Developmental fate and cellular maturity encoded in human regulatory DNA landscapes.

Cellular-state information between generations of developing cells may be propagated via regulatory regions. We report consistent patterns of gain and loss of DNase I-hypersensitive sites (DHSs) as cells progress from embryonic stem cells (ESCs) to terminal fates. DHS patterns alone convey rich information about cell fate and lineage relationships distinct from information conveyed by gene expression. Developing cells share a proportion of their DHS landscapes with ESCs; that proportion decreases continuously in each cell type as differentiation progresses, providing a quantitative benchmark of developmental maturity. Developmentally stable DHSs densely encode binding sites for transcription factors involved in autoregulatory feedback circuits. In contrast to normal cells, cancer cells extensively reactivate silenced ESC DHSs and those from developmental programs external to the cell lineage from which the malignancy derives. Our results point to changes in regulatory DNA landscapes as quantitative indicators of cell-fate transitions, lineage relationships, and dysfunction.

A temporal chromatin signature in human embryonic stem cells identifies regulators of cardiac development.

Directed differentiation of human embryonic stem cells (ESCs) into cardiovascular cells provides a model for studying molecular mechanisms of human cardiovascular development. Although it is known that chromatin modification patterns in ESCs differ markedly from those in lineage-committed progenitors and differentiated cells, the temporal dynamics of chromatin alterations during differentiation along a defined lineage have not been studied. We show that differentiation of human ESCs into cardiovascular cells is accompanied by programmed temporal alterations in chromatin structure that distinguish key regulators of cardiovascular development from other genes. We used this temporal chromatin signature to identify regulators of cardiac development, including the homeobox gene MEIS2. Using the zebrafish model, we demonstrate that MEIS2 is critical for proper heart tube formation and subsequent cardiac looping. Temporal chromatin signatures should be broadly applicable to other models of stem cell differentiation to identify regulators and provide key insights into major developmental decisions.

4HNE Impairs Myocardial Bioenergetics in Congenital Heart Disease-Induced Right Ventricular Failure.

BACKGROUND: In patients with complex congenital heart disease, such as those with tetralogy of Fallot, the right ventricle (RV) is subject to pressure overload stress, leading to RV hypertrophy and eventually RV failure. The role of lipid peroxidation, a potent form of oxidative stress, in mediating RV hypertrophy and failure in congenital heart disease is unknown. METHODS: Lipid peroxidation and mitochondrial function and structure were assessed in right ventricle (RV) myocardium collected from patients with RV hypertrophy with normal RV systolic function (RV fractional area change, 47.3±3.8%) and in patients with RV failure showing decreased RV systolic function (RV fractional area change, 26.6±3.1%). The mechanism of the effect of lipid peroxidation, mediated by 4-hydroxynonenal ([4HNE] a byproduct of lipid peroxidation) on mitochondrial function and structure was assessed in HL1 murine cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: RV failure was characterized by an increase in 4HNE adduction of metabolic and mitochondrial proteins (16 of 27 identified proteins), in particular electron transport chain proteins. Sarcomeric (myosin) and cytoskeletal proteins (desmin, tubulin) also underwent 4HNE adduction. RV failure showed lower oxidative phosphorylation (moderate RV hypertrophy, 287.6±19.75 versus RV failure, 137.8±11.57 pmol/[sec×mL]; P=0.0004), and mitochondrial structural damage. Using a cell model, we show that 4HNE decreases cell number and oxidative phosphorylation (control, 388.1±23.54 versus 4HNE, 143.7±11.64 pmol/[sec×mL]; P<0.0001). Carvedilol, a known antioxidant did not decrease 4HNE adduction of metabolic and mitochondrial proteins and did not improve oxidative phosphorylation. CONCLUSIONS: Metabolic, mitochondrial, sarcomeric, and cytoskeletal proteins are susceptible to 4HNE-adduction in patients with RV failure. 4HNE decreases mitochondrial oxygen consumption by inhibiting electron transport chain complexes. Carvedilol did not improve the 4HNE-mediated decrease in oxygen consumption. Strategies to decrease lipid peroxidation could improve mitochondrial energy generation and cardiomyocyte survival and improve RV failure in patients with congenital heart disease.

Cardiac involvement in classical or hypermobile Ehlers-Danlos syndrome is uncommon.

PURPOSE: Cardiac-valvular and vascular Ehlers-Danlos syndrome (EDS) have significant cardiovascular issues. The prevalence and significance of such abnormalities in classical (cEDS) or hypermobile EDS (hEDS) remain unclear. We report the prevalence of cardiac abnormalities in patients with cEDS and hEDS. METHODS: We identified 532 pediatric patients with potential EDS evaluated at our institution from January 2014 through April 2019 by retrospective chart review. Ninety-five patients (12 cEDS and 83 hEDS patients) met 2017 EDS diagnostic criteria and had an echocardiogram. One patient was excluded due to complex congenital heart disease, and two were excluded due to lack of images. We reviewed echocardiograms for all structural abnormalities. RESULTS: Of these 95 patients, 1 had mild aortic root dilation, and 1 had mild ascending aorta dilation in the setting of a bicuspid aortic valve. Eleven patients (11.6%) had a cardiac valve abnormality, all of which were trivial to mild. None of the patients required cardiac intervention. CONCLUSION: Our results demonstrate that aortic dilation and valvular anomalies are uncommon in cEDS or hEDS patients. Given the lack of evidence, we do not recommend echocardiographic evaluation and surveillance in patients with cEDS and hEDS in the absence of clinical findings or positive family history.

Cardiac Regeneration: Lessons From Development.

Palliative surgery for congenital heart disease has allowed patients with previously lethal heart malformations to survive and, in most cases, to thrive. However, these procedures often place pressure and volume loads on the heart, and over time, these chronic loads can cause heart failure. Current therapeutic options for initial surgery and chronic heart failure that results from failed palliation are limited, in part, by the mammalian heart's low inherent capacity to form new cardiomyocytes. Surmounting the heart regeneration barrier would transform the treatment of congenital, as well as acquired, heart disease and likewise would enable development of personalized, in vitro cardiac disease models. Although these remain distant goals, studies of heart development are illuminating the path forward and suggest unique opportunities for heart regeneration, particularly in fetal and neonatal periods. Here, we review major lessons from heart development that inform current and future studies directed at enhancing cardiac regeneration.

Molecular regulation of cardiomyocyte differentiation.

The heart is the first organ to form during embryonic development. Given the complex nature of cardiac differentiation and morphogenesis, it is not surprising that some form of congenital heart disease is present in ≈1 percent of newborns. The molecular determinants of heart development have received much attention over the past several decades. This has been driven in large part by an interest in understanding the causes of congenital heart disease coupled with the potential of using knowledge from developmental biology to generate functional cells and tissues that could be used for regenerative medicine purposes. In this review, we highlight the critical signaling pathways and transcription factor networks that regulate cardiomyocyte lineage specification in both in vivo and in vitro models. Special focus will be given to epigenetic regulators that drive the commitment of cardiomyogenic cells from nascent mesoderm and their differentiation into chamber-specific myocytes, as well as regulation of myocardial trabeculation.

Transmembrane protein 88: a Wnt regulatory protein that specifies cardiomyocyte development.

Genetic regulation of the cell fate transition from lateral plate mesoderm to the specification of cardiomyocytes requires suppression of Wnt/ß-catenin signaling, but the mechanism for this is not well understood. By analyzing gene expression and chromatin dynamics during directed differentiation of human embryonic stem cells (hESCs), we identified a suppressor of Wnt/ß-catenin signaling, transmembrane protein 88 (TMEM88), as a potential regulator of cardiovascular progenitor cell (CVP) specification. During the transition from mesoderm to the CVP, TMEM88 has a chromatin signature of genes that mediate cell fate decisions, and its expression is highly upregulated in advance of key cardiac transcription factors in vitro and in vivo. In early zebrafish embryos, tmem88a is expressed broadly in the lateral plate mesoderm, including the bilateral heart fields. Short hairpin RNA targeting of TMEM88 during hESC cardiac differentiation increases Wnt/ß-catenin signaling, confirming its role as a suppressor of this pathway. TMEM88 knockdown has no effect on NKX2.5 or GATA4 expression, but 80% of genes most highly induced during CVP development have reduced expression, suggesting adoption of a new cell fate. In support of this, analysis of later stage cell differentiation showed that TMEM88 knockdown inhibits cardiomyocyte differentiation and promotes endothelial differentiation. Taken together, TMEM88 is crucial for heart development and acts downstream of GATA factors in the pre-cardiac mesoderm to specify lineage commitment of cardiomyocyte development through inhibition of Wnt/ß-catenin signaling.

Mechanical Stress Promotes Maturation of Human Myocardium From Pluripotent Stem Cell-Derived Progenitors.

Recent advances in pluripotent stem cell biology and directed differentiation have identified a population of human cardiovascular progenitors that give rise to cardiomyocytes, smooth muscle, and endothelial cells. Because the heart develops from progenitors in 3D under constant mechanical load, we sought to test the effects of a 3D microenvironment and mechanical stress on differentiation and maturation of human cardiovascular progenitors into myocardial tissue. Progenitors were derived from embryonic stem cells, cast into collagen hydrogels, and left unstressed or subjected to static or cyclic mechanical stress. Compared to 2D culture, the unstressed 3D environment increased cardiomyocyte numbers and decreased smooth muscle numbers. Additionally, 3D culture suppressed smooth muscle α-actin content, suggesting diminished cell maturation. Cyclic stress-conditioning increased expression of several cardiac markers, including ß-myosin heavy chain and cardiac troponin T, and the tissue showed enhanced calcium dynamics and force production. There was no effect of mechanical loading on cardiomyocyte or smooth muscle specification. Thus, 3D growth conditions favor cardiac differentiation from cardiovascular progenitors, whereas 2D conditions promote smooth muscle differentiation. Mechanical loading promotes cardiomyocyte structural and functional maturation. Culture in 3-D facilitates understanding how cues such as mechanical stress affect the differentiation and morphogenesis of distinct cardiovascular cell populations into organized, functional human cardiovascular tissue. Stem Cells 2015;33:2148-2157.

Phase 2 Study of Aficamten in Patients With Obstructive Hypertrophic Cardiomyopathy.

BACKGROUND: Left ventricular outflow tract (LVOT) obstruction is a major determinant of heart failure symptoms in obstructive hypertrophic cardiomyopathy (oHCM). Aficamten, a next-in-class cardiac myosin inhibitor, may lower gradients and improve symptoms in these patients. OBJECTIVES: This study aims to evaluate the safety and efficacy of aficamten in patients with oHCM. METHODS: Patients with oHCM and LVOT gradients ≥30 mm Hg at rest or ≥50 mm Hg with Valsalva were randomized 2:1 to receive aficamten (n = 28) or placebo (n = 13) in 2 dose-finding cohorts. Doses were titrated based on gradients and ejection fraction (EF). Safety and changes in gradient, EF, New York Heart Association functional class, and cardiac biomarkers were assessed over a 10-week treatment period and after a 2-week washout. RESULTS: From baseline to 10 weeks, aficamten reduced gradients at rest (mean difference: -40 ± 27 mm Hg, and -43 ± 37 mm Hg in Cohorts 1 and 2, P = 0.0003 and P = 0.0004 vs placebo, respectively) and with Valsalva (-36 ± 27 mm Hg and -53 ± 44 mm Hg, P = 0.001 and <0.0001 vs placebo, respectively). There were modest reductions in EF (-6% ± 7.5% and -12% ± 5.9%, P = 0.007 and P < 0.0001 vs placebo, respectively). Symptomatic improvement in ≥1 New York Heart Association functional class was observed in 31% on placebo, and 43% and 64% on aficamten in Cohorts 1 and 2, respectively (nonsignificant). With aficamten, N-terminal pro-B-type natriuretic peptide was reduced (62% relative to placebo, P = 0.0002). There were no treatment interruptions and adverse events were similar between treatment arms. CONCLUSIONS: Aficamten resulted in substantial reductions in LVOT gradients with most patients experiencing improvement in biomarkers and symptoms. These results highlight the potential of sarcomere-targeted therapy for treatment of oHCM.

Purification of Pluripotent Stem Cell-Derived Cardiomyocytes Using CRISPR/Cas9-Mediated Integration of Fluorescent Reporters.

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have become critically important for the detailed study of cardiac development, disease modeling, and drug screening. However, directed differentiation of hiPSCs into cardiomyocytes often results in mixed populations of cardiomyocytes and other cell types, which may confound experiments that require pure populations of cardiomyocytes. Here, we detail the use of a CRISPR/Cas9 genome editing strategy to develop cardiomyocyte-specific reporters that allow for the isolation of hiPSC-derived cardiomyocytes and chamber-specific myocytes. Moreover, we describe a cardiac differentiation protocol to derive cardiomyocytes from hiPSCs, as well as a strategy to use fluorescence-activated cell sorting to isolate pure populations of fluorescently labeled cardiomyocytes for downstream applications.

Cardiogenesis from human embryonic stem cells.

Over the past decade, the ability to culture and differentiate human embryonic stem cells (ESCs) has offered researchers a novel therapeutic that may, for the first time, repair regions of the damaged heart. Studies of cardiac development in lower organisms have led to identification of the transforming growth factor-ß superfamily (eg, activin A and bone morphogenic protein 4) and the Wnt/ß-catenin pathway as key inducers of mesoderm and cardiovascular differentiation. These factors act in a context-specific manner (eg, Wnt/ß-catenin is required initially to form mesoderm but must be antagonized thereafter to make cardiac muscle). Different lines of ESCs produce different levels of agonists and antagonists for these pathways, but with careful optimization, highly enriched populations of immature cardiomyocytes can be generated. These cardiomyocytes survive transplantation to infarcted hearts of experimental animals, where they create new human myocardial tissue and improve heart function. The grafts generated by cell transplantation have been small, however, leading to an exploration of tissue engineering as an alternate strategy. Engineered tissue generated from preparations of human cardiomyocytes survives poorly after transplantation, most likely because of ischemia. Creation of pre-organized vascular networks in the tissue markedly enhances survival, with human capillaries anastomosed to the host coronary circulation. Thus, pathways controlling formation of the human cardiovascular system are emerging, yielding the building blocks for tissue regeneration that may address the root causes of heart failure.

Intrinsic Endocardial Defects Contribute to Hypoplastic Left Heart Syndrome.

Hypoplastic left heart syndrome (HLHS) is a complex congenital heart disease characterized by abnormalities in the left ventricle, associated valves, and ascending aorta. Studies have shown intrinsic myocardial defects but do not sufficiently explain developmental defects in the endocardial-derived cardiac valve, septum, and vasculature. Here, we identify a developmentally impaired endocardial population in HLHS through single-cell RNA profiling of hiPSC-derived endocardium and human fetal heart tissue with an underdeveloped left ventricle. Intrinsic endocardial defects contribute to abnormal endothelial-to-mesenchymal transition, NOTCH signaling, and extracellular matrix organization, key factors in valve formation. Endocardial abnormalities cause reduced cardiomyocyte proliferation and maturation by disrupting fibronectin-integrin signaling, consistent with recently described de novo HLHS mutations associated with abnormal endocardial gene and fibronectin regulation. Together, these results reveal a critical role for endocardium in HLHS etiology and provide a rationale for considering endocardial function in regenerative strategies.

Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes.

Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3ß (GSK-3ß) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3ß inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3ß inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

Risk factors associated with the development of double-inlet ventricle congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) is the most common birth defect group and a significant contributor to neonatal and infant death. CHD with single ventricle anatomy, including hypoplastic left heart syndrome (HLHS), tricuspid atresia (TA), and various double-inlet ventricle (DIV) malformations, is the most complex with the highest mortality. Prenatal risk factors associated with HLHS have been studied, but such data for DIV are lacking. METHODS: We analyzed DIV cases and nonmalformed controls in the National Birth Defects Prevention Study, a case-control, multicenter population-based study of birth defects. Random forest analysis identified potential predictor variables for DIV, which were included in multivariable models to estimate effect magnitude and directionality. RESULTS: Random forest analysis identified pre-pregnancy diabetes, history of maternal insulin use, maternal total lipid intake, paternal race, and intake of several foods and nutrients as potential predictors of DIV. Logistic regression confirmed pre-pregnancy diabetes, maternal insulin use, and paternal race as risk factors for having a child with DIV. Additionally, higher maternal total fat intake was associated with a reduced risk. CONCLUSIONS: Maternal pre-pregnancy diabetes and history of insulin use were associated with an increased risk of having an infant with DIV, while maternal lipid intake had an inverse association. These novel data provide multiple metabolic pathways for investigation to identify better the developmental etiologies of DIV and suggest that public health interventions targeting diabetes prevention and management in women of childbearing age could reduce CHD risk.

Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair.

Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts.We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBMMNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart.

Radiation-induced edema is dependent on cyclooxygenase 2 activity in mouse brain.

Cerebrovascular dysfunction, characterized by compromise of the blood-brain barrier and formation of cerebral edema, is common during the acute period after brain irradiation and may contribute to delayed pathology (e.g. vascular collapse, white matter necrosis) that leads to functional deficits. Another response of normal brain tissue to radiation is the induction of inflammatory markers, such as cytokine expression and glial activation. In particular, radiation-induced neuroinflammation is associated with an elevation in cyclooxygenase 2 (COX2), one of two isoforms of the obligate enzyme in prostanoid synthesis and the principal target of non-steroid anti-inflammatory drugs. Since prostanoids serve as autocrine and paracrine mediators in numerous physiological and pathological processes, including vasoregulation, we investigated COX2 protein expression and COX2-mediated prostanoid production in radiation-induced cerebral edema in male C57/BL6 mice. We found that radiation induces COX2 protein that is accompanied by specific increases in prostaglandin E(2) and thromboxane A(2) within 4 and 24 h after brain irradiation. Furthermore, we showed that treatment with NS-398, a selective COX2 inhibitor, attenuated prostanoid induction and edema formation. These results suggest that radiation-induced changes in vascular permeability are dependent on COX2 activity, implicating this enzyme and its products as targets for potential therapeutic treatment/protection from the effects of radiation on normal brain tissue.

Engineered biomaterials control differentiation and proliferation of human-embryonic-stem-cell-derived cardiomyocytes via timed Notch activation.

For cell-based treatments of myocardial infarction, a better understanding of key developmental signaling pathways and more robust techniques for producing cardiomyocytes are required. Manipulation of Notch signaling has promise as it plays an important role during cardiovascular development, but previous studies presented conflicting results that Notch activation both positively and negatively regulates cardiogenesis. We developed surface- and microparticle-based Notch-signaling biomaterials that function in a time-specific activation-tunable manner, enabling precise investigation of Notch activation at specific developmental stages. Using our technologies, a biphasic effect of Notch activation on cardiac differentiation was found: early activation in undifferentiated human embryonic stem cells (hESCs) promotes ectodermal differentiation, activation in specified cardiovascular progenitor cells increases cardiac differentiation. Signaling also induces cardiomyocyte proliferation, and repeated doses of Notch-signaling microparticles further enhance cardiomyocyte population size. These results highlight the diverse effects of Notch activation during cardiac development and provide approaches for generating large quantities of cardiomyocytes.

Endogenous Wnt/beta-catenin signaling is required for cardiac differentiation in human embryonic stem cells.

BACKGROUND: Wnt/beta-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4. METHODOLOGY/PRINCIPAL FINDINGS: We tested the hypothesis that Wnt/beta-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/beta-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for beta-myosin heavy chain (beta-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/beta-catenin signaling is required for Smad1 activation by BMP4. CONCLUSIONS/SIGNIFICANCE: Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/beta-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.