The last few years have witnessed remarkable advances in stem cell biology and human genetics, and we have arrived at an era where patient-specific cell and tissue models are now practical. The recent identification of cardiovascular progenitor cells, as well as the identification of genetic variants underlying congenital heart disorders and adult disease, opens the door to the development of human models of human cardiovascular disease. We review the current understanding of the contribution of progenitor cells to cardiogenesis, and we outline how pluripotent stem cells can be applied for the modeling of cardiovascular disorders of genetic origin. A key challenge will be to implement these models in an efficient manner to develop a molecular understanding of how genes lead to disease and to screen for genes and drugs that modify the disease process.
cardiovascular disease; congenital heart disease; cardiac development; stem cells; genetics
The past few years have seen unexpected new developments in direct cardiomyocyte reprogramming. Direct cardiomyocyte reprogramming potentially offers an entirely novel approach to cardiovascular regenerative medicine by converting cardiac fibroblasts into functional cardiomyocytes in situ. There is much to be learned, however, about the mechanisms of direct reprogramming in order that the process can be made more efficient. Early efforts have suggested that this new technology can be technically challenging. Moreover, new methods of inducing heart reprogramming will need to be developed before this approach can be translated to the bedside. Despite this, direct cardiomyocyte reprogramming may lead to new therapeutic options for sufferers of heart disease.
Fibroblasts that form at the site of wound healing in the heart have no contractile ability. The introduction of cardiogenic factors may induce these cells to adopt a cardiomyocyte-like fate.
Targeted deletion of cytoskeletal muscle LIM protein (MLP) in mice consistently leads to dilated cardiomyopathy (DCM) after one or more months. However, next to nothing is known at present about the mechanisms of this process. We investigated whether diastolic performance including passive mechanics and systolic behavior are altered in 2-week-old MLP knockout (MLPKO) mice, in which heart size, fractional shortening and ejection fraction are still normal. Right ventricular trabeculae were isolated from 2-week-old MLPKO and wildtype mice and placed in an apparatus that allowed force measurements and sarcomere length measurements using laser diffraction. During a twitch from the unloaded state at 1 Hz, MLPKO muscles relengthened to slack length more slowly than controls, although the corresponding force relaxation time was unchanged. Active developed stress at a diastolic sarcomere length of 2.00 μm was preserved in MLPKO trabeculae over a wide range of pacing frequencies. Force relaxation under the same conditions was consistently prolonged compared with wildtype controls, whereas time to peak and maximum rate of force generation were not significantly altered. Ca2+ content of the sarcoplasmic reticulum (SR) and the quantities of Ca2+ handling proteins were similar in both genotypes. In summary, young MLPKO mice revealed substantial alterations in passive myocardial properties and relaxation time, but not in most systolic characteristics. These results indicate that the progression to heart failure in the MLPKO model may be driven by diastolic myocardial dysfunction and abnormal passive properties rather than systolic dysfunction.
Cytoskeleton; Sarcomere; Myocardial contraction; Relaxation
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.
Sarco(endo)plasmic reticulum calcium ATPases (SERCA) are cellular pumps that transport Ca2+ into the sarcoplasmic reticulum (SR). Serca2 is the most widely expressed gene family member. The very early embryonic lethality of Serca2null mouse embryos has precluded further evaluation of loss of Serca2 function in the context of organ physiology. We have generated mice carrying a conditional Serca2flox allele which allows disruption of the Serca2 gene in an organ-specific and/or inducible manner. The model was tested by mating Serca2flox mice with MLC-2vwt/Cre mice and with αMHC-Cre transgenic mice. In heterozygous Serca2wt/flox MLC-2vwt/Cre mice, the expression of SERCA2a and SERCA2b proteins were reduced in the heart and slow skeletal muscle, in accordance with the expression pattern of the MLC-2v gene. In Serca2flox/flox Tg(αMHC-Cre) embryos with early homozygous cardiac Serca2 disruption, normal embryonic development and yolk sac circulation was maintained up to at least embryonic stage E10.5. The Serca2flox mouse is the first murine conditional gene disruption model for the SERCA family of Ca2+ ATPases, and should be a powerful tool for investigating specific physiological roles of SERCA2 function in a range of tissues and organs in vivo both in adult and embryonic stages.
Serca2; Calcium ATPase; Endoplasmic reticulum; Sarcoplasmic reticulum; Flox; Transgenic mouse
Studying monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combine patient-derived and genetically engineered iPSCs with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene Tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural, and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS “heart on chip” tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies, and advances iPSC-based in vitro modeling of cardiomyopathy.
The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation.
N-cadherin; anterior heart field; cardiac progenitor cells; premature differentiation; Wnt signaling; microenvironment
The fact that mammals are diploid sets a barrier to rapidly understand the function of non-coding and coding genes in the genome. Recently, Yang et al. reported successful derivation of monkey haploid embryonic stem cells from parthenotes, which provide an effective platform for studying mammalian gene function and enable reverse genetic screening of genes for recessive phenotypes in monkeys.
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 pluripotent stem cell (hPSC)-derived endothelial lineage cells constitutes a promising source for therapeutic revascularization, but progress in this arena has been hampered by a lack of clinically-scalable differentiation protocols and inefficient formation of a functional vessel network integrating with the host circulation upon transplantation. Using a human embryonic stem cell reporter cell line, where green fluorescent protein expression is driven by an endothelial cell-specific VE-cadherin (VEC) promoter, we screened for > 60 bioactive small molecules that would promote endothelial differentiation, and found that administration of BMP4 and a GSK-3β inhibitor in an early phase and treatment with VEGF-A and inhibition of the Notch signaling pathway in a later phase led to efficient differentiation of hPSCs to the endothelial lineage within six days. This sequential approach generated > 50% conversion of hPSCs to endothelial cells (ECs), specifically VEC+CD31+CD34+CD14−KDRhigh endothelial progenitors (EPs) that exhibited higher angiogenic and clonogenic proliferation potential among endothelial lineage cells. Pharmaceutical inhibition or genetical knockdown of Notch signaling, in combination with VEGF-A treatment, resulted in efficient formation of EPs via KDR+ mesodermal precursors and blockade of the conversion of EPs to mature ECs. The generated EPs successfully formed functional capillary vessels in vivo with anastomosis to the host vessels when transplanted into immunocompromised mice. Manipulation of this VEGF-A-Notch signaling circuit in our protocol leads to rapid large-scale production of the hPSC-derived EPs by 12- to 20-fold vs current methods, which may serve as an attractive cell population for regenerative vascularization with superior vessel forming capability compared to mature ECs.
embryonic stem cell; endothelial progenitor; Notch signaling; VEGF
In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) resulted in the expansion and directed differentiation of endogenous heart progenitors in a murine myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors, and temporal control with VEGF inhibitors, documented the markedly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long term organ repair.
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
The gp130 cytokine receptor activates a cardiomyocyte survival pathway during the transition to heart failure following the biomechanical stress of pressure overload. Although gp130 activation is observed transiently during transverse aortic constriction (TAC), its mechanism of inactivation is largely unknown in cardiomyocytes. We show here that suppressor of cytokine signaling 3 (SOCS3), an intrinsic inhibitor of JAK, shows biphasic induction in response to TAC. The induction of SOCS3 was closely correlated with STAT3 phosphorylation, as well as the activation of an embryonic gene program, suggesting that cardiac gp130-JAK signaling is precisely controlled by this endogenous suppressor. In addition to its cytoprotective action, gp130-dependent signaling induces cardiomyocyte hypertrophy. Adenovirus-mediated gene transfer of SOCS3 to ventricular cardiomyocytes completely suppressed both hypertrophy and antiapoptotic phenotypes induced by leukemia inhibitory factor (LIF). To our knowledge, this is the first clear evidence that these two separate cardiomyocyte phenotypes induced by gp130 activation lie downstream of JAK. Three independent signaling pathways, STAT3, MEK1-ERK1/2, and AKT activation, that are coinduced by LIF stimulation were completely suppressed by SOCS3 overexpression. We conclude that SOCS3 is a mechanical stress–inducible gene in cardiac muscle cells and that it directly modulates stress-induced gp130 cytokine receptor signaling as the key molecular switch for a negative feedback circuit for both myocyte hypertrophy and survival.
Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload.
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.
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?
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
Telethonin (also known as titin-cap or t-cap) is a 19 kDa Z-disk protein with a unique β-sheet structure, hypothesized to assemble in a palindromic way with the N-terminal portion of titin and to constitute a signalosome participating in the process of cardio-mechanosensing. In addition, a variety of telethonin mutations are associated with the development of several different diseases; however, little is known about the underlying molecular mechanisms and telethonin’s in vivo function.
Here we aim to investigate the role of telethonin in vivo and to identify molecular mechanisms underlying disease as a result of its mutation.
Methods and Results
By using a variety of different genetically altered animal models and biophysical experiments we show that, contrary to previous views, telethonin is not an indispensable component of the titin-anchoring system, nor is deletion of the gene or cardiac specific overexpression associated with a spontaneous cardiac phenotype. Rather, additional titin-anchorage sites, such as actin-titin crosslinks via α-actinin, are sufficient to maintain Z-disk stability despite the loss of telethonin. We demonstrate that a main novel function of telethonin is to modulate the turnover of the pro-apoptotic tumor suppressor p53 after biomechanical stress in the nuclear compartment, thus linking telethonin, a protein well known to be present at the Z-disk, directly to apoptosis (“mechanoptosis”). In addition, loss of telethonin mRNA and nuclear accumulation of this protein is associated with human heart failure, an effect which may contribute to enhanced rates of apoptosis found in these hearts.
Telethonin knockout mice do not reveal defective heart development or heart function under basal conditions, but develop heart failure following biomechanical stress, owing at least in part to apoptosis of cardiomyocytes, an effect which may also play a role in human heart failure.
Genetics; Mechanosensation; Mechanotransduction; Cardiomyopathy; Heart failure
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
Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.
induced pluripotent stem cells; ventricular cardiomyocyte; regenerative medicine; epigenetic memory
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