Publications by authors named "Milena Furtado"

30 Publications

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Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction.

Nat Commun 2021 01 29;12(1):681. Epub 2021 Jan 29.

Institute for Cardiovascular Regeneration, Goethe University Frankfurt, Frankfurt am Main, Germany.

Endothelial cells play a critical role in the adaptation of tissues to injury. Tissue ischemia induced by infarction leads to profound changes in endothelial cell functions and can induce transition to a mesenchymal state. Here we explore the kinetics and individual cellular responses of endothelial cells after myocardial infarction by using single cell RNA sequencing. This study demonstrates a time dependent switch in endothelial cell proliferation and inflammation associated with transient changes in metabolic gene signatures. Trajectory analysis reveals that the majority of endothelial cells 3 to 7 days after myocardial infarction acquire a transient state, characterized by mesenchymal gene expression, which returns to baseline 14 days after injury. Lineage tracing, using the Cdh5-CreERT2;mT/mG mice followed by single cell RNA sequencing, confirms the transient mesenchymal transition and reveals additional hypoxic and inflammatory signatures of endothelial cells during early and late states after injury. These data suggest that endothelial cells undergo a transient mes-enchymal activation concomitant with a metabolic adaptation within the first days after myocardial infarction but do not acquire a long-term mesenchymal fate. This mesenchymal activation may facilitate endothelial cell migration and clonal expansion to regenerate the vascular network.
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http://dx.doi.org/10.1038/s41467-021-20905-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846794PMC
January 2021

Cross-Priming Dendritic Cells Exacerbate Immunopathology After Ischemic Tissue Damage in the Heart.

Circulation 2021 Feb 10;143(8):821-836. Epub 2020 Dec 10.

National Heart and Lung Institute, Imperial College London, UK (A.S., H.S.K., A.P., C.J., M.A., R.A.C., M.B., M.D.S., S.E.H., F.S.N., N.R., S.S.).

Background: Ischemic heart disease is a leading cause of heart failure and despite advanced therapeutic options, morbidity and mortality rates remain high. Although acute inflammation in response to myocardial cell death has been extensively studied, subsequent adaptive immune activity and anti-heart autoimmunity may also contribute to the development of heart failure. After ischemic injury to the myocardium, dendritic cells (DC) respond to cardiomyocyte necrosis, present cardiac antigen to T cells, and potentially initiate a persistent autoimmune response against the heart. Cross-priming DC have the ability to activate both CD4 helper and CD8 cytotoxic T cells in response to necrotic cells and may thus be crucial players in exacerbating autoimmunity targeting the heart. This study investigates a role for cross-priming DC in post-myocardial infarction immunopathology through presentation of self-antigen from necrotic cardiac cells to cytotoxic CD8 T cells.

Methods: We induced type 2 myocardial infarction-like ischemic injury in the heart by treatment with a single high dose of the β-adrenergic agonist isoproterenol. We characterized the DC population in the heart and mediastinal lymph nodes and analyzed long-term cardiac immunopathology and functional decline in wild type and -depleted mice lacking DC cross-priming function.

Results: A diverse DC population, including cross-priming DC, is present in the heart and activated after ischemic injury. mice deficient in DC cross-priming are protected from persistent immune-mediated myocardial damage and decline of cardiac function, likely because of dampened activation of cytotoxic CD8 T cells.

Conclusion: Activation of cytotoxic CD8 T cells by cross-priming DC contributes to exacerbation of postischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent postischemic immunopathology and heart failure.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.044581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899721PMC
February 2021

Dynamic Interstitial Cell Response during Myocardial Infarction Predicts Resilience to Rupture in Genetically Diverse Mice.

Cell Rep 2020 03;30(9):3149-3163.e6

The Jackson Laboratory, Bar Harbor, ME 04609, USA. Electronic address:

Cardiac ischemia leads to the loss of myocardial tissue and the activation of a repair process that culminates in the formation of a scar whose structural characteristics dictate propensity to favorable healing or detrimental cardiac wall rupture. To elucidate the cellular processes underlying scar formation, here we perform unbiased single-cell mRNA sequencing of interstitial cells isolated from infarcted mouse hearts carrying a genetic tracer that labels epicardial-derived cells. Sixteen interstitial cell clusters are revealed, five of which were of epicardial origin. Focusing on stromal cells, we define 11 sub-clusters, including diverse cell states of epicardial- and endocardial-derived fibroblasts. Comparing transcript profiles from post-infarction hearts in C57BL/6J and 129S1/SvImJ inbred mice, which displays a marked divergence in the frequency of cardiac rupture, uncovers an early increase in activated myofibroblasts, enhanced collagen deposition, and persistent acute phase response in 129S1/SvImJ mouse hearts, defining a crucial time window of pathological remodeling that predicts disease outcome.
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http://dx.doi.org/10.1016/j.celrep.2020.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059115PMC
March 2020

Doxorubicin-Induced Cardiotoxicity in Collaborative Cross (CC) Mice Recapitulates Individual Cardiotoxicity in Humans.

G3 (Bethesda) 2019 08 8;9(8):2637-2646. Epub 2019 Aug 8.

The Jackson Laboratory, Bar Harbor, ME 04609.

Anthracyclines cause progressive cardiotoxicity whose ultimate severity is individual to the patient. Genetic determinants contributing to this variation are difficult to study using current mouse models. Our objective was to determine whether a spectrum of anthracycline induced cardiac disease can be elicited across 10 Collaborative Cross mouse strains given the same dose of doxorubicin. Mice from ten distinct strains were given 5 mg/kg of doxorubicin intravenously once weekly for 5 weeks (total 25 mg/kg). Mice were killed at acute or chronic timepoints. Body weight was assessed weekly, followed by terminal complete blood count, pathology and a panel of biomarkers. Linear models were fit to assess effects of treatment, sex, and sex-by-treatment interactions for each timepoint. Impaired growth and cardiac pathology occurred across all strains. Severity of these varied by strain and sex, with greater severity in males. Cardiac troponin I and myosin light chain 3 demonstrated strain- and sex-specific elevations in the acute phase with subsequent decline despite ongoing progression of cardiac disease. Acute phase cardiac troponin I levels predicted the ultimate severity of cardiac pathology poorly, whereas myosin light chain 3 levels predicted the extent of chronic cardiac injury in males. Strain- and sex-dependent renal toxicity was evident. Regenerative anemia manifested during the acute period. We confirm that variable susceptibility to doxorubicin-induced cardiotoxicity observed in humans can be modeled in a panel of CC strains. In addition, we identified a potential predictive biomarker in males. CC strains provide reproducible models to explore mechanisms contributing to individual susceptibility in humans.
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http://dx.doi.org/10.1534/g3.119.400232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686936PMC
August 2019

Variable outcomes of human heart attack recapitulated in genetically diverse mice.

NPJ Regen Med 2019 4;4. Epub 2019 Mar 4.

1Australian Regenerative Medicine Institute, Monash University, Clayton, VIC Australia.

Clinical variation in patient responses to myocardial infarction (MI) has been difficult to model in laboratory animals. To assess the genetic basis of variation in outcomes after heart attack, we characterized responses to acute MI in the Collaborative Cross (CC), a multi-parental panel of genetically diverse mouse strains. Striking differences in post-MI functional, morphological, and myocardial scar features were detected across 32 CC founder and recombinant inbred strains. Transcriptomic analyses revealed a plausible link between increased intrinsic cardiac oxidative phosphorylation levels and MI-induced heart failure. The emergence of significant quantitative trait loci for several post-MI traits indicates that utilizing CC strains is a valid approach for gene network discovery in cardiovascular disease, enabling more accurate clinical risk assessment and prediction.
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http://dx.doi.org/10.1038/s41536-019-0067-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399323PMC
March 2019

Metformin intervention prevents cardiac dysfunction in a murine model of adult congenital heart disease.

Mol Metab 2019 02 15;20:102-114. Epub 2018 Nov 15.

The Jackson Laboratory, USA; Australian Regenerative Medicine Institute, Monash University, Australia. Electronic address:

Objective: Congenital heart disease (CHD) is the most frequent birth defect worldwide. The number of adult patients with CHD, now referred to as ACHD, is increasing with improved surgical and treatment interventions. However the mechanisms whereby ACHD predisposes patients to heart dysfunction are still unclear. ACHD is strongly associated with metabolic syndrome, but how ACHD interacts with poor modern lifestyle choices and other comorbidities, such as hypertension, obesity, and diabetes, is mostly unknown.

Methods: We used a newly characterized mouse genetic model of ACHD to investigate the consequences and the mechanisms associated with combined obesity and ACHD predisposition. Metformin intervention was used to further evaluate potential therapeutic amelioration of cardiac dysfunction in this model.

Results: ACHD mice placed under metabolic stress (high fat diet) displayed decreased left ventricular ejection fraction. Comprehensive physiological, biochemical, and molecular analysis showed that ACHD hearts exhibited early changes in energy metabolism with increased glucose dependence as main cardiac energy source. These changes preceded cardiac dysfunction mediated by exposure to high fat diet and were associated with increased disease severity. Restoration of metabolic balance by metformin administration prevented the development of heart dysfunction in ACHD predisposed mice.

Conclusions: This study reveals that early metabolic impairment reinforces heart dysfunction in ACHD predisposed individuals and diet or pharmacological interventions can be used to modulate heart function and attenuate heart failure. Our study suggests that interactions between genetic and metabolic disturbances ultimately lead to the clinical presentation of heart failure in patients with ACHD. Early manipulation of energy metabolism may be an important avenue for intervention in ACHD patients to prevent or delay onset of heart failure and secondary comorbidities. These interactions raise the prospect for a translational reassessment of ACHD presentation in the clinic.
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http://dx.doi.org/10.1016/j.molmet.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358551PMC
February 2019

The interstitium in cardiac repair: role of the immune-stromal cell interplay.

Nat Rev Cardiol 2018 10;15(10):601-616

The Jackson Laboratory, Bar Harbor, ME, USA.

Cardiac regeneration, that is, restoration of the original structure and function in a damaged heart, differs from tissue repair, in which collagen deposition and scar formation often lead to functional impairment. In both scenarios, the early-onset inflammatory response is essential to clear damaged cardiac cells and initiate organ repair, but the quality and extent of the immune response vary. Immune cells embedded in the damaged heart tissue sense and modulate inflammation through a dynamic interplay with stromal cells in the cardiac interstitium, which either leads to recapitulation of cardiac morphology by rebuilding functional scaffolds to support muscle regrowth in regenerative organisms or fails to resolve the inflammatory response and produces fibrotic scar tissue in adult mammals. Current investigation into the mechanistic basis of homeostasis and restoration of cardiac function has increasingly shifted focus away from stem cell-mediated cardiac repair towards a dynamic interplay of cells composing the less-studied interstitial compartment of the heart, offering unexpected insights into the immunoregulatory functions of cardiac interstitial components and the complex network of cell interactions that must be considered for clinical intervention in heart diseases.
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http://dx.doi.org/10.1038/s41569-018-0077-xDOI Listing
October 2018

Mutations in the Katnb1 gene cause left-right asymmetry and heart defects.

Dev Dyn 2017 12 5;246(12):1027-1035. Epub 2017 Sep 5.

The Development and Stem Cells Program of Monash Biomedicine Discovery Institute and The Department of Anatomy and Developmental Biology, Monash University, Melbourne, Australia.

Background: The microtubule-severing protein complex katanin is composed two subunits, the ATPase subunit, KATNA1, and the noncatalytic regulatory subunit, KATNB1. Recently, the Katnb1 gene has been linked to infertility, regulation of centriole and cilia formation in fish and mammals, as well as neocortical brain development. KATNB1 protein is expressed in germ cells in humans and mouse, mitotic/meiotic spindles and cilia, although the full expression pattern of the Katnb1 gene has not been described.

Results: Using a knockin-knockout mouse model of Katnb1 dysfunction we demonstrate that Katnb1 is ubiquitously expressed during embryonic development, although a stronger expression is seen in the crown cells of the gastrulation organizer, the murine node. Furthermore, null and hypomorphic Katnb1 gene mutations show a novel correlation between Katnb1 dysregulation and the development of impaired left-right signaling, including cardiac malformations.

Conclusions: Katanin function is a critical regulator of heart development in mice. These findings are potentially relevant to human cardiac development. Developmental Dynamics 246:1027-1035, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24564DOI Listing
December 2017

Properties and Immune Function of Cardiac Fibroblasts.

Adv Exp Med Biol 2017 ;1003:35-70

The Jackson Laboratory, Bar Harbor, ME, 04609, USA.

This chapter will discuss the role of cardiac fibroblasts as a target of various immunological inputs as well as an immunomodulatory hub of the heart through interaction with immune cell types and chemokine or cytokine signaling. While the purpose of this chapter is to explore the immunomodulatory properties of cardiac fibroblasts, it is important to note that cardiac fibroblasts are not a homogeneous cell type, but have a unique embryological origin and molecular identity. Specific properties of cardiac fibroblasts may influence the way they interact with the heart microenvironment to promote healthy homeostatic function or respond to pathological insults. Therefore, we will briefly discuss these aspects of cardiac fibroblast biology and then focus on their immunomodulatory role in the heart.
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http://dx.doi.org/10.1007/978-3-319-57613-8_3DOI Listing
October 2017

Combinatorial Ranking of Gene Sets to Predict Disease Relapse: The Retinoic Acid Pathway in Early Prostate Cancer.

Front Oncol 2017 15;7:30. Epub 2017 Mar 15.

Faculty of Information Technology, Monash University , Melbourne, VIC , Australia.

Background: Quantitative high-throughput data deposited in consortia such as International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) present opportunities and challenges for computational analyses.

Methods: We present a computational strategy to systematically rank and investigate a large number (2-2) of clinically testable gene sets, using combinatorial gene subset generation and disease-free survival (DFS) analyses. This approach integrates protein-protein interaction networks, gene expression, DNA methylation, and copy number data, in association with DFS profiles from patient clinical records.

Results: As a case study, we applied this pipeline to systematically analyze the role of in prostate cancer (PCa). We have previously found this gene to have multiple roles in disease and homeostasis, and here we investigate the role of the associated gene/protein networks in PCa, using our methodology in combination with PCa patient clinical profiles from ICGC and TCGA databases. Relationships between gene signatures and relapse were analyzed using Kaplan-Meier (KM) log-rank analysis and multivariable Cox regression. Relative expression versus pooled mean from diploid population was used for -statistics calculation. Gene/protein interaction network analyses generated 11 core genes associated with ; combinatorial ranking of the power set of these core genes identified two gene sets (out of 2 - 1 = 2,047 combinations) with significant correlation with disease relapse (KM log rank  < 0.05). For the more significant of these two sets, referred to as the optimal gene set (OGS), patients have median survival 62.7 months with OGS alterations compared to >150 months without OGS alterations ( = 0.0248, hazard ratio = 2.213, 95% confidence interval = 1.1-4.098). Two genes comprising OGS ( and ) are strongly associated with in the retinoic acid (RA) pathways, suggesting a major role of RA signaling in early PCa progression. Our pipeline complements human expertise in the search for prognostic biomarkers in large-scale datasets.
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http://dx.doi.org/10.3389/fonc.2017.00030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350134PMC
March 2017

Point mutations in murine phenocopy human congenital heart disease and induce pathogenic Wnt signaling.

JCI Insight 2017 03 23;2(6):e88271. Epub 2017 Mar 23.

The Jackson Laboratory, Bar Harbor, Maine, USA.

Mutations in the gene are a main cause of congenital heart disease. Several studies have addressed the phenotypic consequences of disrupting the gene locus, although animal models to date failed to recapitulate the full spectrum of the human disease. Here, we describe a new point mutation murine model, akin to its human counterpart disease-generating mutation. Our model fully reproduces the morphological and physiological clinical presentations of the disease and reveals an understudied aspect of -driven pathology, a primary right ventricular dysfunction. We further describe the molecular consequences of disrupting the transcriptional network regulated by in the heart and show that -dependent perturbation of the Wnt signaling pathway promotes heart dysfunction through alteration of cardiomyocyte metabolism. Our data provide mechanistic insights on how regulates heart function and metabolism, a link in the study of congenital heart disease, and confirms that our models are the first murine genetic models to our knowledge to present all spectra of clinically relevant adult congenital heart disease phenotypes generated by mutations in patients.
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http://dx.doi.org/10.1172/jci.insight.88271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5358496PMC
March 2017

Congenital valvular defects associated with deleterious mutations in the gene.

J Med Genet 2017 04 31;54(4):278-286. Epub 2016 Oct 31.

Monique and Jacques Roboh Department of Genetic Research, Hadassah, Hebrew University Medical Center, Jerusalem, Israel.

Background: The underlying molecular aetiology of congenital heart defects is largely unknown. The aim of this study was to explore the genetic basis of non-syndromic severe congenital valve malformations in two unrelated families.

Methods: Whole-exome analysis was used to identify the mutations in five patients who suffered from severe valvular malformations involving the pulmonic, tricuspid and mitral valves. The significance of the findings was assessed by studying sporulation of yeast carrying a homologous Phospholipase D () mutation, in situ hybridisation in chick embryo and echocardiography and histological examination of hearts of knockout mice.

Results: Three mutations, p.His442Pro, p.Thr495fs32* and c.2882+2T>C, were identified in the gene. The mutations affected highly conserved sites in the PLD1 protein and the p.His442Pro mutation produced a strong loss of function phenotype in yeast homologous mutant strain. Here we show that in chick embryos expression is confined to the forming heart (E2-E8) and homogeneously expressed all over the heart during days E2-E3. Thereafter its expression decreases, remaining only adjacent to the atrioventricular valves and the right ventricular outflow tract. This pattern of expression follows the known dynamic patterning of apoptosis in the developing heart, consistent with the known role of PLD1 in the promotion of apoptosis. In hearts of knockout mice, we detected marked tricuspid regurgitation, right atrial enlargement, and increased flow velocity, narrowing and thickened leaflets of the pulmonic valve.

Conclusions: The findings support a role for PLD1 in normal heart valvulogenesis.
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http://dx.doi.org/10.1136/jmedgenet-2016-104259DOI Listing
April 2017

An atypical role for the myeloid receptor Mincle in central nervous system injury.

J Cereb Blood Flow Metab 2017 Jun 1;37(6):2098-2111. Epub 2016 Jan 1.

3 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Australia.

The C-type lectin Mincle is implicated in innate immune responses to sterile inflammation, but its contribution to associated pathologies is not well understood. Herein, we show that Mincle exacerbates neuronal loss following ischemic but not traumatic spinal cord injury. Loss of Mincle was beneficial in a model of transient middle cerebral artery occlusion but did not alter outcomes following heart or gut ischemia. High functional scores in Mincle KO animals using the focal cerebral ischemia model were accompanied by reduced lesion size, fewer infiltrating leukocytes and less neutrophil-derived cytokine production than isogenic controls. Bone marrow chimera experiments revealed that the presence of Mincle in the central nervous system, rather than recruited immune cells, was the critical regulator of a poor outcome following transient middle cerebral artery occlusion. There was no evidence for a direct role for Mincle in microglia or neural activation, but expression in a subset of macrophages resident in the perivascular niche provided new clues on Mincle's role in ischemic stroke.
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http://dx.doi.org/10.1177/0271678X16661201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444551PMC
June 2017

Tissue fibroblasts: From bystanders to proactive modulators of homeostasis and disease.

Authors:
Milena B Furtado

Differentiation 2016 09;92(3):65

The Jackson Laboratory, 600 Main St, Bar Harbor, ME 04609, United States. Electronic address:

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http://dx.doi.org/10.1016/j.diff.2016.09.001DOI Listing
September 2016

The cardiac fibroblast: Origin, identity and role in homeostasis and disease.

Differentiation 2016 Sep 12;92(3):93-101. Epub 2016 Jul 12.

The Jackson Laboratory, Bar Harbor, ME, USA; Australian Regenerative Medicine Institute, Monash University, Melbourne, Victoria, Australia; National Heart and Lung Institute, Imperial College London, UK.

The mammalian heart is responsible for supplying blood to two separate circulation circuits in a parallel manner. This design provides efficient oxygenation and nutrients to the whole body through the left-sided pump, while the right-sided pump delivers blood to the pulmonary circulation for re-oxygenation. In order to achieve this demanding job, the mammalian heart evolved into a highly specialised organ comprised of working contractile cells or cardiomyocytes, a directional and insulated conduction system, capable of independently generating and conducting electric impulses that synchronises chamber contraction, valves that allow the generation of high pressure and directional blood flow into the circulation, coronary circulation, that supplies oxygenated blood for the heart muscle high metabolically active pumping role and inlet/outlet routes, as the venae cavae and pulmonary veins, aorta and pulmonary trunk. This organization highlights the complexity and compartmentalization of the heart. This review will focus on the cardiac fibroblast, a cell type until recently ignored, but that profoundly influences heart function in its various compartments. We will discuss current advances on definitions, molecular markers and function of cardiac fibroblasts in heart homeostasis and disease.
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http://dx.doi.org/10.1016/j.diff.2016.06.004DOI Listing
September 2016

A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels.

Differentiation 2016 Jan-Mar;91(1-3):29-41. Epub 2016 Feb 17.

Australian Regenerative Medicine Institute, Monash University, Clayton, Vic 3800, Australia; The Jackson Laboratory, ME 04609, United States. Electronic address:

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies.
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http://dx.doi.org/10.1016/j.diff.2015.12.003DOI Listing
December 2016

View from the heart: cardiac fibroblasts in development, scarring and regeneration.

Development 2016 Feb;143(3):387-97

Australian Regenerative Medicine Institute, Monash University, Clayton, Victoria 3800, Australia Systems Biology Institute (SBI) Australia, Monash University, Clayton, Victoria 3800, Australia National Heart and Lung Institute, Imperial College London, London SW3 6LY, UK The Jackson Laboratory, Bar Harbor, ME 04609, USA.

In the adult, tissue repair after injury is generally compromised by fibrosis, which maintains tissue integrity with scar formation but does not restore normal architecture and function. The process of regeneration is necessary to replace the scar and rebuild normal functioning tissue. Here, we address this problem in the context of heart disease, and discuss the origins and characteristics of cardiac fibroblasts, as well as the crucial role that they play in cardiac development and disease. We discuss the dual nature of cardiac fibroblasts, which can lead to scarring, pathological remodelling and functional deficit, but can also promote heart function in some contexts. Finally, we review current and proposed approaches whereby regeneration could be fostered by interventions that limit scar formation.
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http://dx.doi.org/10.1242/dev.120576DOI Listing
February 2016

CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts.

PLoS One 2015 16;10(12):e0143274. Epub 2015 Dec 16.

Australian Regenerative Medicine Institute, Monash University, Clayton, VIC, 3800, Australia.

The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143274PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684407PMC
June 2016

Microarray profiling to analyse adult cardiac fibroblast identity.

Genom Data 2014 Dec 12;2:345-50. Epub 2014 Oct 12.

Australian Regenerative Medicine Institute, Monash University, VIC 3800, Australia ; Systems Biology Institute (SBI) Australia, Monash University, VIC 3800, Australia.

Heart failure is one of the leading causes of death worldwide [1-4]. Current therapeutic strategies are inefficient and cannot cure this chronic and debilitating condition [5]. Ultimately, heart transplants are required for patient survival, but donor organs are scarce in availability and only prolong the life-span of patients for a limited time. Fibrosis is one of the main pathological features of heart failure [6,7], caused by inappropriate stimulation of fibroblasts and excessive extracellular matrix production. Therefore, an in-depth understanding of the cardiac fibroblast is essential to underpin effective therapeutic treatments for heart failure [5]. Fibroblasts in general have been an underappreciated cell type, regarded as relatively inert and providing only basic functionality; they are usually referred to as the  'biological glue' of all tissues in the body. However, more recent literature suggests that they actively participate in organ homeostasis and disease [7,8]. We have recently uncovered a unique molecular identity for fibroblasts isolated from the heart [9], expressing a set of cardiogenic transcription factors that have been previously associated with cardiomyocyte ontogenesis. This signature suggests that cardiac fibroblasts may be ideal for use in stem cell replacement therapies, as they may retain the memory of where they derive from embryologically. Our data also revealed that about 90% of fibroblasts from both tail and heart origins share a cell surface signature that has previously been described for mesenchymal stem cells (MSCs), raising the possibility that fibroblasts and MSCs may in fact be the same cell type. Thus, our findings carry profound implications for the field of regenerative medicine. Here, we describe detailed methodology and quality controls related to the gene expression profiling of cardiac fibroblasts, deposited at the Gene Expression Omnibus (GEO) under the accession number GSE50531. We also provide the R code to easily reproduce the data quantification and analysis processes.
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http://dx.doi.org/10.1016/j.gdata.2014.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536021PMC
December 2014

VISIONET: intuitive visualisation of overlapping transcription factor networks, with applications in cardiogenic gene discovery.

BMC Bioinformatics 2015 May 1;16:141. Epub 2015 May 1.

Systems Biology Institute (SBI) Australia, Monash University, Clayton, VIC, 3800, Australia.

Background: Existing de novo software platforms have largely overlooked a valuable resource, the expertise of the intended biologist users. Typical data representations such as long gene lists, or highly dense and overlapping transcription factor networks often hinder biologists from relating these results to their expertise.

Results: VISIONET, a streamlined visualisation tool built from experimental needs, enables biologists to transform large and dense overlapping transcription factor networks into sparse human-readable graphs via numerically filtering. The VISIONET interface allows users without a computing background to interactively explore and filter their data, and empowers them to apply their specialist knowledge on far more complex and substantial data sets than is currently possible. Applying VISIONET to the Tbx20-Gata4 transcription factor network led to the discovery and validation of Aldh1a2, an essential developmental gene associated with various important cardiac disorders, as a healthy adult cardiac fibroblast gene co-regulated by cardiogenic transcription factors Gata4 and Tbx20.

Conclusions: We demonstrate with experimental validations the utility of VISIONET for expertise-driven gene discovery that opens new experimental directions that would not otherwise have been identified.
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http://dx.doi.org/10.1186/s12859-015-0578-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4426166PMC
May 2015

Cardiogenic genes expressed in cardiac fibroblasts contribute to heart development and repair.

Circ Res 2014 Apr 20;114(9):1422-34. Epub 2014 Mar 20.

From the Australian Regenerative Medicine Institute (M.B.F., M.W.C., E.A.P., E.S., A.R.P., A.C., N.A.R.), Department of Anatomy and Developmental Biology (A.R.P., R.B.), and Monash Biomedical Imaging (J.P.), Monash University, Melbourne, Victoria, Australia; Baker IDI Heart and Diabetes Institute, Melbourne, Victoria, Australia (N.T.L., D.M.K.); Department of Pediatrics, Indiana University School of Medicine, Indianapolis (P.S., S.J.C.); and Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia (R.P.H.).

Rationale: Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment, but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers.

Objective: To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration.

Methods And Results: High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical mesenchymal stem cell and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. While genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, T-box 20, caused marked myocardial dysmorphology and perturbations in scar formation on myocardial infarction.

Conclusions: The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs, and direct contribution to cardiac development and repair provoke alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.
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http://dx.doi.org/10.1161/CIRCRESAHA.114.302530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083003PMC
April 2014

Effect of oxygen on cardiac differentiation in mouse iPS cells: role of hypoxia inducible factor-1 and Wnt/beta-catenin signaling.

PLoS One 2013 12;8(11):e80280. Epub 2013 Nov 12.

Heart Failure Research Group, Baker IDI Heart and Diabetes Institute, Melbourne, Australia.

Background: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.

Objective: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process.

Methods: Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.

Results: At 14 days of differentiation, 59 ± 2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.

Conclusion: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0080280PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827186PMC
July 2014

Functional characterization of a novel mutation in NKX2-5 associated with congenital heart disease and adult-onset cardiomyopathy.

Circ Cardiovasc Genet 2013 Jun 9;6(3):238-47. Epub 2013 May 9.

Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia.

Background: The transcription factor NKX2-5 is crucial for heart development, and mutations in this gene have been implicated in diverse congenital heart diseases and conduction defects in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown.

Methods And Results: Mutation screening was performed in 220 probands with adult-onset dilated cardiomyopathy. Six NKX2-5 coding sequence variants were identified, including 3 nonsynonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain, was identified in 1 family. A subset of family members had congenital heart disease, but there was an unexpectedly high prevalence of dilated cardiomyopathy. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes because of reduced degradation by the Ubiquitin-proteasome system. In functional assays, DNA-binding activity of I184M was reduced, resulting in impaired activation of target genes despite increased expression levels of mutant protein.

Conclusions: Certain NKX2-5 homeodomain mutations show abnormal protein degradation via the Ubiquitin-proteasome system and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function and contribute to NKX2-5-related cardiomyopathies with graded severity.
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http://dx.doi.org/10.1161/CIRCGENETICS.113.000057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3816146PMC
June 2013

Complex SUMO-1 regulation of cardiac transcription factor Nkx2-5.

PLoS One 2011 12;6(9):e24812. Epub 2011 Sep 12.

Developmental and Stem Cell Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a "shifting" site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024812PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3171482PMC
February 2012

Loss of Cited2 causes congenital heart disease by perturbing left-right patterning of the body axis.

Hum Mol Genet 2011 Mar 28;20(6):1097-110. Epub 2010 Dec 28.

Developmental Biology Division, Victor Chang Cardiac Research Institute, Sydney, Australia.

Cited2 is a transcriptional coactivator that is required for normal development of the embryo and placenta. Cited2-null mice die during gestation with fully penetrant heart defects and partially penetrant laterality defects. The laterality defects occur due to the loss of Nodal expression in the left lateral plate mesoderm (LPM). The cause of the heart defects that arise independently of laterality defects is unknown; they might occur due to an intrinsic requirement for Cited2 in the developing heart, or to disturbances in left-right patterning of the early embryo. Herein it is established that deletion of Cited2 from the heart progenitors does not alter development, and that heart defects in Cited2-null embryos arise due to an extra-cardiac requirement for Cited2 in establishing the left-right body axis. In addition, we provide evidence supporting a role for Cited2 in tissues of the embryo vital for left-right patterning (the node and LPM). Molecular and genetic analysis reveals that Cited2 is required for the initiation, but not propagation of, the left-sided determinant Nodal in the LPM. Moreover, a new role for Cited2 is identified as a potentiator of bone morphogenetic protein (BMP) signalling, counteracting the initiation of Nodal expression in the LPM. These data define Cited2 as a key regulator of left-right patterning in the mammalian embryo, and reveal that the role of Cited2 in cardiac development lies in its extra-cardiac functions. The clinical relevance of these findings lies in the fact that heterozygous mutation of human CITED2 is associated with congenital heart disease and laterality defects.
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http://dx.doi.org/10.1093/hmg/ddq554DOI Listing
March 2011

Characterization of Pitx2c expression in the mouse heart using a reporter transgene.

Dev Dyn 2011 Jan;240(1):195-203

Victor Chang Cardiac Research Institute, Darlinghurst, Sydney, Australia.

To aid in detection and tracking of cells targeted by the left-right (LR) pathway in the heart throughout morphogenesis, expression from a Pitx2c-lacZ transgene (P2Ztg) was analysed in detail. β-galactosidase expression from P2Ztg was robust, allowing reliable visualisation of low-level Pitx2c expression, and was virtually entirely dependent upon NODAL signalling in the heart. P2Ztg showed expression in trabecular and septal, as well as non-trabecular, myocardium, and a strong expression bias in myocardium associated with individual endocardial cushions of the atrioventricular canal and outflow tract, which are essential for cardiac septation. Expression on the ventral surface of the outflow tract evolved to a specific stripe that could be used to track the future aorta during outflow tract spiralling and remodelling. Our data show that the P2Ztg transgene is a useful resource for detection of molecular disturbances in the LR cascade, as well as morphogenetic defects associated with other cardiac congenital disorders.
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http://dx.doi.org/10.1002/dvdy.22492DOI Listing
January 2011

BMP/SMAD1 signaling sets a threshold for the left/right pathway in lateral plate mesoderm and limits availability of SMAD4.

Genes Dev 2008 Nov;22(21):3037-49

Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

Bistability in developmental pathways refers to the generation of binary outputs from graded or noisy inputs. Signaling thresholds are critical for bistability. Specification of the left/right (LR) axis in vertebrate embryos involves bistable expression of transforming growth factor beta (TGFbeta) member NODAL in the left lateral plate mesoderm (LPM) controlled by feed-forward and feedback loops. Here we provide evidence that bone morphogenetic protein (BMP)/SMAD1 signaling sets a repressive threshold in the LPM essential for the integrity of LR signaling. Conditional deletion of Smad1 in the LPM led to precocious and bilateral pathway activation. NODAL expression from both the left and right sides of the node contributed to bilateral activation, indicating sensitivity of mutant LPM to noisy input from the LR system. In vitro, BMP signaling inhibited NODAL pathway activation and formation of its downstream SMAD2/4-FOXH1 transcriptional complex. Activity was restored by overexpression of SMAD4 and in embryos, elevated SMAD4 in the right LPM robustly activated LR gene expression, an effect reversed by superactivated BMP signaling. We conclude that BMP/SMAD1 signaling sets a bilateral, repressive threshold for NODAL-dependent Nodal activation in LPM, limiting availability of SMAD4. This repressive threshold is essential for bistable output of the LR system.
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http://dx.doi.org/10.1101/gad.1682108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2577791PMC
November 2008

An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation.

Cell 2007 Mar;128(5):947-59

Victor Chang Cardiac Research Institute, Sydney 2010, Australia.

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.
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http://dx.doi.org/10.1016/j.cell.2007.01.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2092439PMC
March 2007

A tyrosine-rich domain within homeodomain transcription factor Nkx2-5 is an essential element in the early cardiac transcriptional regulatory machinery.

Development 2006 Apr 1;133(7):1311-22. Epub 2006 Mar 1.

Victor Chang Cardiac Research Institute, Darlinghurst, Sydney 2010, Australia.

Homeodomain factor Nkx2-5 is a central component of the transcription factor network that guides cardiac development; in humans, mutations in NKX2.5 lead to congenital heart disease (CHD). We have genetically defined a novel conserved tyrosine-rich domain (YRD) within Nkx2-5 that has co-evolved with its homeodomain. Mutation of the YRD did not affect DNA binding and only slightly diminished transcriptional activity of Nkx2-5 in a context-specific manner in vitro. However, the YRD was absolutely essential for the function of Nkx2-5 in cardiogenesis during ES cell differentiation and in the developing embryo. Furthermore, heterozygous mutation of all nine tyrosines to alanine created an allele with a strong dominant-negative-like activity in vivo: ES cell<-->embryo chimaeras bearing the heterozygous mutation died before term with cardiac malformations similar to the more severe anomalies seen in NKX2.5 mutant families. These studies suggest a functional interdependence between the NK2 class homeodomain and YRD in cardiac development and evolution, and establish a new model for analysis of Nkx2-5 function in CHD.
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http://dx.doi.org/10.1242/dev.02305DOI Listing
April 2006

Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.

Development 2005 May 20;132(10):2451-62. Epub 2005 Apr 20.

Victor Chang Cardiac Research Institute, St Vincent's Hospital, 384 Victoria Street, Darlinghurst 2010, New South Wales, Australia.

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.
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http://dx.doi.org/10.1242/dev.01799DOI Listing
May 2005