulted in a significant loss of contractile function, injection of an antisense oligonucleotide (antagomiR) against miR-25 markedly halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR oligonucleotide. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggest that it might be targeted therapeutically to restore function.
View details for DOI 10.1038/nature13073
View details for Web of Science ID 000334741600038
View details for PubMedID 24670661
View details for PubMedCentralID PMC4131725
HDAC-regulated myomiRs control BAF60 variant exchange and direct the functional phenotype of fibro-adipogenic progenitors in dystrophic muscles
GENES & DEVELOPMENT
2014; 28 (8): 841-857
Fibro-adipogenic progenitors (FAPs) are important components of the skeletal muscle regenerative environment. Whether FAPs support muscle regeneration or promote fibro-adipogenic degeneration is emerging as a key determinant in the pathogenesis of muscular diseases, including Duchenne muscular dystrophy (DMD). However, the molecular mechanism that controls FAP lineage commitment and activity is currently unknown. We show here that an HDAC-myomiR-BAF60 variant network regulates the fate of FAPs in dystrophic muscles of mdx mice. Combinatorial analysis of gene expression microarray, genome-wide chromatin remodeling by nuclease accessibility (NA) combined with next-generation sequencing (NA-seq), small RNA sequencing (RNA-seq), and microRNA (miR) high-throughput screening (HTS) against SWI/SNF BAF60 variants revealed that HDAC inhibitors (HDACis) derepress a "latent" myogenic program in FAPs from dystrophic muscles at early stages of disease. Specifically, HDAC inhibition induces two core components of the myogenic transcriptional machinery, MYOD and BAF60C, and up-regulates the myogenic miRs (myomiRs) (miR-1.2, miR-133, and miR-206), which target the alternative BAF60 variants BAF60A and BAF60B, ultimately directing promyogenic differentiation while suppressing the fibro-adipogenic phenotype. In contrast, FAPs from late stage dystrophic muscles are resistant to HDACi-induced chromatin remodeling at myogenic loci and fail to activate the promyogenic phenotype. These results reveal a previously unappreciated disease stage-specific bipotency of mesenchimal cells within the regenerative environment of dystrophic muscles. Resolution of such bipotency by epigenetic intervention with HDACis provides a molecular rationale for the in situ reprogramming of target cells to promote therapeutic regeneration of dystrophic muscles.
View details for DOI 10.1101/gad.234468.113
View details for Web of Science ID 000334585400005
View details for PubMedID 24682306
View details for PubMedCentralID PMC4003277
- Reprogramming the Cardiac Field CIRCULATION RESEARCH 2014; 114 (3): 409-411
Technical Variations in Low-Input RNA-seq Methodologies
Recent advances in RNA-seq methodologies from limiting amounts of mRNA have facilitated the characterization of rare cell-types in various biological systems. So far, however, technical variations in these methods have not been adequately characterized, vis-à-vis sensitivity, starting with reduced levels of mRNA. Here, we generated sequencing libraries from limiting amounts of mRNA using three amplification-based methods, viz. Smart-seq, DP-seq and CEL-seq, and demonstrated significant technical variations in these libraries. Reduction in mRNA levels led to inefficient amplification of the majority of low to moderately expressed transcripts. Furthermore, noise in primer hybridization and/or enzyme incorporation was magnified during the amplification step resulting in significant distortions in fold changes of the transcripts. Consequently, the majority of the differentially expressed transcripts identified were either high-expressed and/or exhibited high fold changes. High technical variations ultimately masked subtle biological differences mandating the development of improved amplification-based strategies for quantitative transcriptomics from limiting amounts of mRNA.
View details for DOI 10.1038/srep03678
View details for Web of Science ID 000329846100007
View details for PubMedID 24419370
Coordinate Nodal and BMP inhibition directs Baf60c-dependent cardiomyocyte commitment
GENES & DEVELOPMENT
2013; 27 (21): 2332-2344
A critical but molecularly uncharacterized step in heart formation and regeneration is the process that commits progenitor cells to differentiate into cardiomyocytes. Here, we show that the endoderm-derived dual Nodal/bone morphogenetic protein (BMP) antagonist Cerberus-1 (Cer1) in embryonic stem cell cultures orchestrates two signaling pathways that direct the SWI/SNF chromatin remodeling complex to cardiomyogenic loci in multipotent (KDR/Flk1+) progenitors, activating lineage-specific transcription. Transient inhibition of Nodal by Cer1 induces Brahma-associated factor 60c (Baf60c), one of three Baf60 variants (a, b, and c) that are mutually exclusively assembled into SWI/SNF. Blocking Nodal and BMP also induces lineage-specific transcription factors Gata4 and Tbx5, which interact with Baf60c. siRNA to Cer1, Baf60c, or the catalytic SWI/SNF subunit Brg1 prevented the developmental opening of chromatin surrounding the Nkx2.5 early cardiac enhancer and cardiomyocyte differentiation. Overexpression of Baf60c fully rescued these deficits, positioning Baf60c and SWI/SNF function downstream from Cer1. Thus, antagonism of Nodal and BMP coordinates induction of the myogenic Baf60c variant and interacting transcription factors to program the developmental opening of cardiomyocyte-specific loci in chromatin. This is the first demonstration that cues from the progenitor cell environment direct the subunit variant composition of SWI/SNF to remodel the transcriptional landscape for lineage-specific differentiation.
View details for DOI 10.1101/gad.225144.113
View details for Web of Science ID 000326921900005
View details for PubMedID 24186978
- Jumonji and Cardiac Fate CIRCULATION RESEARCH 2013; 113 (7): 837-839
Quantitative Transcriptomics using Designed Primer-based Amplification
We developed a novel Designed Primer-based RNA-sequencing strategy (DP-seq) that uses a defined set of heptamer primers to amplify the majority of expressed transcripts from limiting amounts of mRNA, while preserving their relative abundance. Our strategy reproducibly yielded high levels of amplification from as low as 50 picograms of mRNA while offering a dynamic range of over five orders of magnitude in RNA concentrations. We also demonstrated the potential of DP-seq to selectively suppress the amplification of the highly expressing ribosomal transcripts by more than 70% in our sequencing library. Using lineage segregation in embryonic stem cell cultures as a model of early mammalian embryogenesis, DP-seq revealed novel sets of low abundant transcripts, some corresponding to the identity of cellular progeny before they arise, reflecting the specification of cell fate prior to actual germ layer segregation.
View details for DOI 10.1038/srep01740
View details for Web of Science ID 000318145200001
View details for PubMedID 23624976
Induced Pluripotent Stem Cells in Cardiovascular Drug Discovery
2013; 112 (3): 534-548
The unexpected discovery that somatic cells can be reprogrammed to a pluripotent state yielding induced pluripotent stem cells has made it possible to produce cardiovascular cells exhibiting inherited traits and disorders. Use of these cells in high throughput analyses should broaden our insight into fundamental disease mechanisms and provide many benefits for patients, including new therapeutics and individually tailored therapies. Here we review recent progress in generating induced pluripotent stem cell-based models of cardiovascular disease and their multiple applications in drug development.
View details for DOI 10.1161/CIRCRESAHA.111.250266
View details for Web of Science ID 000314356700021
View details for PubMedID 23371902
Developing microRNA screening as a functional genomics tool for disease research.
Frontiers in physiology
2013; 4: 223-?
Originally discovered as regulators of developmental timing in C. elegans, microRNAs (miRNAs) have emerged as modulators of nearly every cellular process, from normal development to pathogenesis. With the advent of whole genome libraries of miRNA mimics suitable for high throughput screening, it is possible to comprehensively evaluate the function of each member of the miRNAome in cell-based assays. Since the relatively few microRNAs in the genome are thought to directly regulate a large portion of the proteome, miRNAome screening, coupled with the identification of the regulated proteins, might be a powerful new approach to gaining insight into complex biological processes.
View details for DOI 10.3389/fphys.2013.00223
View details for PubMedID 23986717
- CARDIOVASCULAR BIOLOGY A boost for heart regeneration NATURE 2012; 492 (7429): 360-362
BAF60 A, B, and Cs of muscle determination and renewal
GENES & DEVELOPMENT
2012; 26 (24): 2673-2683
Developmental biologists have defined many of the diffusible and transcription factors that control muscle differentiation, yet we still have only rudimentary knowledge of the mechanisms that dictate whether a myogenic progenitor cell forms muscle versus alternate lineages, including those that can be pathological in a state of disease or degeneration. Clues about the molecular basis for lineage determination in muscle progenitors are only now emerging from studies of chromatin modifications that avail myogenic genes for transcription, together with analysis of the composition and activities of the chromatin-modifying complexes themselves. Here we review recent progress on muscle determination and explore a unifying theme that environmental cues from the stem or progenitor niche control the selection of specific subunit variants of the switch/sucrose nonfermentable (SWI/SNF) chromatin-modifying complex, creating a combinatorial code that dictates whether cells adopt myogenic versus nonmyogenic cell fates. A key component of the code appears to be the mutually exclusive usage of the a, b, and c variants of the 60-kD structural subunit BAF60 (BRG1/BRM-associated factor 60), of which BAF60c is essential to activate both skeletal and cardiac muscle programs. Since chromatin remodeling governs myogenic fate, the combinatorial assembly of the SWI/SNF complex might be targeted to develop drugs aimed at the therapeutic reduction of compensatory fibrosis and fatty deposition in chronic muscular disorders.
View details for DOI 10.1101/gad.207415.112
View details for Web of Science ID 000312775700002
View details for PubMedID 23222103
Whole-genome microRNA screening identifies let-7 and mir-18 as regulators of germ layer formation during early embryogenesis
GENES & DEVELOPMENT
2012; 26 (23): 2567-2579
Tight control over the segregation of endoderm, mesoderm, and ectoderm is essential for normal embryonic development of all species, yet how neighboring embryonic blastomeres can contribute to different germ layers has never been fully explained. We postulated that microRNAs, which fine-tune many biological processes, might modulate the response of embryonic blastomeres to growth factors and other signals that govern germ layer fate. A systematic screen of a whole-genome microRNA library revealed that the let-7 and miR-18 families increase mesoderm at the expense of endoderm in mouse embryonic stem cells. Both families are expressed in ectoderm and mesoderm, but not endoderm, as these tissues become distinct during mouse and frog embryogenesis. Blocking let-7 function in vivo dramatically affected cell fate, diverting presumptive mesoderm and ectoderm into endoderm. siRNA knockdown of computationally predicted targets followed by mutational analyses revealed that let-7 and miR-18 down-regulate Acvr1b and Smad2, respectively, to attenuate Nodal responsiveness and bias blastomeres to ectoderm and mesoderm fates. These findings suggest a crucial role for the let-7 and miR-18 families in germ layer specification and reveal a remarkable conservation of function from amphibians to mammals.
View details for DOI 10.1101/gad.200758.112
View details for Web of Science ID 000311944000002
View details for PubMedID 23152446
Synthesis and SAR of b-Annulated 1,4-Dihydropyridines Define Cardiomyogenic Compounds as Novel Inhibitors of TGF beta Signaling
JOURNAL OF MEDICINAL CHEMISTRY
2012; 55 (22): 9946-9957
A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17000 small molecules for cardiogenesis led to the identification of a b-annulated 1,4-dihydropyridine (1,4-DHP) that inhibited transforming growth factor β (TGFβ)/Smad signaling by clearing the type II TGFβ receptor from the cell surface. Because this is an unprecedented mechanism of action, we explored the series' structure-activity relationship (SAR) based on TGFβ inhibition, and evaluated SAR aspects for cell-surface clearance of TGFβ receptor II (TGFBR2) and for biological activity in mESCs. We determined a pharmacophore and generated 1,4-DHPs with IC(50)s for TGFβ inhibition in the nanomolar range (e.g., compound 28, 170 nM). Stereochemical consequences of a chiral center at the 4-position was evaluated, revealing 10- to 15-fold more potent TGFβ inhibition for the (+)- than the (-) enantiomer. This stereopreference was not observed for the low level inhibition against Activin A signaling and was reversed for effects on calcium handling in HL-1 cells.
View details for DOI 10.1021/jm301144g
View details for Web of Science ID 000311461500045
View details for PubMedID 23130626
High throughput measurement of Ca2+ dynamics for drug risk assessment in human stem cell-derived cardiomyocytes by kinetic image cytometry
JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS
2012; 66 (3): 246-256
Current methods to measure physiological properties of cardiomyocytes and predict fatal arrhythmias that can cause sudden death, such as Torsade de Pointes, lack either the automation and throughput needed for early-stage drug discovery and/or have poor predictive value. To increase throughput and predictive power of in vitro assays, we developed kinetic imaging cytometry (KIC) for automated cell-by-cell analyses via intracellular fluorescence Ca²⁺ indicators. The KIC instrument simultaneously records and analyzes intracellular calcium concentration [Ca²⁺](i) at 30-ms resolution from hundreds of individual cells/well of 96-well plates in seconds, providing kinetic details not previously possible with well averaging technologies such as plate readers. Analyses of human embryonic stem cell and induced pluripotent stem cell-derived cardiomyocytes revealed effects of known cardiotoxic and arrhythmogenic drugs on kinetic parameters of Ca²⁺ dynamics, suggesting that KIC will aid in the assessment of cardiotoxic risk and in the elucidation of pathogenic mechanisms of heart disease associated with drugs treatment and/or genetic background.
View details for DOI 10.1016/j.vascn.2012.08.167
View details for Web of Science ID 000311489100006
View details for PubMedID 22926323
TGF beta-Dependent Epithelial-to-Mesenchymal Transition Is Required to Generate Cardiospheres from Human Adult Heart Biopsies
STEM CELLS AND DEVELOPMENT
2012; 21 (17): 3081-3090
Autologous cardiac progenitor cells (CPCs) isolated as cardiospheres (CSps) represent a promising candidate for cardiac regenerative therapy. A better understanding of the origin and mechanisms underlying human CSps formation and maturation is undoubtedly required to enhance their cardiomyogenic potential. Epithelial-to-mesenchymal transition (EMT) is a key morphogenetic process that is implicated in the acquisition of stem cell-like properties in different adult tissues, and it is activated in the epicardium after ischemic injury to the heart. We investigated whether EMT is involved in the formation and differentiation of human CSps, revealing that an up-regulation of the expression of EMT-related genes accompanies CSps formation that is relative to primary explant-derived cells and CSp-derived cells grown in a monolayer. EMT and CSps formation is enhanced in the presence of transforming growth factor β1 (TGFβ1) and drastically blocked by the type I TGFβ-receptor inhibitor SB431452, indicating that TGFβ-dependent EMT is essential for the formation of these niche-like 3D-multicellular clusters. Since TGFβ is activated in the myocardium in response to injury, our data suggest that CSps formation mimics an adaptive mechanism that could potentially be enhanced to increase in vivo or ex vivo regenerative potential of adult CPCs.
View details for DOI 10.1089/scd.2012.0277
View details for Web of Science ID 000310840500002
View details for PubMedID 22765842
View details for PubMedCentralID PMC4146498
Serum-free generation of multipotent mesoderm (Kdr+) progenitor cells in mouse embryonic stem cells for functional genomics screening.
Current protocols in stem cell biology
2012; Chapter 1: Unit 1F 13-?
This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions, and for functional genomics screening using these cells. Kdr-positive cells are able to differentiate into a wide array of mesodermal derivatives, including vascular endothelial cells, cardiomyocytes, hematopoietic progenitors, and smooth muscle cells. The efficient generation of such progenitor cells is of particular interest because it permits subsequent steps in cardiovascular development to be analyzed in detail, including deciphering the mechanisms that direct differentiation. In addition, the oligonucleotide transfection protocol used to functionally screen siRNA and miRNA libraries is a powerful tool to reveal networks of genes, signaling proteins, and miRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. Technical limitations, troubleshooting, and potential applications of these methods are discussed.
View details for DOI 10.1002/9780470151808.sc01f13s23
View details for PubMedID 23154934
View details for PubMedCentralID PMC3562707
Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (40): 16196-16201
Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of selection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line-specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differentiated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming-specific epigenetic signature comprised of nine aberrantly methylated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state.
View details for DOI 10.1073/pnas.1202352109
View details for Web of Science ID 000309611400053
View details for PubMedID 22991473
A Nodal-to-TGF beta Cascade Exerts Biphasic Control Over Cardiopoiesis
2012; 111 (7): 876-?
The transforming growth factor-β (TGFβ) family member Nodal promotes cardiogenesis, but the mechanism is unclear despite the relevance of TGFβ family proteins for myocardial remodeling and regeneration.To determine the function(s) of TGFβ family members during stem cell cardiogenesis.Murine embryonic stem cells were engineered with a constitutively active human type I Nodal receptor (caACVR1b) to mimic activation by Nodal and found to secrete a paracrine signal that promotes cardiogenesis. Transcriptome and gain- and loss-of-function studies identified the factor as TGFβ2. Both Nodal and TGFβ induced early cardiogenic progenitors in embryonic stem cell cultures at day 0 to 2 of differentiation. However, Nodal expression declines by day 4 due to feedback inhibition, whereas TGFβ persists. At later stages (days 4-6), TGFβ suppresses the formation of cardiomyocytes from multipotent Kdr(+) progenitors while promoting the differentiation of vascular smooth muscle and endothelial cells.Nodal induces TGFβ, and both stimulate the formation of multipotent cardiovascular Kdr(+) progenitors. TGFβ, however, becomes uniquely responsible for controlling subsequent lineage segregation by stimulating vascular smooth muscle and endothelial lineages and simultaneously blocking cardiomyocyte differentiation.
View details for DOI 10.1161/CIRCRESAHA.112.270272
View details for Web of Science ID 000308868800014
View details for PubMedID 22872153
View details for PubMedCentralID PMC3766357
APJ acts as a dual receptor in cardiac hypertrophy
2012; 488 (7411): 394-398
Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.
View details for DOI 10.1038/nature11263
View details for Web of Science ID 000307501000045
View details for PubMedID 22810587
View details for PubMedCentralID PMC3422434
Transcription factors ETS2 and MESP1 transdifferentiate human dermal fibroblasts into cardiac progenitors
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2012; 109 (32): 13016-13021
Unique insights for the reprograming of cell lineages have come from embryonic development in the ascidian Ciona, which is dependent upon the transcription factors Ci-ets1/2 and Ci-mesp to generate cardiac progenitors. We tested the idea that mammalian v-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and mesoderm posterior (MESP) homolog may be used to convert human dermal fibroblasts into cardiac progenitors. Here we show that murine ETS2 has a critical role in directing cardiac progenitors during cardiopoiesis in embryonic stem cells. We then use lentivirus-mediated forced expression of human ETS2 to convert normal human dermal fibroblasts into replicative cells expressing the cardiac mesoderm marker KDR(+). However, although neither ETS2 nor the purported cardiac master regulator MESP1 can by themselves generate cardiac progenitors de novo from fibroblasts, forced coexpression of ETS2 and MESP1 or cell treatment with purified proteins reprograms fibroblasts into cardiac progenitors, as shown by the de novo appearance of core cardiac transcription factors, Ca(2+) transients, and sarcomeres. Our data indicate that ETS2 and MESP1 play important roles in a genetic network that governs cardiopoiesis.
View details for DOI 10.1073/pnas.1120299109
View details for Web of Science ID 000307551700041
View details for PubMedID 22826236
Small Molecule-Mediated TGF-beta Type II Receptor Degradation Promotes Cardiomyogenesis in Embryonic Stem Cells
CELL STEM CELL
2012; 11 (2): 242-252
The cellular signals controlling the formation of cardiomyocytes, vascular smooth muscle, and endothelial cells from stem cell-derived mesoderm are poorly understood. To identify these signals, a mouse embryonic stem cell (ESC)-based differentiation assay was screened against a small molecule library resulting in a 1,4-dihydropyridine inducer of type II TGF-β receptor (TGFBR2) degradation-1 (ITD-1). ITD analogs enhanced proteasomal degradation of TGFBR2, effectively clearing the receptor from the cell surface and selectively inhibiting intracellular signaling (IC(50) ~0.4-0.8 μM). ITD-1 was used to evaluate TGF-β involvement in mesoderm formation and cardiopoietic differentiation, which occur sequentially during early development, revealing an essential role in both processes in ESC cultures. ITD-1 selectively enhanced the differentiation of uncommitted mesoderm to cardiomyocytes, but not to vascular smooth muscle and endothelial cells. ITD-1 is a highly selective TGF-β inhibitor and reveals an unexpected role for TGF-β signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.
View details for DOI 10.1016/j.stem.2012.04.025
View details for Web of Science ID 000307432600014
View details for PubMedID 22862949
RBPJ Controls the Angiogenic Response of Mouse Adult Cardiomyocytes
LIPPINCOTT WILLIAMS & WILKINS. 2012
View details for Web of Science ID 000312506400259
HNF4 alpha Antagonists Discovered by a High-Throughput Screen for Modulators of the Human Insulin Promoter
CHEMISTRY & BIOLOGY
2012; 19 (7): 806-818
Hepatocyte nuclear factor (HNF)4α is a central regulator of gene expression in cell types that play a critical role in metabolic homeostasis, including hepatocytes, enterocytes, and pancreatic β cells. Although fatty acids were found to occupy the HNF4α ligand-binding pocket and were proposed to act as ligands, there is controversy about both the nature of HNF4α ligands as well as the physiological role of the binding. Here, we report the discovery of potent synthetic HNF4α antagonists through a high-throughput screen for effectors of the human insulin promoter. These molecules bound to HNF4α with high affinity and modulated the expression of known HNF4α target genes. Notably, they were found to be selectively cytotoxic to cancer cell lines in vitro and in vivo, although in vivo potency was limited by suboptimal pharmacokinetic properties. The discovery of bioactive modulators for HNF4α raises the possibility that diseases involving HNF4α, such as diabetes and cancer, might be amenable to pharmacologic intervention by modulation of HNF4α activity.
View details for DOI 10.1016/j.chembiol.2012.05.014
View details for Web of Science ID 000307261100007
View details for PubMedID 22840769
Wnt Inhibition Correlates with Human Embryonic Stem Cell Cardiomyogenesis: A Structure-Activity Relationship Study Based on Inhibitors for the Wnt Response
JOURNAL OF MEDICINAL CHEMISTRY
2012; 55 (2): 697-708
Human embryonic stem cell-based high-content screening of 550 known signal transduction modulators showed that one "lead" (1, a recently described inhibitor of the proteolytic degradation of Axin) stimulated cardiomyogenesis. Because Axin controls canonical Wnt signaling, we conducted an investigation to determine whether the cardiogenic activity of 1 is Wnt-dependent, and we developed a structure-activity relationship to optimize the cardiogenic properties of 1. We prepared analogues with a range of potencies (low nanomolar to inactive) for Wnt/β-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (r(2) = 0.72). The optimal compounds induced cardiogenesis 1.5-fold greater than 1 at 30-fold lower concentrations. In contrast, no correlation was observed for cardiogenesis and modulation of transforming growth factor β (TGFβ)/Smad signaling that prominently influences cardiogenesis. Taken together, these data show that Wnt signaling inhibition is essential for cardiogenic activity and that the pathway can be targeted for the design of druglike cardiogenic molecules.
View details for DOI 10.1021/jm2010223
View details for Web of Science ID 000299453300013
View details for PubMedID 22191557
Fine-Tuning of Drp1/Fis1 Availability by AKAP121/Siah2 Regulates Mitochondrial Adaptation to Hypoxia
2011; 44 (4): 532-544
Defining the mechanisms underlying the control of mitochondrial fusion and fission is critical to understanding cellular adaptation to diverse physiological conditions. Here we demonstrate that hypoxia induces fission of mitochondrial membranes, dependent on availability of the mitochondrial scaffolding protein AKAP121. AKAP121 controls mitochondria dynamics through PKA-dependent inhibitory phosphorylation of Drp1 and PKA-independent inhibition of Drp1-Fis1 interaction. Reduced availability of AKAP121 by the ubiquitin ligase Siah2 relieves Drp1 inhibition by PKA and increases its interaction with Fis1, resulting in mitochondrial fission. High AKAP121 levels, seen in cells lacking Siah2, attenuate fission and reduce apoptosis of cardiomyocytes under simulated ischemia. Infarct size and degree of cell death were reduced in Siah2(-/-) mice subjected to myocardial infarction. Inhibition of Siah2 or Drp1 in hatching C. elegans reduces their life span. Through modulating Fis1/Drp1 complex availability, our studies identify Siah2 as a key regulator of hypoxia-induced mitochondrial fission and its physiological significance in ischemic injury and nematode life span.
View details for DOI 10.1016/j.molcel.2011.08.045
View details for Web of Science ID 000297387800006
View details for PubMedID 22099302
Small-Molecule Inhibitors of the Wnt Pathway Potently Promote Cardiomyocytes From Human Embryonic Stem Cell-Derived Mesoderm
2011; 109 (4): 360-364
Human embryonic stem cells can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that promote for cardiac lineages are poorly understood and limit the yield of cardiomyocytes.Our aim was to develop a human embryonic stem cell-based high-content screening assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology involved. Screening of libraries of small-molecule pathway modulators was predicted to provide insight into the cellular proteins and signaling pathways that control stem cell cardiogenesis.Approximately 550 known pathway modulators were screened in a high-content screening assay, with hits being called out by the appearance of a red fluorescent protein driven by the promoter of the cardiac-specific MYH6 gene. One potent small molecule was identified that inhibits transduction of the canonical Wnt response within the cell, which demonstrated that Wnt inhibition alone was sufficient to generate cardiomyocytes from human embryonic stem cell-derived mesoderm cells. Transcriptional profiling of inhibitor-treated compared with vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors were very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, which confirmed that Wnt inhibition was the relevant biological activity.Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes; this could yield novel tools for the benefit of pharmaceutical and clinical applications.
View details for DOI 10.1161/CIRCRESAHA.111.249540
View details for Web of Science ID 000293504100004
View details for PubMedID 21737789
Cardiac muscle regeneration: lessons from development
GENES & DEVELOPMENT
2011; 25 (4): 299-309
The adult human heart is an ideal target for regenerative intervention since it does not functionally restore itself after injury yet has a modest regenerative capacity that could be enhanced by innovative therapies. Adult cardiac cells with regenerative potential share gene expression signatures with early fetal progenitors that give rise to multiple cardiac cell types, suggesting that the evolutionarily conserved regulatory networks that drive embryonic heart development might also control aspects of regeneration. Here we discuss commonalities of development and regeneration, and the application of the rich developmental biology heritage to achieve therapeutic regeneration of the human heart.
View details for DOI 10.1101/gad.2018411
View details for Web of Science ID 000287365000003
View details for PubMedID 21325131
View details for PubMedCentralID PMC3042154
What Your Heart Doth Know
CELL STEM CELL
2011; 8 (2): 124-126
Combining embryological insight with careful analysis of early stage cardiomyocyte differentiation, Kattman et al. (2011) in this issue of Cell Stem Cell have defined minimal culture conditions to efficiently produce cardiomyocytes from hESCs and hiPSCs. The lessons learned are applicable to the derivation of other organotypic cell types.
View details for DOI 10.1016/j.stem.2011.01.003
View details for Web of Science ID 000287633400003
View details for PubMedID 21295266
Phenothiazine Neuroleptics Signal to the Human Insulin Promoter as Revealed by a Novel High-Throughput Screen
JOURNAL OF BIOMOLECULAR SCREENING
2010; 15 (6): 663-670
A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic beta-cell. A cell line from human islets in which the expression of insulin and other beta-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of beta-cell differentiated function.
View details for DOI 10.1177/1087057110372257
View details for Web of Science ID 000279950400006
View details for PubMedID 20547533
View details for PubMedCentralID PMC3374493
Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (23): 10514-10519
Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.
View details for DOI 10.1073/pnas.0912260107
View details for Web of Science ID 000278549300035
View details for PubMedID 20498046
Non-Cardiomyocytes Influence the Electrophysiological Maturation of Human Embryonic Stem Cell-Derived Cardiomyocytes During Differentiation
STEM CELLS AND DEVELOPMENT
2010; 19 (6): 783-795
Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca(2+)) imaging, and intracellular recording techniques, we found that intracellular Ca(2+)-cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca(2+)-handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to influence early cardiac development. Our study is the first to demonstrate that non-cardiomyocytes influence electrophysiological maturation of early hESC-CMs in cultures. Defining the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.
View details for DOI 10.1089/scd.2009.0349
View details for Web of Science ID 000279033900004
View details for PubMedID 20001453
Hybrid Median Filter Background Estimator for Correcting Distortions in Microtiter Plate Data
ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES
2010; 8 (2): 238-250
Microtiter plate (MTP) assays often exhibit distortions, such as caused by edge-dependent drying and robotic fluid handling variation. Distortions vary by assay system but can have both systematic patterns (predictable from plate to plate) and random (sporadic and unpredictable) components. Random errors can be especially difficult to resolve by assay optimization alone, and postassay algorithms reported to date have smoothing effects that often blunt hits. We implemented a 5 x 5 bidirectional hybrid median filter (HMF) as a local background estimator to scale each data point to the MTP global background median and compared it with a recently described Discrete Fourier Transform (DFT) technique for correcting errors on computationally and experimentally generated MTP datasets. Experimental data were generated from a 384-well format fluorescent bioassay using cells engineered to express eGFP and DsRED. MTP arrays were produced with and without control treatments used to simulate hits in random wells. The HMF demonstrated the greatest improvements in MTP coefficients of variation and dynamic range (defined by the ratio of average hit amplitude to standard deviation, SD) for all synthetic and experimental MTPs examined. After HMF application to a MTP of eGFP signal from mouse insulinoma (MIN6) cells obtained by a plate-reader, the assay coefficient of variation (CV) decreased from 8.0% in the raw dataset to 5.1% and the hit amplitudes were reduced by only 1% while the DFT method increased the CV by 36.0% and reduced the hit amplitude by 21%. Thus, our results show that the bidirectional HMF provides superior corrections of MTP data distortions while at the same time preserving hit amplitudes and improving dynamic range. The software to perform hybrid median filter MTP corrections is available at http://bccg.burnham.org/HTS/HMF_Download_Page.aspx, password is pbushway.
View details for DOI 10.1089/adt.2009.0242
View details for Web of Science ID 000277283900010
View details for PubMedID 20230301
View details for PubMedCentralID PMC3096555
- Electrophysiological Challenges of Cell-Based Myocardial Repair CIRCULATION 2009; 120 (24): 2496-2508
Natural and Synthetic Regulators of Embryonic Stem Cell Cardiogenesis
2009; 30 (5): 635-642
Debilitating cardiomyocyte loss underlies the progression to heart failure. Although there have been significant advances in treatment, current therapies are intended to improve or preserve heart function rather than regenerate lost myocardium. A major hurdle in implementing a cell-based regenerative therapy is the inefficient differentiation of cardiomyocytes from either endogenous or exogenous stem cell sources. Moreover, cardiomyocytes that develop in human embryonic stem cell (hESC) or human-induced pluripotent stem cell (hIPSC) cultures are comparatively immature, even after prolonged culture, and differences in their calcium handling, ion channel, and force generation properties relative to adult cardiomyocytes raise concerns of improper integration and function after transplantation. Thus, the discovery of natural and novel small molecule synthetic regulators of differentiation and maturation would accelerate the development of stem-cell-based myocardial therapies. Here, we document recent advances in defining natural signaling pathways that direct the multistep cardiomyogenic differentiation program and the development of small molecules that might be used to enhance differentiation as well as the potential characteristics of lead candidates for pharmaceutical stimulation of endogenous myocardial replacement.
View details for DOI 10.1007/s00246-009-9409-2
View details for Web of Science ID 000267033500009
View details for PubMedID 19319460
View details for PubMedCentralID PMC3478151
Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes
2009; 4 (4)
Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications.
View details for DOI 10.1371/journal.pone.0005046
View details for Web of Science ID 000265505700004
View details for PubMedID 19352491
Deletion of Shp2 Tyrosine Phosphatase in Muscle Leads to Dilated Cardiomyopathy, Insulin Resistance, and Premature Death
MOLECULAR AND CELLULAR BIOLOGY
2009; 29 (2): 378-388
The intracellular signaling mechanisms underlying the pathogenesis of cardiac diseases are not fully understood. We report here that selective deletion of Shp2, an SH2-containing cytoplasmic tyrosine phosphatase, in striated muscle results in severe dilated cardiomyopathy in mice, leading to heart failure and premature mortality. Development of cardiomyopathy in this mouse model is coupled with insulin resistance, glucose intolerance, and impaired glucose uptake in striated muscle cells. Shp2 deficiency leads to upregulation of leukemia inhibitory factor-stimulated phosphatidylinositol 3-kinase/Akt, Erk5, and Stat3 pathways in cardiomyocytes. Insulin resistance and impaired glucose uptake in Shp2-deficient mice are at least in part due to impaired protein kinase C-zeta/lambda and AMP-kinase activities in striated muscle. Thus, we have generated a mouse line modeling human patients suffering from cardiomyopathy and insulin resistance. This study reinforces a concept that a compound disease with multiple cardiovascular and metabolic disturbances can be caused by a defect in a single molecule such as Shp2, which modulates multiple signaling pathways initiated by cytokines and hormones.
View details for DOI 10.1128/MCB.01661-08
View details for Web of Science ID 000262045800007
View details for PubMedID 19001090
A novel activity of the Dickkopf-1 amino terminal domain promotes axial and heart development independently of canonical Wnt inhibition
2008; 324 (1): 131-138
The secreted Dickkopf-1 (Dkk1) protein mediates numerous cell fate decisions and morphogenetic processes. Its carboxyl terminal cysteine-rich region (termed C1) binds LRP5/6 and inhibits canonical Wnt signaling. Paradoxically, the isolated C1 domain of Dkk1 as well as Wnt antagonists that act by sequestering Wnts, such as Frz-B, WIF-1 and Crescent, are poor mimics of the inductive and patterning activities of Dkk1 critical for heart and axial development. To understand the basis for the unique properties of Dkk1, we investigated the function of its amino terminal cysteine-rich region (N1). N1 does not bind LRP or Kremen nor inhibit Wnt signaling and has had no known function. We show that it can synergize with BMP antagonism to induce prechordal and axial mesoderm when expressed as an independent protein in Xenopus embryos. Moreover, we show that it can function in trans to complement the activity of C1 protein to mediate two embryologic functions of Dkk1: induction of chordal and prechordal mesoderm and specification of heart tissue from non-cardiogenic mesoderm. Remarkably, N1 also synergizes with WIF-1 and Crescent, indicating that N1 signals independently of C1 and its interactions with LRP. Since cleavage of Dkk1 is not detected, these results define N1 as a novel signaling domain within the intact protein that is responsible for the potent effects of Dkk1 on the induction and patterning of the body axis and heart. We conclude that this new activity is also likely to synergize with canonical Wnt inhibitory in the numerous developmental and disease processes that involve Dkk1.
View details for DOI 10.1016/j.ydbio.2008.09.012
View details for Web of Science ID 000261837200013
View details for PubMedID 18840423
View details for PubMedCentralID PMC3038239
Notch activates cell cycle reentry and progression in quiescent cardiomyocytes
JOURNAL OF CELL BIOLOGY
2008; 183 (1): 129-141
The inability of heart muscle to regenerate by replication of existing cardiomyocytes has engendered considerable interest in identifying developmental or other stimuli capable of sustaining the proliferative capacity of immature cardiomyocytes or stimulating division of postmitotic cardiomyocytes. Here, we demonstrate that reactivation of Notch signaling causes embryonic stem cell-derived and neonatal ventricular cardiomyocytes to enter the cell cycle. The proliferative response of neonatal ventricular cardiomyocytes declines as they mature, such that late activation of Notch triggers the DNA damage checkpoint and G2/M interphase arrest. Notch induces recombination signal-binding protein 1 for Jkappa (RBP-Jkappa)-dependent expression of cyclin D1 but, unlike other inducers, also shifts its subcellular distribution from the cytosol to the nucleus. Nuclear localization of cyclin D1 is independent of RBP-Jkappa. Thus, the influence of Notch on nucleocytoplasmic localization of cyclin D1 is an unanticipated property of the Notch intracellular domain that is likely to regulate the cell cycle in multiple contexts, including tumorigenesis as well as cardiogenesis.
View details for DOI 10.1083/jcb.200806104
View details for Web of Science ID 000259985000013
View details for PubMedID 18838555
- Chemical probes of neural stem cell self-renewal NATURE CHEMICAL BIOLOGY 2007; 3 (5): 246-247
Multiple functions of Cerberus cooperate to induce heart downstream of Nodal
2007; 303 (1): 57-65
The TGFbeta family member Nodal has been implicated in heart induction through misexpression of a dominant negative version of the type I Nodal receptor (Alk4) and targeted deletion of the co-receptor Cripto in murine ESCs and mouse embryos; however, whether Nodal acts directly or indirectly to induce heart tissue or interacts with other signaling molecules or pathways remained unclear. Here we present Xenopus embryological studies demonstrating an unforeseen role for the DAN family protein Cerberus within presumptive foregut endoderm as essential for differentiation of cardiac mesoderm in response to Nodal. Ectopic activation of Nodal signaling in non-cardiogenic ventroposterior mesendoderm, either by misexpression of the Nodal homologue XNr1 together with Cripto or by a constitutively active Alk4 (caAlk4), induced both cardiac markers and Cerberus. Mosaic lineage tracing studies revealed that Nodal/Cripto and caAlk4 induced cardiac markers cell non-autonomously, thus supporting the idea that Cerberus or another diffusible factor is an essential mediator of Nodal-induced cardiogenesis. Cerberus alone was found sufficient to initiate cardiogenesis at a distance from its site of synthesis. Conversely, morpholino-mediated specific knockdown of Cerberus reduced both endogenous cardiomyogenesis and ectopic heart induction resulting from misactivation of Nodal/Cripto signaling. Since the specific knockdown of Cerberus did not abrogate heart induction by the Wnt antagonist Dkk1, Nodal/Cripto and Wnt antagonists appear to initiate cardiogenesis through distinct pathways. This idea was further supported by the combinatorial effect of morpholino-medicated knockdown of Cerberus and Hex, which is required for Dkk1-induced cardiogenesis, and the differential roles of essential downstream effectors: Nodal pathway activation did not induce the transcriptional repressor Hex while Dkk-1 did not induce Cerberus. These studies demonstrated that cardiogenesis in mesoderm depends on Nodal-mediated induction of Cerberus in underlying endoderm, and that this pathway functions in a pathway parallel to cardiogenesis initiated through the induction of Hex by Wnt antagonists. Both pathways operate in endoderm to initiate cardiogenesis in overlying mesoderm.
View details for DOI 10.1016/j.ydbio.2006.10.033
View details for Web of Science ID 000244542800005
View details for PubMedID 17123501
View details for PubMedCentralID PMC1855199
Developmental patterning of the cardiac atrioventricular canal by Notch and Hairy-related transcription factors
2006; 133 (21): 4381-4390
Mutations in Notch2, Jagged1 or homologs of the Hairy-related transcriptional repressor Hey2 cause congenital malformations involving the non-chamber atrioventricular canal (AVC) and inner curvature (IC) regions of the heart, but the underlying mechanisms have not been investigated. By manipulating signaling directly within the developing chick heart, we demonstrated that Notch2, Hey1 and Hey2 initiate a signaling cascade that delimits the non-chamber AVC and IC regions. Specifically, misactivation of Notch2 signaling, or misexpression of either Hey1 or Hey2, repressed Bmp2. Because Jagged (also known as Serrate in non-mammalian species) ligands were found to be present in prospective chamber myocardium, these data support the model that Notch2 and Hey proteins cause the progressive restriction of Bmp2 expression to within the developing AVC and IC, where it is essential for differentiation. Misactivation or inhibition of Notch2 specifically induced or inhibited Hey1, respectively, but these manipulations did not affect Hey2, implicating Hey1 as the direct mediator of Notch2. Bmp2 within the developing AVC and IC has been shown to induce Tbx2, and we found that Tbx2 misexpression inhibited the expression of both Hey1 and Hey2. Tbx2, therefore, is envisaged to constitute a feedback loop that sharpens the border with the developing AVC and IC by delimiting Hey gene expression to within prospective chamber regions. Analysis of the loss-of-function phenotype in mouse embryos homozygous for targeted disruption of Hey2 revealed an expanded AVC domain of Bmp2. Similarly, zebrafish gridlock (Hey2 homolog) mutant embryos showed ectopic expression of Bmp4, which normally marks AVC myocardium in this species. Thus, Hey pathway regulation of cardiac Bmp appears to be an evolutionarily conserved mechanism to delimit AVC and IC fate, and provides a potential mechanistic explanation for cardiac malformations caused by mutations in Serrate/Jagged1 and Notch signaling components.
View details for DOI 10.1242/dev.02607
View details for Web of Science ID 000241217400023
View details for PubMedID 17021042
Beta-cell differentiation from nonendocrine epithelial cells of the adult human pancreas
2006; 12 (3): 310-316
The nature and even existence of adult pancreatic endocrine stem or progenitor cells is a subject of controversy in the field of beta-cell replacement for diabetes. One place to search for such cells is in the nonendocrine fraction of cells that remain after islet isolation, which consist of a mixture of epithelia and mesenchyme. Culture in G418 resulted in elimination of the mesenchymal cells, leaving a highly purified population of nonendocrine pancreatic epithelial cells (NEPECs). To evaluate their differentiation potential, NEPECs were heritably marked and transplanted under the kidney capsule of immunodeficient mice. When cotransplanted with fetal pancreatic cells, NEPECs were capable of endocrine differentiation. We found no evidence of beta-cell replication or cell fusion that could have explained the appearance of insulin positive cells from a source other than NEPECs. Nonendocrine-to-endocrine differentiation of NEPECs supports the existence of endocrine stem or progenitor cells within the epithelial compartment of the adult human pancreas.
View details for DOI 10.1038/nm1367
View details for Web of Science ID 000235802900030
View details for PubMedID 16491084
Heart induction by Wnt antagonists depends on the homeodomain transcription factor Hex
GENES & DEVELOPMENT
2005; 19 (3): 387-396
Inhibition of canonical Wnt/beta-catenin signaling by Dickkopf-1 (Dkk-1) or Crescent initiates cardiogenesis in vertebrate embryos. However, nearly nothing is known about the downstream effectors of these secreted Wnt antagonists or the mechanism by which they activate heart formation. Here we show that Wnt antagonists in Xenopus stimulate cardiogenesis non-cell-autonomously, up to several cells away from those in which canonical Wnt/beta-catenin signaling is blocked, indicative of an indirect role in heart induction. A screen for downstream mediators revealed that Dkk-1 and other inhibitors of the canonical Wnt pathway induce the homeodomain transcription factor Hex, which is normally expressed in endoderm underlying the presumptive cardiac mesoderm in amphibian, bird, and mammalian embryos. Loss of Hex function blocks both endogenous heart development and ectopic heart induction by Dkk-1. As with the canonical Wnt pathway antagonists, ectopic Hex induces expression of cardiac markers non-cell-autonomously. Thus, to initiate cardiogenesis, Wnt antagonists act on endoderm to up-regulate Hex, which, in turn, controls production of a diffusible heart-inducing factor. This novel function for Hex suggests an etiology for the cardiac malformations in Hex mutant mice and will make possible the isolation of factors that induce heart directly in the mesoderm.
View details for DOI 10.1101/gad.1279405
View details for Web of Science ID 000226800100010
View details for PubMedID 15687261
View details for PubMedCentralID PMC546516
- No pancreatic endocrine stem cells? NEW ENGLAND JOURNAL OF MEDICINE 2004; 351 (10): 1024-1026
Asymmetries in H+/K+-ATPase and cell membrane potentials comprise a very early step in left-right patterning
2002; 111 (1): 77-89
A pharmacological screen identified the H+ and K+ ATPase transporter as obligatory for normal orientation of the left-right body axis in Xenopus. Maternal H+/K+-ATPase mRNA is symmetrically expressed in the 1-cell Xenopus embryo but becomes localized during the first two cell divisions, demonstrating that asymmetry is generated within two hours postfertilization. Although H+/K+-ATPase subunit mRNAs are symmetrically localized in chick embryos, an endogenous H+/K+-ATPase-dependent difference in membrane voltage potential exists between the left and right sides of the primitive streak. In both species, pharmacologic or genetic perturbation of endogenous H+/K+-ATPase randomized the sided pattern of asymmetrically expressed genes and induced organ heterotaxia. Thus, LR asymmetry determination depends on a very early differential ion flux created by H+/K+-ATPase activity.
View details for Web of Science ID 000178461900010
View details for PubMedID 12372302