[Show abstract][Hide abstract] ABSTRACT: Correct regulation of troponin and myosin contractile protein gene isoforms is a critical determinant of cardiac and skeletal striated muscle development and function, with misexpression frequently associated with impaired contractility or disease. Here we reveal a novel requirement for Prospero-related homeobox factor 1 (Prox1) during mouse heart development in the direct transcriptional repression of the fast-twitch skeletal muscle genes troponin T3, troponin I2, and myosin light chain 1. A proportion of cardiac-specific Prox1 knockout mice survive beyond birth with hearts characterized by marked overexpression of fast-twitch genes and postnatal development of a fatal dilated cardiomyopathy. Through conditional knockout of Prox1 from skeletal muscle, we demonstrate a conserved requirement for Prox1 in the repression of troponin T3, troponin I2, and myosin light chain 1 between cardiac and slow-twitch skeletal muscle and establish Prox1 ablation as sufficient to cause a switch from a slow- to fast-twitch muscle phenotype. Our study identifies conserved roles for Prox1 between cardiac and skeletal muscle, specifically implicated in slow-twitch fiber-type specification, function, and cardiomyopathic disease.
Proceedings of the National Academy of Sciences 06/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epicardium is a cellular source with the potential to reconstitute lost cardiovascular tissue following myocardial infarction. Here we show that the adult epicardium contains a population of CD45+ haematopoietic cells (HCs), which are located proximal to coronary vessels and encased by extracellular matrix (ECM). This complex tertiary structure is established during the regenerative window between post-natal days 1 and 7. We show that these HCs proliferate within the first 24 h and are released between days 2 and 7 after myocardial infarction. The ECM subsequently reforms to encapsulate HCs after 21 days. Vav1-tdTomato labelling reveals an integral contribution of CD45+ HCs to the developing epicardium, which is not derived from the proepicardial organ. Transplantation experiments with either whole bone marrow or a Vav1+ subpopulation of cells confirm a contribution of HCs to the intact adult epicardium, which is elevated during the first 24 weeks of adult life but depleted in aged mice.
[Show abstract][Hide abstract] ABSTRACT: In the last decade, cell replacement therapy has emerged as a potential approach to treat patients suffering from myocardial infarction (MI). The transplantation or local stimulation of progenitor cells with the ability to form new cardiac tissue provides a novel strategy to overcome the massive loss of myocardium after MI. In this regard the epicardium, the outer layer of the heart, is a tractable local progenitor cell population for therapeutic pursuit. The epicardium has a crucial role in formation of the embryonic heart. After activation and migration into the developing myocardium, epicardial cells differentiate into several cardiac cells types. Additionally, the epicardium provides instructive signals for the growth of the myocardium and coronary angiogenesis. In the adult heart, the epicardium is quiescent, but recent evidence suggests that it becomes reactivated upon
damage and recapitulates at least part of its embryonic functions. In this review we provide an update on the current knowledge regarding the contribution of epicardial cells to the adult mammalian heart during the injury response.
[Show abstract][Hide abstract] ABSTRACT: Cardiovascular disease remains the major cause of mortality and cardiac cell therapy has recently emerged as a paradigm for heart repair. The epicardium is a layer of mesothelial cells covering the heart that during development contributes to different cardiovascular lineages, including cardiomyocytes, but becomes quiescent after birth. We previously revealed that the peptide thymosin beta 4 (Tβ4) can reactivate the adult epicardium-derived cells (EPDCs) following myocardial infarction (MI) to proliferate and differentiate into cardiovascular derivatives. The aim of this study was to provide a lineage characterisation of the adult EPDCs relative to the embryonic epicardial lineage and to determine prospective cell fate biases within the activated adult population during cardiovascular repair. Wt1GFPCre/+ mice were primed with Tβ4 and MI induced by ligation of the left anterior descending coronary artery. Adult WT1+ GFP+ EPDCs were FACS-sorted 2, 4 and 7 days after MI. Embryonic WT1+ GFP+ EPDCs were isolated from embryonic hearts (E12.5) by FACS and sorted cells were characterised by real time qRT-PCR and immunostaining. Adult WT1+ GFP+ EPDCs were highly heterogeneous, expressing cardiac progenitor and mesenchymal stem markers. Based on the expression of Sca-1, CD44 and CD90 we identified different subpopulations of EPDCs of varying cardiovascular potential, based on marker gene profiles, with a molecular phenotype distinct from the source embryonic epicardial cells at E12.5. Thus, the activated adult WT1+ GFP+ cells are a heterogeneous population which when activated can restore an embryonic gene programme but do not revert entirely to adopt an embryonic phenotype. Potential biases in cardiovascular cell fate suggest discrete subpopulations of EPDCs might be clinically relevant for regenerative therapy.
Stem cells and development 04/2014; · 4.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The downstream consequences of inflammation in the adult mammalian heart are formation of a non-functional scar, pathological remodelling and heart failure. In zebrafish, hydrogen peroxide released from a wound is the initial instructive chemotactic cue for the infiltration of inflammatory cells, however, the identity of a subsequent resolution signal(s), to attenuate chronic inflammation, remains unknown. Here we reveal that thymosin β4-sulfoxide lies downstream of hydrogen peroxide in the wounded fish and triggers depletion of inflammatory macrophages at the injury site. This function is conserved in the mouse and observed after cardiac injury, where it promotes wound healing and reduced scarring. In human T-cell/CD14+ monocyte co-cultures, thymosin β4-sulfoxide inhibits interferon-γ, and increases monocyte dispersal and cell death, likely by stimulating superoxide production. Thus, thymosin β4-sulfoxide is a putative target for therapeutic modulation of the immune response, resolution of fibrosis and cardiac repair.
[Show abstract][Hide abstract] ABSTRACT: Efficient cardiac regeneration postinfarction (MI) requires the replacement of lost cardiomyocytes, formation of new coronary vessels and appropriate modulation of the inflammatory response. However, insight into how to stimulate repair of the human heart is currently limited. Using the embryonic paradigm of regeneration, we demonstrated that the actin-binding peptide thymosin β4 (Tβ4), required for epicardium-derived coronary vasculogenesis, can recapitulate its embryonic role and activate quiescent adult epicardial cells (EPDCs). Once stimulated, EPDCs facilitate neovascularization of the ischemic adult heart and, moreover, contribute bona fide cardiomyocytes. EPDC-derived cardiomyocytes structurally and functionally integrate with resident muscle to regenerate functional myocardium, limiting pathological remodeling, and effecting an improvement in cardiac function. Alongside pro-survival and anti-inflammatory properties, these regenerative roles, via EPDCs, markedly expand the range of therapeutic benefits of Tβ4 to sustain and repair the myocardium after ischemic damage.
Annals of the New York Academy of Sciences 10/2012; 1269(1):92-101. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: While cardiovascular diseases remain the major worldwide cause of mortality and morbidity, there is an urgent need to tackle the clinical and economic burden of heart failure. Since the mammalian heart is unable to adequately regenerate beyond early postnatal stages, individuals surviving acute myocardial infarction are at risk of heart failure. Understanding the embryonic mechanisms of vasculogenesis and cardiogenesis, as well as the mechanisms retained for regeneration in species such as the zebrafish, will inform on strategies for human myocardial repair. Due to their fundamental role in heart development, epicardium-derived cells (EPDCs) have emerged as a population with potential to restore myocardium and coronary vasculature. The ability to revive ordinarily dormant EPDCs lies in the identification of key molecular cues used in the embryo to orchestrate cardiovascular development. One such stimulatory factor, Thymosin β4 (Tβ4), restores the quiescent adult epicardium to its pluripotent embryonic state. Tβ4 treatment of infarcted hearts induces dramatic EPDC proliferation and formation of a network of perfused, functional vessels to enhance blood flow to the ischaemic myocardium. Moreover, Tβ4 facilitates an epicardial contribution of mature de novo cardiomyocytes, structurally and functionally coupled with resident myocardium, which may contribute towards the functional improvement of Tβ4-treated hearts post-MI.
[Show abstract][Hide abstract] ABSTRACT: Compromised development of blood vessel walls leads to vascular instability that may predispose to aneurysm with risk of rupture and lethal hemorrhage. There is currently a lack of insight into developmental insults that may define the molecular and cellular characteristics of initiating and perpetrating factors in adult aneurismal disease.
To investigate a role for the actin-binding protein thymosin β4 (Tβ4), previously shown to be proangiogenic, in mural cell development and vascular wall stability.
Phenotypic analyses of both global and endothelial-specific loss-of-function Tβ4 mouse models revealed a proportion of Tβ4-null embryos with vascular hemorrhage coincident with a reduction in smooth muscle cell coverage of their developing vessels. Mechanistic studies revealed that extracellular Tβ4 can stimulate differentiation of mesodermal progenitor cells to a mature mural cell phenotype through activation of the transforming growth factor-beta (TGFβ) pathway and that reduced TGFβ signaling correlates with the severity of hemorrhagic phenotype in Tβ4-null vasculature.
Tβ4 is a novel endothelial secreted trophic factor that functions synergistically with TGFβ to regulate mural cell development and vascular wall stability. These findings have important implications for understanding congenital anomalies that may be causative for adult-onset vascular instability.
Circulation Research 06/2012; 111(4):e89-102. · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Across biomedicine, there is a major drive to develop stem cell (SC) treatments for debilitating diseases. Most effective treatments restore an embryonic phenotype to adult SCs. This has led to two emerging paradigms in SC biology: the application of developmental biology studies and the manipulation of the SC niche. Developmental studies can reveal how SCs are orchestrated to build organs, the understanding of which is important in order to instigate tissue repair in the adult. SC niche studies can reveal cues that maintain SC 'stemness' and how SCs may be released from the constraints of the niche to differentiate and repopulate a 'failing' organ. The haematopoietic system provides an exemplar whereby characterisation of the blood lineages during development and the bone marrow niche has resulted in therapeutics now routinely used in the clinic. Ischaemic heart disease is a major cause of morbidity and mortality in humans and the question remains as to whether these principles can be applied to the heart, in order to exploit the potential of adult SCs for use in cardiovascular repair and regeneration.
Journal of Cardiovascular Translational Research 06/2012; 5(5):631-40. · 3.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nkx2.5 is one of the most widely studied cardiac-specific transcription factors, conserved from flies to man, with multiple essential roles in both the developing and adult heart. Specific dominant mutations in NKX2.5 have been identified in adult congenital heart disease patients presenting with conduction system anomalies and recent genome-wide association studies implicate the NKX2.5 locus, as causative for lethal arrhythmias ("sudden cardiac death") that occur at a frequency in the population of 1 in 1000 per annum worldwide. Haploinsufficiency for Nkx2.5 in the mouse phenocopies human conduction disease pathology yet the phenotypes, described in both mouse and man, are highly pleiotropic, implicit of unknown modifiers and/or factors acting in epistasis with Nkx2.5/NKX2.5.
To identify bone fide upstream genetic modifier(s) of Nkx2.5/NKX2.5 function and to determine epistatic effects relevant to the manifestation of NKX2.5-dependent adult congenital heart disease.
A study of cardiac function in prospero-related homeobox protein 1 (Prox1) heterozygous mice, using pressure-volume loop and micromannometry, revealed rescue of hemodynamic parameters in Nkx2.5(Cre/+); Prox1(loxP/+) animals versus Nkx2.5(Cre/+) controls. Anatomic studies, on a Cx40(EGFP) background, revealed Cre-mediated knock-down of Prox1 restored the anatomy of the atrioventricular node and His-Purkinje network both of which were severely hypoplastic in Nkx2.5(Cre/+) littermates. Steady state surface electrocardiography recordings and high-speed multiphoton imaging, to assess Ca(2+) handling, revealed atrioventricular conduction and excitation-contraction were also normalized by Prox1 haploinsufficiency, as was expression of conduction genes thought to act downstream of Nkx2.5. Chromatin immunoprecipitation on adult hearts, in combination with both gain and loss-of-function reporter assays in vitro, revealed that Prox1 recruits the corepressor HDAC3 to directly repress Nkx2.5 via a proximal upstream enhancer as a mechanism for regulating Nkx2.5 function in adult cardiac conduction.
Here we identify Prox1 as a direct upstream modifier of Nkx2.5 in the maintenance of the adult conduction system and rescue of Nkx2.5 conduction disease phenotypes. This study is the first example of rescue of Nkx2.5 function and establishes a model for ensuring electrophysiological function within the adult heart alongside insight into a novel Prox1-HDAC3-Nkx2.5 signaling pathway for therapeutic targeting in conduction disease.
Circulation Research 05/2012; 111(2):e19-31. · 11.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Clinical interventions leading to improved survival in patients with acute myocardial infarction have, paradoxically, increased the need for cardiac regenerative strategies as more people are living with heart failure. Over the last 10-15 years there have been significant advances in our understanding of cell-based therapy for cardiac repair. Evidence that paracrine stimulation largely underlies the functional benefits in cell transplantation has led to a paradigm shift in regenerative medicine: from cell therapy to factor/protein-based therapy. Although, future regenerative approaches may likely involve a synergistic protein cocktail, this review will focus on the role of a promising candidate, thymosin beta 4 (Tβ4) in cardioprotection, neovascularization, tissue regeneration and inflammation - all essential components in cardiac repair.
Current pharmaceutical design 02/2012; 18(6):799-806. · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epicardium is a mesothelial cell layer which contributes to the coronary vessels and myocardium and acts as an important source of trophic signals to maintain continued growth and differentiation of the developing heart. The precise lineage potential of the embryonic epicardium has come under recent scrutiny with notable questions around its capacity to give rise to derivative vascular endothelial cells and cardiomyocytes. The importance of the epicardium is not restricted to heart formation. Recent studies in the adult heart have highlighted a paracrine role in modulating injury and have begun to realize its potential as a source of progenitor cells (EPDCs) which may be reactivated toward facilitating neovascularization and myocardial repair after ischemic injury. Thus, the adult epicardium has an embryological origin and emerges as a prime exemplar of the paradigm of activated resident stem cell therapy in regenerative medicine, whereupon a major goal is to restore embryonic plasticity to otherwise dormant adult progenitors and facilitate organ repair. In this review, we will explore current thinking on the origins of the epicardium, its role as a signaling center, lineage heterogeneity, and controversy around epicardial potential within the developing heart. We will extrapolate to the adult injury setting, drawing on key studies in zebrafish and mouse which establish the basis for the adult epicardium as a target for cardiovascular regeneration. Finally, we will consider translation of this potential to the human lineage alongside the prospects for discovery of target cell-based therapeutics.
Current Topics in Developmental Biology 01/2012; 100:233-51. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mammalian heart loses its regenerative capacity during early postnatal stages; consequently, individuals surviving myocardial infarction are at risk of heart failure due to excessive fibrosis and maladaptive remodeling. There is an urgent need, therefore, to develop novel therapies for myocardial and coronary vascular regeneration. The epicardium-derived cells present a tractable resident progenitor source with the potential to stimulate neovasculogenesis and contribute de novo cardiomyocytes. The ability to revive ordinarily dormant epicardium-derived cells lies in the identification of key stimulatory factors, such as Tβ4, and elucidation of the molecular cues used in the embryo to orchestrate cardiovascular development. myocardial infarction injury signaling reactivates the adult epicardium; understanding the timing and magnitude of these signals will enlighten strategies for myocardial repair.
[Show abstract][Hide abstract] ABSTRACT: As the developing heart grows and the chamber walls thicken, passive diffusion of oxygen and nutrients is replaced by a vascular plexus which remodels and expands to form a mature coronary vascular system. The coronary arteries and veins ensure the continued development of the heart and facilitate cardiac output with progression towards birth. Many aspects of coronary vessel development are surprisingly not well understood and recently there has been much debate surrounding both the developmental origin and tissue contribution of cardiovascular cells alongside the specific signals that determine their fate and function. What is clear is that an understanding of the cellular and molecular cues to vascularize the heart of the embryo has significant implications for adult heart disease and regeneration, as we move towards targeted cell-based therapies for neovascularization and coronary bypass engraftment. This review will focus on the proposed cellular origins for the coronary endothelium with due consideration to the pro-epicardial organ/epicardium, sinus venosus and endocardium as potential sources, and we will explore the outstanding questions and technical limitations with respect to accurate labelling and lineage tracing of the developing coronaries. We will briefly document canonical vascular signalling that induces vessels in the heart alongside a focus on the potential for developmental reprogramming and putative mechanisms underpinning venous vs. arterial cell fate. Finally, we will extrapolate directly from development to address adult maintenance of the coronaries, vascular homeostasis and remodelling in response to pathology, aligned with the potential for revascularizing the injured adult heart.
Cardiovascular Research 06/2011; 91(2):260-8. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.