Stem cell-derived cardiomyocytes represent unique tools for cell- and tissue-based regenerative therapies, drug discovery and safety, and studies of fundamental heart-failure mechanisms. However, the degree to which stem cell-derived cardiomyocytes compare to mature cardiomyocytes is often debated. We reasoned that physiological metrics of engineered cardiac tissues offer a means of comparison. We built laminar myocardium engineered from cardiomyocytes that were differentiated from mouse embryonic stem cell-derived cardiac progenitors or harvested directly from neonatal mouse ventricles, and compared their anatomy and physiology in vitro. Tissues assembled from progenitor-derived myocytes and neonate myocytes demonstrated similar cytoskeletal architectures but different gap junction organization and electromechanical properties. Progenitor-derived myocardium had significantly less contractile stress and slower longitudinal conduction velocity than neonate-derived myocardium, indicating that the developmental state of the cardiomyocytes affects the electromechanical function of the resultant engineered tissue. These data suggest a need to establish performance metrics for future stem cell applications.
•Engineered myocardium was compared with progenitor-derived and neonatal myocytes•Progenitor-derived and neonatal myocytes had similar sarcomere and actin structure•Progenitor-derived tissue had significantly slower conduction velocity than neonate•Progenitor-derived tissue generated significantly less contractile stress than neonate
Parker and colleagues engineered myocardium from cardiomyocytes either differentiated from mouse embryonic stem cell-derived cardiac progenitors or harvested directly from neonatal mouse ventricles, and compared their anatomy and physiology in vitro. Results show the ability to build structurally similar tissues, but the progenitor-derived myocardium had significantly lower conduction velocity and contractile stress generation than the neonatal myocardium.
The term “lineage reprogramming” is typically used to describe the conversion of one differentiated somatic cell type into another without transit through a pluripotent intermediate. Two recent reports in Nature demonstrate that such a conversion can be achieved in the heart in situ, and suggest a novel, regenerative approach for the development of cardiac therapeutics.
Distinct families of multipotent heart progenitors play a central role in the generation of diverse cardiac, smooth muscle and endothelial cell lineages during mammalian cardiogenesis. The identification of precise paracrine signals that drive the cell-fate decision of these multipotent progenitors, and the development of novel approaches to deliver these signals in vivo, are critical steps towards unlocking their regenerative therapeutic potential. Herein, we have identified a family of human cardiac endothelial intermediates located in outflow tract of the early human fetal hearts (OFT-ECs), characterized by coexpression of Isl1 and CD144/vWF. By comparing angiocrine factors expressed by the human OFT-ECs and non-cardiac ECs, vascular endothelial growth factor (VEGF)-A was identified as the most abundantly expressed factor, and clonal assays documented its ability to drive endothelial specification of human embryonic stem cell (ESC)-derived Isl1+ progenitors in a VEGF receptor-dependent manner. Human Isl1-ECs (endothelial cells differentiated from hESC-derived ISL1+ progenitors) resemble OFT-ECs in terms of expression of the cardiac endothelial progenitor- and endocardial cell-specific genes, confirming their organ specificity. To determine whether VEGF-A might serve as an in vivo cell-fate switch for human ESC-derived Isl1-ECs, we established a novel approach using chemically modified mRNA as a platform for transient, yet highly efficient expression of paracrine factors in cardiovascular progenitors. Overexpression of VEGF-A promotes not only the endothelial specification but also engraftment, proliferation and survival (reduced apoptosis) of the human Isl1+ progenitors in vivo. The large-scale derivation of cardiac-specific human Isl1-ECs from human pluripotent stem cells, coupled with the ability to drive endothelial specification, engraftment, and survival following transplantation, suggest a novel strategy for vascular regeneration in the heart.
cardiovascular progenitors; endothelial cells; Isl1; mRNA; VEGF-A
Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1±0.13 versus 1.66±0.06; P<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.
gap junction; connexin43; arrhythmia; conduction
The ability to create new functional cardiomyocytes is the holy grail of cardiac regenerative medicine. From studies using model organisms, new insights into the fundamental pathways that drive heart muscle regeneration have begun to arise as well as a growing knowledge of the distinct families of multipotent cardiovascular progenitors that generate diverse lineages during heart development. In this Review, we highlight this intersection of the “pregenerative” biology of heart progenitor cells and heart regeneration and discuss the longer term challenges and opportunities in moving toward a therapeutic goal of regenerative cardiovascular medicine.
Several clinical trials of bone marrow stem cell therapy for myocardial infarction are ongoing, but the mechanistic basis for any potential therapeutic effect is currently unclear. A growing body of evidence suggests that the potential improvement in cardiac function is largely independent of cardiac muscle regeneration. A study by Fazel et al. in this issue of the JCI provides evidence that bone marrow–derived c-kit+ cells can lead to an improvement in cardiac function in mutant hypomorphic c-kit mice that is independent of transdifferentiation into either cardiac muscle or endothelial cells, but rather is associated with the release of angiogenic cytokines and associated neovascularization in the infarct border zone (see the related article beginning on page 1865). These findings suggest the potential therapeutic effect of specific paracrine pathways for angiogenesis in improving cardiac function in the injured heart.
Human congenital heart disease linked to mutations in the homeobox transcription factor, NKX2-5, is characterized by cardiac anomalies, including atrial and ventricular septal defects as well as conduction and occasional defects in contractility. In the mouse, homozygous germline deletion of Nkx2-5 gene results in death around E10.5. It is, however, not established whether Nkx2-5 is necessary for cardiac development beyond this embryonic stage. Because human NKX2-5 mutations are related to septum secundum type atrial septal defects (ASD), we hypothesized that Nkx2-5 deficiency during the processes of septum secundum formation may cause cardiac anomalies; thus, we analysed mice with tamoxifen-inducible Nkx2-5 ablation beginning at E12.5 when the septum secundum starts to develop.
Methods and results
Using tamoxifen-inducible Nkx2-5 gene-targeted mice, this study demonstrates that Nkx2-5 ablation beginning at E12.5 results in embryonic death by E17.5. Analysis of mutant embryos at E16.5 shows arrhythmias, contraction defects, and cardiac malformations, including ASD. Quantitative measurements using serial section histology and three-dimensional reconstruction demonstrate growth retardation of the septum secundum and enlarged foramen ovale in Nkx2-5-ablated embryos. Functional cardiac defects may be attributed to abnormal expression of transcripts critical for conduction and contraction, including cardiac voltage-gated Na+ channel pore-forming α-subunit (Nav1.5-α), gap junction protein connexin40, cardiac myosin light chain kinase, and sarcolipin within 4 days after tamoxifen injection.
Nkx2-5 is necessary for survival after the mid-embryonic stage for cardiac function and formation by regulating the expression of its downstream target genes.
Genetics; Heart defects; Congenital; Conduction; Contractility
Z-line protein have important structural functions. Recent publications point to additional, previously unexpected functions and new views are now emerging. Z-line proteins are involved in important intra- and intercellular signalling pathways. They translocate into the nucleus, they interact with a variety of signalling molecules including kinases and transcription factors, and they have the ability to form macromolecular protein complexes indicating furthermore their multifuntionality. The muscle LIM protein (MLP) is muscle specific, and is expressed and located at the z-line. MLP’s physiological role at the z-line and in the nucleus may be better understood by precise investigations of specific mutations in specific domains of this protein.
Cardiomyopathy; Heart failure; Muscle LIM protein; Z-line proteins
Multipotent Isl1+ heart progenitors give rise to three major cardiovascular cell types; cardiac, smooth muscle, and endothelial cells, and play a pivotal role in lineage diversification during cardiogenesis. A critical question is pinpointing when this cardiac-vascular lineage decision is made, and how this plasticity serves to coordinate cardiac chamber and vessel growth. The posterior domain of the Isl1-positive second heart field contributes to the SLN-positive atrial myocardium and myocardial sleeves in the cardiac inflow tract, where myocardial and vascular smooth muscle layers form anatomical and functional continuity. Herein, using a new atrial specific SLN-Cre knockin mouse line, we report that an Isl1+/SLN+ transient cell population contributes to cardiac as well as smooth muscle cells at the heart-vessel junction in cardiac inflow tract. The Isl1+/SLN+ cells are capable of giving rise to cardiac and smooth muscle cells until late gestational stages. These data suggest that the cardiac and smooth muscle cells in the cardiac inflow tract share a common developmental origin.
cardiogenesis; myogenic progenitor; smooth muscle; great vessel; plasticity
Several studies have claimed to identify cardiac stem cells. But what criteria do such cells have to fulfil before we can be confident about their true potential?
The mammalian heart is formed from distinct sets of first (FHF) and second (SHF) heart field progenitors. Although multipotent progenitors have been previously shown to give rise to cardiomyocytes, smooth muscle, and endothelial cells, the mechanism governing the generation of large numbers of differentiated progeny remains poorly understood. Herein, we have employed a two-colored fluorescent reporter system to isolate FHF and SHF progenitors from developing mouse embryos and embryonic stem cells. Genome wide profiling of coding and non-coding transcripts revealed distinct molecular signatures of these progenitor populations. We further identify a committed ventricular progenitor cell in the Islet 1 lineage that is capable of in vitro expansion, differentiation, and assembly into functional ventricular muscle tissue. These results represent a novel approach combining tissue-engineering with stem cell biology for the generation of functional ventricular tissue.
Mutations in homeoprotein NKX2–5 are linked to human congenital heart disease resulting in various cardiac anomalies, as well as postnatal progressive conduction defects and occasional left ventricular dysfunction, yet the function of Nkx2–5 in the postnatal period is largely unexplored. In the heart, the majority of cardiomyocytes are believed to complete cell-cycle withdrawal shortly after birth, which is generally accompanied by re-organization of chromatin structure demonstrated in other tissues. We reasoned that effects of loss of Nkx2–5 in mice may be different after cell-cycle withdrawal compared to perinatal loss of Nkx2–5, which results in rapid conduction and contraction defects within 4 days after deletion of Nkx2–5 alleles (Circ Res. 2008;103:580).
In this study, floxed-Nkx2–5 alleles were deleted using tamoxifen-inducible Cre transgene (Cre-ER™) beginning at 2 weeks of age. Loss of Nkx2–5 beginning at 2 weeks of age resulted in conduction and contraction defects similar to perinatal loss of Nkx2–5, however with substantially slower disease progression demonstrated by 1° atrioventricular block at 6 weeks of age (4 weeks after tamoxifen injections), and heart enlargement after 12 weeks of age (10 weeks after tamoxifen injections). The phenotypes were accompanied by slower and smaller degree of reduction of several critical Nkx2–5 downstream targets that were observed in mice with perinatal loss of Nkx2–5. These results suggest that Nkx2–5 is necessary for proper conduction and contraction after 2 weeks of age, but with substantially distinct level of necessity at 2 weeks of age compared to the perinatal period.
Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the homeobox transcription factor Prox1 results in disruption of the expression and localisation of sarcomeric proteins, gross myofibril disarray and growth retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of structural proteins α-actinin, N-RAP and Zyxin which collectively function to maintain an actin-α-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study demonstrates an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling fetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.
Prox1; heart development; myocardium; sarcomere; hypertrophy; myopathy
The heart is formed from cardiogenic progenitors expressing the transcription factors Nkx2-5 and Isl1 1,2. These multipotent progenitors give rise to cardiomyocyte, smooth muscle, and endothelial cells, the major lineages of the mature heart 3,4. Here we identify a novel cardiogenic precursor marked by expression of the transcription factor Wt1 and located within the epicardium, an epithelial sheet overlying the heart. During normal heart development, a subset of these Wt1+ precursors differentiated into fully functional cardiomyocytes. Wt1+ proepicardial cells arose from progenitors that express Nkx2-5 and Isl1, suggesting that they share a developmental origin with multipotent Nkx2-5+/Isl1+ progenitors (Suppl. Fig 1). These results identify Wt1+ epicardial cells as previously unrecognized cardiomyocyte progenitors, and lay the foundation for future efforts to harness the cardiogenic potential of these progenitors for cardiac regeneration and repair.
During cardiogenesis, perturbation of a key transition at midgestation from cardiac patterning to cardiac growth and chamber maturation often leads to diverse types of congenital heart disease, such as ventricular septal defect (VSD), myocardium noncompaction, and ventricular hyper-trabeculation. This transition, which occurs at embryonic day (E) 9.0-9.5 in murine and E24-28 in human embryos, is crucial for the developing heart to maintain normal cardiac growth and function in response to an increasing haemodynamic load. Although, ventricular trabeculation and compaction are key morphogenetic events associated with this transition, its molecular and cellular mechanism is currently unclear. Initially, cardiac restricted cytokine Bone Morphogenetic Protein 10 (BMP-10) was identified as being up-regulated in hypertrabeculated hearts from mutant embryos deficient in FK506 Binding Protein 12 (FKBP12). To determine the biological function of BMP-10 during cardiac development, we generated BMP-10-deficient mice. Here we describe an essential role of BMP-10 in regulating cardiac growth and chamber maturation. BMP-10 null mice display ectopic and elevated expression of p57kip2 and a dramatic reduction in proliferative activity in cardiomyocytes at E9.0-E9.5. BMP-10 is also required for maintaining normal expression levels of several key cardiogenic factors (e.g., Nkx2.5 and MEF2C) in the developing myocardium at midgestation. Furthermore, BMP-10 conditioned medium is able to rescue BMP-10-deficient hearts in culture. Our data suggest an important pathway that involves a genetic interaction between BMP-10, cell cycle regulatory proteins, and several major cardiac transcription factors in orchestrating this transition in cardiogenesis at midgestation. This may provide an underlying mechanism for understanding the pathogenesis of both structural and functional congenital heart defects.
BMP-10; p57kip2; Nkx2.5 and MEF2C; cardiac growth and development; ventricular trabeculation and compaction
Homeobox transcription factor Nkx2-5, highly expressed in heart, is a critical factor during early embryonic cardiac development. In this study, using tamoxifen-inducible Nkx2-5 knockout mice, we demonstrate the role of Nkx2-5 in conduction and contraction in neonates within 4 days after perinatal tamoxifen injection. Conduction defect was accompanied by reduction in ventricular expression of the cardiac voltage-gated Na+ channel pore-forming α-subunit (Nav1.5-α), the largest ion channel in the heart responsive for rapid depolarization of the action potential, which leads to increased intracellular Ca2+ for contraction (conduction-contraction coupling). In addition, expression of ryanodine receptor 2, through which Ca2+ is released from sarcoplasmic reticulum, was substantially reduced in Nkx2-5 knockout mice. These results indicate that Nkx2-5 function is critical not only during cardiac development but also in perinatal hearts, by regulating expression of several important gene products involved in conduction and contraction.
conduction; contraction; gene targeting; transcription
Multipotent cardiac progenitor cells are found in the fetal and adult heart of many mammalian species including humans and form as intermediates during the differentiation of embryonic stem cells. Despite similar biological properties, the molecular identities of these different cardiac progenitor cell populations appear to be distinct. Elucidating the origins and lineage relationships of these cell populations will accelerate clinical applications such as drug screening and cell therapy as well as shedding light on the pathogenic mechanisms underlying cardiac diseases.
Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca2+/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.
kinase; transcription; contraction
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 up-regulated, leading initially to progenitor over-specification, 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.
Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat–containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5–deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171–175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168–171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.
In this report, an antisense RNA strategy has allowed us to show that disruption of ALP expression affects the expression of the muscle transcription factors myogenin and MyoD, resulting in the inhibition of muscle differentiation. Introduction of a MyoD expression construct into ALP-antisense cells is sufficient to restore the capacity of the cells to differentiate, illustrating that ALP function occurs upstream of MyoD. It is known that MyoD is under the control of serum response factor (SRF), a transcriptional regulator whose activity is modulated by actin dynamics. A dramatic reduction of actin filament bundles is observed in ALP-antisense cells and treatment of these cells with the actin-stabilizing drug jasplakinolide stimulates SRF activity and restores the capacity of the cells to differentiate. Furthermore, we show that modulation of ALP expression influences SRF activity, the level of its coactivator, MAL, and muscle differentiation. Collectively, these results suggest a critical role of ALP on muscle differentiation, likely via cytoskeletal regulation of SRF.
Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2TrxR2−/−minus;/TrxR2−/−minus; embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2TrxR2−/−minus;/TrxR2−/−minus; embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.