Networks that Govern Cardiomyocyte Proliferation to Facilitate Repair of the Injured Mammalian Heart.

Cardiovascular diseases are the number one cause of death worldwide and in the United States (US). Cardiovascular diseases frequently progress to end-stage heart failure, and curative therapies are extremely limited. Intense interest has focused on deciphering the cascades and networks that govern cardiomyocyte proliferation and regeneration of the injured heart. For example, studies have shown that lower organisms such as the adult newt and adult zebrafish have the capacity to completely regenerate their injured heart with restoration of function. Similarly, the neonatal mouse and pig are also able to completely regenerate injured myocardium due to cardiomyocyte proliferation from preexisting cardiomyocytes. Using these animal models and transcriptome analyses, efforts have focused on the definition of factors and signaling pathways that can reactivate and induce cardiomyocyte proliferation in the adult mammalian injured heart. These studies and discoveries have the potential to define novel therapies to promote cardiomyocyte proliferation and repair of the injured, mammalian heart.

FOXK1 regulates Wnt signalling to promote cardiogenesis.

AIMS: Congenital heart disease (CHD) is the most common genetic birth defect, which has considerable morbidity and mortality. We focused on deciphering key regulators that govern cardiac progenitors and cardiogenesis. FOXK1 is a forkhead/winged helix transcription factor known to regulate cell cycle kinetics and is restricted to mesodermal progenitors, somites, and heart. In the present study, we define an essential role for FOXK1 during cardiovascular development. METHODS AND RESULTS: We used the mouse embryoid body system to differentiate control and Foxk1 KO embryonic stem cells into mesodermal, cardiac progenitor cells and mature cardiac cells. Using flow cytometry, immunohistochemistry, cardiac beating, transcriptional and chromatin immunoprecipitation quantitative polymerase chain reaction assays, bulk RNA sequencing (RNAseq) and assay for transposase-accessible chromatin using sequencing (ATACseq) analyses, FOXK1 was observed to be an important regulator of cardiogenesis. Flow cytometry analyses revealed perturbed cardiogenesis in Foxk1 KO embryoid bodies (EBs). Bulk RNAseq analysis at two developmental stages showed a significant reduction of the cardiac molecular program in Foxk1 KO EBs compared to the control EBs. ATACseq analysis during EB differentiation demonstrated that the chromatin landscape nearby known important regulators of cardiogenesis was significantly relaxed in control EBs compared to Foxk1 KO EBs. Furthermore, we demonstrated that in the absence of FOXK1, cardiac differentiation was markedly impaired by assaying for cardiac Troponin T expression and cardiac contractility. We demonstrate that FOXK1 is an important regulator of cardiogenesis by repressing the Wnt/ß-catenin signalling pathway and thereby promoting differentiation. CONCLUSION: These results identify FOXK1 as an essential transcriptional and epigenetic regulator of cardiovascular development. Mechanistically, FOXK1 represses Wnt signalling to promote the development of cardiac progenitor cells.

ETV2 and VEZF1 interaction and regulation of the hematoendothelial lineage during embryogenesis.

Ets variant 2 (Etv2), a member of the Ets factor family, has an essential role in the formation of endothelial and hematopoietic cell lineages during embryonic development. The functional role of ETS transcription factors is, in part, dependent on the interacting proteins. There are relatively few studies exploring the coordinated interplay between ETV2 and its interacting proteins that regulate mesodermal lineage determination. In order to identify novel ETV2 interacting partners, a yeast two-hybrid analysis was performed and the C2H2 zinc finger transcription factor VEZF1 (vascular endothelial zinc finger 1) was identified as a binding factor, which was specifically expressed within the endothelium during vascular development. To confirm this interaction, co-immunoprecipitation and GST pull down assays demonstrated the direct interaction between ETV2 and VEZF1. During embryoid body differentiation, Etv2 achieved its peak expression at day 3.0 followed by rapid downregulation, on the other hand Vezf1 expression increased through day 6 of EB differentiation. We have previously shown that ETV2 potently activated Flt1 gene transcription. Using a Flt1 promoter-luciferase reporter assay, we demonstrated that VEZF1 co-activated the Flt1 promoter. Electrophoretic mobility shift assay and Chromatin immunoprecipitation established VEZF1 binding to the Flt1 promoter. Vezf1 knockout embryonic stem cells had downregulation of hematoendothelial marker genes when undergoing embryoid body mediated mesodermal differentiation whereas overexpression of VEZF1 induced the expression of hematoendothelial genes during differentiation. These current studies provide insight into the co-regulation of the hemato-endothelial lineage development via a co-operative interaction between ETV2 and VEZF1.

Xenotransplantation and exotransplantation: Strategies to expand the number of donor organs.

Cardiovascular disease is common and has a high mortality. Due to the limited number of organs available for orthotopic heart transplantation, alternative therapies have received intense interest. In this commentary we contrast xenotransplantation and blastocyst complementation to produce pigs that will serve as donors for organ transplantation. These strategies hold tremendous promise and have the potential to provide an unlimited number of organs for chronic, terminal diseases.

Blastocyst complementation and interspecies chimeras in gene edited pigs.

The only curative therapy for many endstage diseases is allograft organ transplantation. Due to the limited supply of donor organs, relatively few patients are recipients of a transplanted organ. Therefore, new strategies are warranted to address this unmet need. Using gene editing technologies, somatic cell nuclear transfer and human induced pluripotent stem cell technologies, interspecies chimeric organs have been pursued with promising results. In this review, we highlight the overall technical strategy, the successful early results and the hurdles that need to be addressed in order for these approaches to produce a successful organ that could be transplanted in patients with endstage diseases.

Emerging technologies and ethics-exogenic chimeric humanized organs.

Organ transplantation is limited due to the scarcity of donor organs. In order to expand the supply of organs for transplantation, interspecies chimeras have been examined as a potential future source of humanized organs. Recent studies using gene editing technologies in combination with somatic cell nuclear transfer technology and hiPSCs successfully engineered humanized skeletal muscle in the porcine embryo. As these technologies progress, there are ethical issues that warrant consideration and dialogue.

Human muscle in gene edited pigs for treatment of volumetric muscle loss.

Focusing on complex extremity trauma and volumetric muscle loss (VML) injuries, this review highlights: 1) the current pathophysiologic limitations of the injury sequela; 2) the gene editing strategy of the pig as a model that provides a novel treatment approach; 3) the notion that human skeletal muscle derived from gene edited, humanized pigs provides a groundbreaking treatment option; and 4) the impact of this technologic platform and how it will advance to far more multifaceted applications. This review seeks to shed insights on a novel treatment option using gene edited pigs as a platform which is necessary to overcome the clinical challenges and limitations in the field.

Mechanisms and strategies to promote cardiac xenotransplantation.

End stage heart failure is a terminal disease, and the only curative therapy is orthotopic heart transplantation. Due to limited organ availability, alternative strategies have received intense interest for treatment of patients with advanced heart failure. Recent studies using gene-edited porcine organs suggest that cardiac xenotransplantation may provide a future source of organs. In this review, we highlight the historical milestones for cardiac xenotransplantation and the gene editing strategies designed to overcome immunological barriers, which have culminated in a recent cardiac pig-to-human xenotransplant. We also discuss recent results of studies on the engineering of human-porcine chimeric organs that may provide an alternative and complementary strategy to overcome some of the major immunological barriers to producing a new source of transplantable organs.

ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage.

The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and is thus relevant to the treatment of ischaemic diseases, injury-induced regeneration and solid tumour growth. Previously, we demonstrated that ETV2 is an essential transcription factor for the development of cardiac, endothelial and haematopoietic lineages. Here we report that ETV2 functions as a pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with multi-omics techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and recruit BRG1. BRG1 recruitment remodelled chromatin around endothelial genes and helped to maintain an open configuration, resulting in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular diseases and solid tumours.

Interspecies chimeras as a platform for exogenic organ production and transplantation.

Chronic diseases are associated with considerable morbidity and mortality. Therefore, new therapeutic strategies are warranted. Here, we provide a brief review outlining the rationale and feasibility for the generation of intraspecies and interspecies chimeras, which one day may serve as a platform for organ transplantation. These strategies are further associated with consideration of scientific and ethical issues.

Biologically Derived, Three-Dimensional, Embryonic Scaffolds for Long-Term Cardiomyocyte Culture.

The ability to maintain viable cultures of mature, primary cardiomyocytes is challenging. The lack of viable cardiomyocyte cultures severely limits in vitro biochemical assays, toxicology assays, drug screening assays, and other analyses. Here, we describe a novel three-dimensional (3D) embryonic scaffold, which supports the culture of postnatal day 7 murine cardiomyocytes within the embryonic heart for, at least, 28 days. We have observed that these cardiomyocytes display normal differentiation, protein expression, and function after extended culture. This novel culture system will allow for prolonged treatment of cardiomyocytes in a natural 3D orientation and has the potential for providing a superior tool for the screening of therapeutic compounds.

Humanized skeletal muscle in MYF5/MYOD/MYF6-null pig embryos.

Because post-mortem human skeletal muscle is not viable, autologous muscle grafts are typically required in tissue reconstruction after muscle loss due to disease or injury. However, the use of autologous tissue often leads to donor-site morbidity. Here, we show that intraspecies and interspecies chimaeric pig embryos lacking native skeletal muscle can be produced by deleting the MYF5, MYOD and MYF6 genes in the embryos via CRISPR, followed by somatic-cell nuclear transfer and the delivery of exogenous cells (porcine blastomeres or human induced pluripotent stem cells) via blastocyst complementation. The generated intraspecies chimaeras were viable and displayed normal histology, morphology and function. Human:pig chimaeras generated with TP53-null human induced pluripotent stem cells led to higher chimaerism efficiency, with embryos collected at embryonic days 20 and 27 containing humanized muscle, as confirmed by immunohistochemical and molecular analyses. Human:pig chimaeras may facilitate the production of exogenic organs for research and xenotransplantation.

ETV2 (Ets Variant Transcription Factor 2)-Rhoj Cascade Regulates Endothelial Progenitor Cell Migration During Embryogenesis.

OBJECTIVE: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. The regulatory mechanisms that govern their migration are not completely defined. Here, we describe a novel role for ETV2 (Ets variant transcription factor 2) in cell migration and provide evidence for an ETV2-Rhoj network as a mechanism responsible for this process. Approach and Results: Analysis of RNAseq datasets showed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 chromatin immunoprecipitation-seq and assay for transposase accessible chromatin-seq datasets, which showed enrichment of chromatin immunoprecipitation-seq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Migratory assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells, embryoid bodies, and mouse embryonic fibroblasts. Knockout of Etv2 led to migratory defects of Etv2-EYFP+ angioblasts to their predefined regions of developing embryos relative to wild-type controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds the promoter region of Rhoj serving as an upstream regulator of cell migration. Single-cell RNAseq analysis of Etv2-EYFP+ sorted cells revealed coexpression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both embryoid bodies and mouse embryonic fibroblasts, whereas, its expression was abolished in the Etv2 knockout embryoid bodies. Finally, shRNA-mediated knockdown of Rhoj resulted in migration defects, which were partially rescued by overexpression of ETV2. CONCLUSIONS: These results define an ETV2-Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration.

A murine model of the exercise pressor reflex.

KEY POINTS: The decerebrate mouse provides a novel working model of the exercise pressor reflex (EPR). The decerebrate mouse model of the EPR is similar to the previously described decerebrate rat model. Studying the EPR in transgenic mouse models can define exact mechanisms of the EPR in health and disease. ABSTRACT: The exercise pressor reflex (EPR) is defined by a rise in mean arterial pressure (MAP) and heart rate (HR) in response to exercise and is necessary to match metabolic demand and prevent premature fatigue. While this reflex is readily tested in humans, mechanistic studies are largely infeasible. Here, we have developed a novel murine model of the EPR to allow for mechanistic studies in various mouse models. We observed that ventral root stimulation (VRS) in an anaesthetized mouse causes a depressor response and a reduction in HR. In contrast, the same stimulation in a decerebrate mouse causes a rise in MAP and HR which is abolished by dorsal rhizotomy or by neuromuscular blockade. Moreover, we demonstrate a reduced MAP response to VRS using TRPV1 antagonism or in Trpv1 null mice while the response to passive stretch remains intact. Additionally, we demonstrate that intra-arterial infusion of capsaicin results in a dose-related rise in MAP and HR that is significantly reduced by a selective and potent TRPV1 antagonist or is completely abolished in Trpv1 null mice. These data serve to validate the development of a decerebrate mouse model for the study of cardiovascular responses to exercise and further define the role of the TRPV1 receptor in mediating the EPR. This novel model will allow for extensive study of the EPR in unlimited transgenic and mutant mouse lines, and for an unprecedented exploration of the molecular mechanisms that control cardiovascular responses to exercise in health and disease.

Chimeric Humanized Vasculature and Blood: The Intersection of Science and Ethics.

The only curative therapy for diseases such as organ failure is orthotopic organ transplantation. Organ transplantation has been limited due to the shortage of donor organs. The huge disparity between those who need and those who receive transplantation therapy drives the pursuit of alternative treatments. Therefore, novel therapies are warranted. Recent studies support the feasibility of generating human-porcine chimeras that one day would provide humanized vasculature and blood for transplantation and serve as important research models. The ethical issues they raise require open discussion and dialog lest promising lines of inquiry flounder due to unfounded fears or compromised public trust.

Stem Cell-Derived Cardiomyocytes and Beta-Adrenergic Receptor Blockade in Duchenne Muscular Dystrophy Cardiomyopathy.

BACKGROUND: Although cardiomyopathy has emerged as a leading cause of death in Duchenne muscular dystrophy (DMD), limited studies and therapies have emerged for dystrophic heart failure. OBJECTIVES: The purpose of this study was to model DMD cardiomyopathy using DMD patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and to identify physiological changes and future drug therapies. METHODS: To explore and define therapies for DMD cardiomyopathy, the authors used DMD patient-specific hiPSC-derived cardiomyocytes to examine the physiological response to adrenergic agonists and ß-blocker treatment. The authors further examined these agents in vivo using wild-type and mdx mouse models. RESULTS: At baseline and following adrenergic stimulation, DMD hiPSC-derived cardiomyocytes had a significant increase in arrhythmic calcium traces compared to isogenic controls. Furthermore, these arrhythmias were significantly decreased with propranolol treatment. Using telemetry monitoring, the authors observed that mdx mice, which lack dystrophin, had an arrhythmic death when stimulated with isoproterenol; the lethal arrhythmias were rescued, in part, by propranolol pre-treatment. Using single-cell and bulk RNA sequencing (RNA-seq), the authors compared DMD and control hiPSC-derived cardiomyocytes, mdx mice, and control mice (in the presence or absence of propranolol and isoproterenol) and defined pathways that were perturbed under baseline conditions and pathways that were normalized after propranolol treatment in the mdx model. The authors also undertook transcriptome analysis of human DMD left ventricle samples and found that DMD hiPSC-derived cardiomyocytes have dysregulated pathways similar to the human DMD heart. The authors further determined that relatively few patients with DMD see a cardiovascular specialist or receive ß-blocker therapy. CONCLUSIONS: The results highlight mechanisms and therapeutic interventions from human to animal and back to human in the dystrophic heart. These results may serve as a prelude for an adequately powered clinical study that examines the impact of ß-blocker therapy in patients with dystrophinopathies.

Generation of human endothelium in pig embryos deficient in ETV2.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney1,2. However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages3-7. ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.

ETV2-null porcine embryos survive to post-implantation following incomplete enucleation.

Blind enucleation is used in porcine somatic cell nuclear transfer (SCNT) to remove the metaphase II (MII) spindle from the oocyte. Deviation of the MII spindle location, however, leads to incomplete enucleation (IE). Here, we report that the rate of complete enucleation (CE) using the blind method was 80.2 ± 1.7%, although this significantly increased when the polar body-MII deviation was minimized (≦45°). While it is established that IE embryos will not survive to full term, the effect of IE on early stage development is unknown. We have previously demonstrated in mice and pigs that ETV2 deletion results in embryonic lethality due to the lack of hematoendothelial lineages. We observed that ETV2-null cloned embryos derived from blindly and incompletely enucleated oocytes had both WT and mutant sequences at E18 and, using FISH analysis, we observed triploidy. We also compared SCNT embryos generated from either CE or intentionally IE oocytes using the spindle viewer system. We observed a higher in vitro blastocyst rate in the IE versus the CE-SCNT embryos (31.9 ± 3.2% vs 21.0 ± 2.1%). Based on known processes in normal fertilization, we infer that the IE-SCNT embryos extruded the haploid second PB after fusion with donor fibroblasts and formed a near-triploid aneuploid nucleus in each blastomere. These studies demonstrate the peri-implantation survival of residual haploid nuclei following IE and emphasize the need for complete enucleation especially for the analysis of SCNT embryos in the peri-implantation stage and will, further, impact the field of reverse xenotransplantation.