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1.  Inhibition of miR-25 Improves Cardiac Contractility in the Failing Heart 
Nature  2014;508(7497):531-535.
Heart failure is characterized by a debilitating decline in cardiac function1, and recent clinical trial results indicate that improving the contractility of heart muscle cells by boosting intracellular calcium handling might be an effective therapy2,3. microRNAs (miRs) are dysregulated with heart failure4,5 but whether they control contractility or constitute therapeutic targets remain speculative. Using high throughput, functional screening of the human microRNAome, we identified miRs that suppress intracellular calcium handling in heart muscle by interacting with mRNA encoding the sarcoplasmic reticulum calcium uptake pump SERCA2a. Of 875 miRs tested, miR-25 potently delayed calcium uptake kinetics in cardiomyocytes in vitro and was upregulated in heart failure, both in mice and humans. Whereas 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 dramatically halted established heart failure in a mouse model, improving cardiac function and survival relative to a control antagomiR. These data reveal that increased expression of endogenous miR-25 contributes to declining cardiac function during heart failure and suggests that it might be targeted therapeutically to restore function.
doi:10.1038/nature13073
PMCID: PMC4131725  PMID: 24670661
heart failure; microRNAs; miRNAs; sarcoplasmic reticulum; SERCA2a; calcium
2.  Jumonji and cardiac fate 
Circulation research  2013;113(7):837-839.
Considerable information is known about the extracellular signals that drive pluripotent stem cells through a stepwise program to become cardiomyocytes. A paper by Ohtani et al. in this issue now links extrinsic signals to histone demethylation needed to form mesoderm, the first step in cardiogenesis.
doi:10.1161/CIRCRESAHA.113.302202
PMCID: PMC3974910  PMID: 24030015
3.  Zebrafish narrowminded suggests a genetic link between formation of neural crest and primary sensory neurons 
Development (Cambridge, England)  1999;126(18):3969-3979.
SUMMARY
In the developing vertebrate nervous system, both neural crest and sensory neurons form at the boundary between non-neural ectoderm and the neural plate. From an in situ hybridization based expression analysis screen, we have identified a novel zebrafish mutation, narrowminded (nrd), which reduces the number of early neural crest cells and eliminates Rohon-Beard (RB) sensory neurons. Mosaic analysis has shown that the mutation acts cell autonomously suggesting that nrd is involved in either the reception or interpretation of signals at the lateral neural plate boundary. Characterization of the mutant phenotype indicates that nrd is required for a primary wave of neural crest cell formation during which progenitors generate both RB sensory neurons and neural crest cells. Moreover, the early deficit in neural crest cells in nrd homozygotes is compensated later in development. Thus, we propose that a later wave can compensate for the loss of early neural crest cells but, interestingly, not the RB sensory neurons. We discuss the implications of these findings for the possibility that RB sensory neurons and neural crest cells share a common evolutionary origin.
PMCID: PMC4059008  PMID: 10457007
narrowminded; Neural crest; Sensory neurons; Cell signalling; Zebrafish
4.  Reprogramming the Cardiac Field 
Circulation research  2014;114(3):409-411.
Two recent reports show that it is possible to reprogram somatic cells to a stem cell state using only a chemical cocktail or by inducing genes in tissues of engineered mice. These studies herald a time when cellular plasticity can be manipulated directly in the body to regenerate damaged tissues and organs. How do we go from these milestones to therapeutic heart regeneration?
doi:10.1161/CIRCRESAHA.113.302946
PMCID: PMC4046860  PMID: 24481840
5.  Zebrafish narrowminded disrupts the transcription factor prdm1 and is required for neural crest and sensory neuron specification 
Developmental biology  2005;278(2):347-357.
Specification of both neural crest cells and Rohon–Beard (RB) sensory neurons involves a complex series of interactions between the neural and non-neural ectoderm. The molecular mechanisms directing this process are not well understood. The zebrafish narrowminded (nrd) mutation is unique, since it is one of two mutations in which defects are observed in both cell populations: it leads to a complete absence of RB neurons and a reduction in neural crest cells and their derivatives. Here, we show that nrd is a mutation in prdm1, a SET/zincfinger domain transcription factor. A Morpholino-mediated depletion of prdm1 phenocopies the nrd mutation, and conversely overexpression of prdm1 mRNA rescues the nrd RB sensory neuron and neural crest phenotype. prdm1 is expressed at the border of the neural plate within the domain where neural crest cells and RB sensory neurons form. Analysis of prdm1 function by overexpression indicates that prdm1 functions to promote the cell fate specification of both neural crest cells and RB sensory neurons, most likely as a downstream effector of the BMP signaling pathway.
doi:10.1016/j.ydbio.2004.11.014
PMCID: PMC4028833  PMID: 15680355
Narrowminded; Neural crest; Rohon–Beard sensory neurons; Blimp-1
6.  Dlx proteins position the neural plate border and determine adjacent cell fates 
Development (Cambridge, England)  2003;130(2):331-342.
Summary
The lateral border of the neural plate is a major source of signals that induce primary neurons, neural crest cells and cranial placodes as well as provide patterning cues to mesodermal structures such as somites and heart. Whereas secreted BMP, FGF and Wnt proteins influence the differentiation of neural and non-neural ectoderm, we show here that members of the Dlx family of transcription factors position the border between neural and non-neural ectoderm and are required for the specification of adjacent cell fates. Inhibition of endogenous Dlx activity in Xenopus embryos with an EnR-Dlx homeodomain fusion protein expands the neural plate into non-neural ectoderm tissue whereas ectopic activation of Dlx target genes inhibits neural plate differentiation. Importantly, the stereotypic pattern of border cell fates in the adjacent ectoderm is re-established only under conditions where the expanded neural plate abuts Dlx-positive non-neural ectoderm. Experiments in which presumptive neural plate was grafted to ventral ectoderm reiterate induction of neural crest and placodal lineages and also demonstrate that Dlx activity is required in non-neural ectoderm for the production of signals needed for induction of these cells. We propose that Dlx proteins regulate intercellular signaling across the interface between neural and non-neural ectoderm that is critical for inducing and patterning adjacent cell fates.
PMCID: PMC4018238  PMID: 12466200
Dlx; Neural crest; Neural induction; Xenopus; hairy2a; slug; snail; msx
7.  iPSCs in Cardiovascular Drug Discovery 
Circulation research  2013;112(3):534-548.
The unexpected discovery that somatic cells can be reprogrammed to a pluripotent state yielding induced pluripotent stem cells (iPSCs) 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 iPSC-based models of cardiovascular disease and their multiple applications in drug development.
doi:10.1161/CIRCRESAHA.111.250266
PMCID: PMC3706265  PMID: 23371902
induced pluripotent stem cells; high throughput screening; functional genomics; chemical biology
8.  Technical Variations in Low-Input RNA-seq Methodologies 
Scientific Reports  2014;4:3678.
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.
doi:10.1038/srep03678
PMCID: PMC3890974  PMID: 24419370
9.  Synthesis and SAR of b-Annulated 1,4-Dihydropyridines Define Cardiomyogenic Compounds as Novel Inhibitors of TGFβ Signaling 
Journal of medicinal chemistry  2012;55(22):9946-9957.
A medium-throughput murine embryonic stem cell (mESC)-based high-content screening of 17,000 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. Since 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 IC50'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.
doi:10.1021/jm301144g
PMCID: PMC3518459  PMID: 23130626
1,4-dihydropyridines; enantiomers; TGFβ inhibition; inducers of TGFβ type II receptor degradation; ITD-1; structure-activity-relationship; cardiomyogenesis; murine embryonic stem cells
10.  TGFβ-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.
doi:10.1089/scd.2012.0277
PMCID: PMC4146498  PMID: 22765842
11.  Serum-Free Generation of Multipotent Mesoderm (Kdr-positive) Progenitor Cells in Mouse Embryonic Stem Cells For Functional Genomics Screening 
Current protocols in stem cell biology  2012;CHAPTER:Unit-1F.13.
This unit describes a robust protocol for producing multipotent Kdr-expressing mesoderm progenitor cells in serum-free conditions and functional genomics screening using these cells. Kdr-positive cells are known to be able to differentiate into a wide array of mesoderm derivatives including, vascular endothelial cells, cardiomyocytes, hematopietic 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. The oligonucleotide transfection protocol used to functionally screen siRNA and microRNA libraries is a powerful tool to reveal networks of genes, signaling proteins and microRNAs that control the diversification of cardiovascular lineages from multipotent progenitors. The discussion addresses technical limitations, troubleshooting and potential applications.
doi:10.1002/9780470151808.sc01f13s23
PMCID: PMC3562707  PMID: 23154934
mouse embryonic stem cells; mesendoderm; mesoderm; endoderm; siRNA transfection; Kdr; Foxa2
12.  High Throughput Measurement of Ca2+ Dynamics for Drug Risk Assessment in Human Stem Cell-derived Cardiomyocytes by Kinetic Image Cytometry 
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.
doi:10.1016/j.vascn.2012.08.167
PMCID: PMC3667588  PMID: 22926323
13.  A Nodal to TGFβ Cascade Exerts Biphasic Control Over Cardiopoiesis 
Circulation research  2012;111(7):876-881.
Rationale
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.
Objective
Determine the function(s) of TGFβ family members during stem cell cardiogenesis.
Methods and Results
Murine embryonic stem cells (mESCs) 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 ESC cultures at day 0–2 of differentiation. However, Nodal expression declines by day 4 due to feedback inhibition whereas TGFβ persists. At later stages (day 4–6), TGFβ suppresses the formation of cardiomyocytes from multipotent Kdr+ progenitors, while promoting the differentiation of vascular smooth muscle and endothelial cells.
Conclusions
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.
doi:10.1161/CIRCRESAHA.112.270272
PMCID: PMC3766357  PMID: 22872153
Nodal; Cripto; TGFβ2; Kdr; cardiogenesis
14.  Developing microRNA screening as a functional genomics tool for disease research 
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.
doi:10.3389/fphys.2013.00223
PMCID: PMC3753477  PMID: 23986717
systems biology and network biology; microRNA target; protein-protein interaction; functional genomics; functional screens; proteomics
15.  Small molecule-mediated TGFβ Type II receptor degradation promotes cardiomyogenesis in embryonic stem cells 
Cell stem cell  2012;11(2):242-252.
Summary
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 novel 1,4-dihydropyridine inducer of type II TGFβ 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 (IC50 ~ 0.4-0.8μM). ITD-1 was used to evaluate TGFβ 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. Together, ITD-1 is the first selective TGFβ inhibitor and reveals an unexpected role for TGFβ signaling in controlling cardiomyocyte differentiation from multipotent cardiovascular precursors.
doi:10.1016/j.stem.2012.04.025
PMCID: PMC3419596  PMID: 22862949
Small molecule inhibitor; TGFβ; proteasomal degradation; embryonic stem cells; mesoderm; cardiogenesis; 1,4-dihydropyridine
16.  Developmental patterning of the cardiac atrioventricular canal by Notch and Hairy-related transcription factors 
Development (Cambridge, England)  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.
doi:10.1242/dev.02607
PMCID: PMC3619037  PMID: 17021042
Notch; Hairy-related transcription factor; HRT; HES; Hey; Gridlock; Atrioventricular canal; T-box; Tbx
17.  APJ ACTS AS A DUAL RECEPTOR IN CARDIAC HYPERTROPHY 
Nature  2012;488(7411):394-398.
Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues1. Here we report that genetic loss of APJ confers resistance to chronic pressure overload by dramatically 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 mechano-sensor. 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 for the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.
doi:10.1038/nature11263
PMCID: PMC3422434  PMID: 22810587
18.  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 developed a structure activity relationship to optimize the cardiogenic properties of 1. We prepared analogs with a range of potencies (low nanomolar to inactive) for Wnt/β-catenin inhibition and for cardiogenic induction. Both functional activities correlated positively (r2 = 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 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 drug-like cardiogenic molecules.
doi:10.1021/jm2010223
PMCID: PMC3335202  PMID: 22191557
Human Embryonic Stem Cell; Wnt inhibition; SAR; cardiomyogenesis; IWR-1; inhibitors
19.  Characterization of a novel angiogenic model based on stable, fluorescently labeled endothelial cell lines amenable to scale-up for high content screening 
Background
Blood vessel formation is important for many physiological and pathological processes, and is therefore a critical target for drug development. Inhibiting angiogenesis to starve a tumor or promoting “normalization” of tumor blood vessels in order to facilitate delivery of anticancer drugs are both areas of active research. Recapitulation of vessel formation by human cells in vitro allows investigating cell-cell and cell-matrix interactions in a controlled environment, and is thereby a crucial step in developing high content (HC) and high throughput (HT) screening assays to search for modulators of blood vessel formation. Human umbilical vein endothelial cells (HUVECs) exemplify primary cells used in angiogenesis assays. However, primary cells have significant limitations that include phenotypic decay and/or senescence by 6–8 passages in culture, making stable integration of fluorescent markers and large-scale expansion for high throughput screening problematic. To overcome these limitations for HTS, we developed a novel angiogenic model system that employs stable fluorescent endothelial cell lines based on immortalized human microvascular endothelial cells (HMEC-1, hereafter HMECs). We then evaluated HMEC cultures, both alone and co-cultured with an epicardial mesothelial cell (EMC) line that contributes vascular smooth muscle cells, to determine suitability for HTS or HCS.
Results
The endothelial and epicardial lines were engineered to express a panel of nuclear- and cytoplasm-localized fluorescent proteins to be mixed and matched to suit particular experimental goals. HMECs retained their angiogenic potential and stably expressed fluorescent proteins for at least 13 passages after transduction. Within 8 hours upon plating on Matrigel, the cells migrated and coalesced into networks of vessel-like structures. If co-cultured with EMCs, the branches formed cylindrical-shaped structures of HMECs surrounded by EMC-derivatives reminiscent of vessels. Network formation measurements revealed responsiveness to media composition and control compounds.
Conclusions
HMEC-based lines retain most of the angiogenic features of primary endothelial cells, yet possess long-term stability and ease of culture, making them intriguing candidates for large-scale primary HC and HT screening (of ~10,000–1,000,000 molecules). Furthermore, inclusion of EMCs demonstrates the feasibility of using epicardial-derived cells, which normally contribute to smooth muscle, to model large vessel formation. In summary, the immortalized fluorescent HMEC and EMC lines and straightforward culture conditions will enable assay development for HCS of angiogenesis.
doi:10.1042/BC20100146
PMCID: PMC3538155  PMID: 21732911
Angiogenesis; microvascular; fluorescent proteins; time-lapse microscopy of live cells in culture
20.  Fine-Tuning of Drp1/Fis1 Availability by AKAP121/Siah2 Regulates Mitochondrial Adaptation to Hypoxia 
Molecular Cell  2011;44(4):532-544.
SUMMARY
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.
doi:10.1016/j.molcel.2011.08.045
PMCID: PMC3360955  PMID: 22099302
21.  Natural and Synthetic Regulators of Embryonic Stem Cell Cardiogenesis 
Pediatric cardiology  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.
doi:10.1007/s00246-009-9409-2
PMCID: PMC3478151  PMID: 19319460
Cardiomyocyte; Embryonic stem cell; Chemical screening
22.  HNF4α Antagonists Discovered by a High-Throughput Screen for Modulators of the Human Insulin Promoter 
Chemistry & biology  2012;19(7):806-818.
SUMMARY
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 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.
doi:10.1016/j.chembiol.2012.05.014
PMCID: PMC3447631  PMID: 22840769
23.  Small molecule inhibitors of the Wnt pathway potently promote cardiomyocytes from human embryonic stem cell derived mesoderm 
Circulation Research  2011;109(4):360-364.
Rationale
Human embryonic stem cells (hESCs) can form cardiomyocytes when cultured under differentiation conditions. Although the initiating step of mesoderm formation is well characterized, the subsequent steps that enrich for cardiac lineages are poorly understood and limit the yield of cardiomyocytes.
Objective
Our aim was to develop a hESC-based high content screening (HCS) assay to discover small molecules that drive cardiogenic differentiation after mesoderm is established to improve our understanding of the biology. 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.
Methods and results
About 550 known pathway modulators were screened in a HCS 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, demonstrating that Wnt inhibition alone is sufficient for deriving cardiomyocytes from hESC originating mesoderm cells. Transcriptional profiling of inhibitor-treated compared to vehicle-treated samples further indicated that inhibition of Wnt does not induce other mesoderm lineages. Notably, several other Wnt inhibitors are very efficient in inducing cardiogenesis, including a molecule that prevents Wnts from being secreted by the cell, confirming Wnt inhibition as the relevant biological activity.
Conclusions
Pharmacological inhibition of Wnt signaling is sufficient to drive human mesoderm cells to form cardiomyocytes, yielding novel tools for the benefit of pharmaceutical and clinical applications.
doi:10.1161/CIRCRESAHA.111.249540
PMCID: PMC3327303  PMID: 21737789
human embryonic stem cells; small molecule Wnt inhibitors; cardiogenesis
24.  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 β-cell. A cell line from human islets in which the expression of insulin and other β-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 β-cell differentiated function.
doi:10.1177/1087057110372257
PMCID: PMC3374493  PMID: 20547533
diabetes; chlorpromazine; ethopropazine
25.  Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential 
Stem Cells International  2012;2012:926463.
Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.
doi:10.1155/2012/926463
PMCID: PMC3369526  PMID: 22701128

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