Accelerated cardiomyocyte senescence contributes to late-onset doxorubicin-induced cardiotoxicity.

Children surviving cancer and chemotherapy are at risk for adverse health events including heart failure that may be delayed by years. Although the early effects of doxorubicin-induced cardiotoxicity may be attributed to a direct effect on the cardiomyocytes, the mechanisms underlying the delayed or late effects (8-20 yr) are unknown. The goal of this project was to develop a model of late-onset doxorubicin-induced cardiotoxicity to better delineate the underlying pathophysiology responsible. The underlying hypothesis was that doxorubicin-induced "late-onset cardiotoxicity" was the result of mitochondrial dysfunction leading to cell failure and death. Wistar rats, 3-4 wk of age, were randomly assigned to vehicle or doxorubicin injection groups (1-45 mg/kg). Cardiovascular function was unaltered at the lower dosages (1-15 kg/mg), but beginning at 6 mo after injection significant cardiac degradation was observed in the 45 mg/kg group. Doxorubicin significantly increased myocardial mitochondrial DNA (mtDNA) damage. In contrast, in isolated c-kit left ventricular (LV) cells, doxorubicin treatment did not increase mtDNA damage. Biomarkers of senescence within the LV were significantly increased, suggesting accelerated aging of the LV. Doxorubicin also significantly increased LV histamine content suggestive of mast cell activation. With the use of flow cytometry, a significant expansion of the c-kit and stage-specific embryonic antigen 1 cell populations within the LV were concomitant with significant decreases in the circulating peripheral blood population of these cells. These results are consistent with the concept that doxorubicin induced significant damage to the cardiomyocyte population and that although the heart attempted to compensate it eventually succumbed to an inability for self-repair.

Nuclear Gene 33/Mig6 regulates the DNA damage response through an ATM serine/threonine kinase-dependent mechanism.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently linked Gene 33 to the DNA damage response (DDR) induced by hexavalent chromium (Cr(VI)), but the molecular mechanism remains unknown. Here we show that ectopic expression of Gene 33 triggers DDR in an ATM serine/threonine kinase (ATM)-dependent fashion and through pathways dependent or not dependent on ABL proto-oncogene 1 non-receptor tyrosine kinase (c-Abl). We observed the clear presence of Gene 33 in the nucleus and chromatin fractions of the cell. We also found that the nuclear localization of Gene 33 is regulated by its 14-3-3-binding domain and that the chromatin localization of Gene 33 is partially dependent on its ErbB-binding domain. Our data further indicated that Gene 33 may regulate the targeting of c-Abl to chromatin. Moreover, we observed a clear association of Gene 33 with histone H2AX and that ectopic expression of Gene 33 promotes the interaction between ATM and histone H2AX without triggering DNA damage. In summary, our results reveal nuclear functions of Gene 33 that regulate DDR. The nuclear localization of Gene 33 also provides a spatial explanation of the previously reported regulation of apoptosis by Gene 33 via the c-Abl/p73 pathway. On the basis of these findings and our previous studies, we propose that Gene 33 is a proximal regulator of DDR that promotes DNA repair.

Notch signaling modulates the electrical behavior of cardiomyocytes.

Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K+ Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na+ currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. NEW & NOTEWORTHY We investigated the effects of Notch receptor signaling on the electrical properties of cardiomyocytes. Our results indicate that the Notch transduction pathway interferes with outward K+ Kv currents, critical determinants of the electrical repolarization of myocytes.

Inhibition of heart formation by lithium is an indirect result of the disruption of tissue organization within the embryo.

Lithium is a commonly used drug for the treatment of bipolar disorder. At high doses, lithium becomes teratogenic, which is a property that has allowed this agent to serve as a useful tool for dissecting molecular pathways that regulate embryogenesis. This study was designed to examine the impact of lithium on heart formation in the developing frog for insights into the molecular regulation of cardiac specification. Embryos were exposed to lithium at the beginning of gastrulation, which produced severe malformations of the anterior end of the embryo. Although previous reports characterized this deformity as a posteriorized phenotype, histological analysis revealed that the defects were more comprehensive, with disfigurement and disorganization of all interior tissues along the anterior-posterior axis. Emerging tissues were poorly segregated and cavity formation was decreased within the embryo. Lithium exposure also completely ablated formation of the heart and prevented myocardial cell differentiation. Despite the complete absence of cardiac tissue in lithium treated embryos, exposure to lithium did not prevent myocardial differentiation of precardiac dorsal marginal zone explants. Moreover, precardiac tissue freed from the embryo subsequent to lithium treatment at gastrulation gave rise to cardiac tissue, as demonstrated by upregulation of cardiac gene expression, display of sarcomeric proteins, and formation of a contractile phenotype. Together these data indicate that lithium's effect on the developing heart was not due to direct regulation of cardiac differentiation, but an indirect consequence of disrupted tissue organization within the embryo.

GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling.

This study examined transgenic mice whose expression of a ß-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that <1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial ß-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states.

Large-Scale Generation and Characterization of Homogeneous Populations of Migratory Cortical Interneurons from Human Pluripotent Stem Cells.

During development, cortical interneurons (cINs) are generated from the ventral telencephalon, robustly migrate to the dorsal telencephalon, make local synaptic connections, and critically regulate brain circuitry by inhibiting other neurons. Thus, their abnormality is associated with various brain disorders. Human pluripotent stem cell (hPSC)-derived cINs can provide unlimited sources with which to study the pathogenesis mechanism of these disorders as well as provide a platform to develop novel therapeutics. By employing spinner culture, we could obtain a >10-fold higher yield of cIN progenitors compared to conventional culture without affecting their phenotype. Generated cIN spheres can be maintained feeder-free up to 10 months and are optimized for passaging and cryopreservation. In addition, we identified a combination of chemicals that synchronously matures generated progenitors into SOX6+KI67- migratory cINs and extensively characterized their maturation in terms of metabolism, migration, arborization, and electrophysiology. When transplanted into mouse brains, chemically matured migratory cINs generated grafts that efficiently disperse and integrate into the host circuitry without uncontrolled growth, making them an optimal cell population for cell therapy. Efficient large-scale generation of homogeneous migratory cINs without the need of feeder cells will play a critical role in the full realization of hPSC-derived cINs for development of novel therapeutics.

Dysregulated protocadherin-pathway activity as an intrinsic defect in induced pluripotent stem cell-derived cortical interneurons from subjects with schizophrenia.

We generated cortical interneurons (cINs) from induced pluripotent stem cells derived from 14 healthy controls and 14 subjects with schizophrenia. Both healthy control cINs and schizophrenia cINs were authentic, fired spontaneously, received functional excitatory inputs from host neurons, and induced GABA-mediated inhibition in host neurons in vivo. However, schizophrenia cINs had dysregulated expression of protocadherin genes, which lie within documented schizophrenia loci. Mice lacking protocadherin-α showed defective arborization and synaptic density of prefrontal cortex cINs and behavioral abnormalities. Schizophrenia cINs similarly showed defects in synaptic density and arborization that were reversed by inhibitors of protein kinase C, a downstream kinase in the protocadherin pathway. These findings reveal an intrinsic abnormality in schizophrenia cINs in the absence of any circuit-driven pathology. They also demonstrate the utility of homogenous and functional populations of a relevant neuronal subtype for probing pathogenesis mechanisms during development.

Evaluating the role of Wnt signal transduction in promoting the development of the heart.

Wnts are a family of secreted signaling proteins that are encoded by 19 distinct genes in the vertebrate genome. These molecules initiate several signal transduction pathways: the canonical Wnt, Wnt/Ca2+, and Wnt/planar cell polarity pathways. Wnt proteins have major impact on embryonic development, tumor progression, and stem cell differentiation. Wnt signal transduction also influences the formation of the heart, yet many issues concerning the involvement of Wnt regulation in initiating cardiac development remain unresolved. In this review, we will examine the published record to discern (a) what has been shown by experimental studies on the participation of Wnt signaling in cardiogenesis, and (b) what are the important questions that need to be addressed to understand the importance and function of Wnt signal transduction in facilitating the development of the heart.

Inhibition of Histone Methyltransferase, Histone Deacetylase, and ß-Catenin Synergistically Enhance the Cardiac Potential of Bone Marrow Cells.

Previously, we reported that treatment with the G9a histone methyltransferase inhibitor BIX01294 causes bone marrow mesenchymal stem cells (MSCs) to exhibit a cardiocompetent phenotype, as indicated by the induction of the precardiac markers Mesp1 and brachyury. Here, we report that combining the histone deacetylase inhibitor trichostatin A (TSA) with BIX01294 synergistically enhances MSC cardiogenesis. Although TSA by itself had no effect on cardiac gene expression, coaddition of TSA to MSC cultures enhanced BIX01294-induced levels of Mesp1 and brachyury expression 5.6- and 7.2-fold. Moreover, MSCs exposed to the cardiogenic stimulus Wnt11 generated 2.6- to 5.6-fold higher levels of the cardiomyocyte markers GATA4, Nkx2.5, and myocardin when pretreated with TSA in addition to BIX01294. MSC cultures also showed a corresponding increase in the prevalence of sarcomeric protein-positive cells when treated with these small molecule inhibitors. These results correlated with data showing synergism between (1) TSA and BIX01294 in promoting acetylation of lysine 27 on histone H3 and (2) BIX01294 and Wnt11 in decreasing ß-catenin accumulation in MSCs. The implications of these findings are discussed in light of observations in the early embryo on the importance of ß-catenin signaling and histone modifications for cardiomyocyte differentiation and heart development.

Blood-borne stem cells differentiate into vascular and cardiac lineages during normal development.

Recent investigations have indicated that hematopoietic stem cells (HSCs) have the potential to differentiate into multiple non-blood cell lineages and contribute to the cellular regeneration of various tissues and multiple organs. Most studies to date on HSC potential have examined the adult, focusing on their potential to repair tissue under pathological conditions (e.g., ischemic injury, organ failure). Comparatively little is known about the physiological role of HSCs in normal tissue homeostasis in the adult, and even less of their contribution to organogenesis during prenatal development. This study reports the contribution of blood-borne cells to various organ systems of the developing embryo using a quail-chick parabiosis model. Under these conditions, the developing circulatory systems fuse between ED6-ED8, resulting in free exchange of circulating cells. Cells of quail origin, identified by quail-specific antibodies at ED15, were found in numerous organs of the parabiotic chick embryo. Circulating cells contributed to developing vasculature, where they differentiated into endothelial, smooth muscle, and adventitial tissues. In the heart, differentiation of circulating cells into cardiomyocytes was demonstrated using double immunolabeling for QCPN and sarcomeric actin or myosin. These results were confirmed by intramyocardial injection of quail bone marrow cells that were found to express markers of myocytes, coronary smooth muscle, and epicardium. Experiments using lacZ-transgenic chick embryos for a second positive cellular marker showed that fusion between chick and quail cells was a rare event. These results suggest that during development, multipotent cells are present in the embryonic circulation and home into different organs where they undergo tissue-specific differentiation. Moreover, the demonstration that blood-borne cells contribute to the development of various organs lends credence to claims that hematopoietic stem cells have utility for treating diseased or damaged tissues in the adult.

Embryonic myocardium shows increased longevity as a functional tissue when cultured in the presence of a noncardiac tissue layer.

A major aim of regenerative medicine is the construction of bioengineered organs and tissue for transplantation into human patients; yet living tissue is dynamic, and thus arranging cellular and extracellular constituents into an architecture resembling normal adult organs may not be sufficient to maintain tissue stability. In this study, we used cultures of embryonic chick heart tissue as a model to explore how newly formed cardiac tissue constructs can sustain their morphological structure and functional capabilities over extended periods. During the initial days of incubation, embryonic cardiac explants will thrive as beating three-dimensional tissue aggregates. However, within the first week of culture, cardiac aggregates lose their contractile function and flatten. After 2 weeks of incubation, the cardiac cells will have spread out into a homogeneous monolayer and dedifferentiated to a noncardiac phenotype. In contrast, when the embryonic heart tissue was co-cultured with a noncardiac cell layer obtained from adult bone marrow, the cardiac aggregates maintained their contractile function, three-dimensional tissue morphology, and myocyte phenotype for a full month of incubation. The capacity of this noncardiac cell layer to sustain the phenotype and morphology of the cardiac explants was partially replicated by treatment of the heart tissue with conditioned media from bone marrow cells. These findings are discussed in regard to the importance of adjacent cell layers for facilitating organogenesis in the developing embryo and having potential utility in producing stable bioengineered tissue constructs.

Stem cells: shibboleths of development, part II: Toward a functional definition.

Our previous discourse on stem cell characteristics led to the conclusion that the qualities deemed essential for a cell to be considered a "stem cell" are neither firmly established nor universally accepted, and this we accept as editorial policy. In that study, self-renewal, asymmetric division, phenotypic markers, and other attributes touted as being indicative of cells being stem cells were critically questioned as fundamental to the definition of a stem cell, leading us to seek a functional definition instead. Here, we offer further considerations, and elaborate on the characteristics that diverse investigators feel are essential for a cell to function as a stem cell, either in development or body maintenance. We hope that this discourse will promote further reflection, culminating with a definition that is widely accepted and universally applicable. We confess this goal has not been reached, neither here nor elsewhere. The outstanding goal of understanding what stem cells are, a prerequisite of characterizing what stem cells do and how they do it, is still outstanding.

Multiple stem cell populations contribute to the formation of the myocardium.

Owing to the very rapid growth of the vertebrate embryo following fertilization, an efficient circulatory system needs to be established during the initial stages of development. For that reason, the first functional organ that develops in both the bird and mammalian embryo is the heart. Until recently, the narrative of cardiac development was portrayed in a straightforward manner, with all the myocardium in the mature heart being generated from the expansion of an original pool of myocardial cells present in the early gastrula. It is now known that the story of the developing myocardium is more dynamic, as it is comprises cellular components of multiple ancestries. The de novo addition of myocytes to the developing heart occurs at various points during embryogenesis, as cardiac muscle takes on new members by the absorption of cells that either reside in neighboring nonmuscle tissue or come into contact with the myocardium by entering the heart upon migration or via the circulation. This article reviews what is presently known about cellular populations that contribute to the myocardium and examine reasons why the embryo utilizes multiple cellular sources for forming the cardiac muscle.

Inhibition of G9a Histone Methyltransferase Converts Bone Marrow Mesenchymal Stem Cells to Cardiac Competent Progenitors.

The G9a histone methyltransferase inhibitor BIX01294 was examined for its ability to expand the cardiac capacity of bone marrow cells. Inhibition of G9a histone methyltransferase by gene specific knockdown or BIX01294 treatment was sufficient to induce expression of precardiac markers Mesp1 and brachyury in bone marrow cells. BIX01294 treatment also allowed bone marrow mesenchymal stem cells (MSCs) to express the cardiac transcription factors Nkx2.5, GATA4, and myocardin when subsequently exposed to the cardiogenic stimulating factor Wnt11. Incubation of BIX01294-treated MSCs with cardiac conditioned media provoked formation of phase bright cells that exhibited a morphology and molecular profile resembling similar cells that normally form from cultured atrial tissue. Subsequent aggregation and differentiation of BIX01294-induced, MSC-derived phase bright cells provoked their cardiomyogenesis. This latter outcome was indicated by their widespread expression of the primary sarcomeric proteins muscle α-actinin and titin. MSC-derived cultures that were not initially treated with BIX01294 exhibited neither a commensurate burst of phase bright cells nor stimulation of sarcomeric protein expression. Collectively, these data indicate that BIX01294 has utility as a pharmacological agent that could enhance the ability of an abundant and accessible stem cell population to regenerate new myocytes for cardiac repair.

Orexin Receptor Activation Generates Gamma Band Input to Cholinergic and Serotonergic Arousal System Neurons and Drives an Intrinsic Ca(2+)-Dependent Resonance in LDT and PPT Cholinergic Neurons.

A hallmark of the waking state is a shift in EEG power to higher frequencies with epochs of synchronized intracortical gamma activity (30-60 Hz) - a process associated with high-level cognitive functions. The ascending arousal system, including cholinergic laterodorsal (LDT) and pedunculopontine (PPT) tegmental neurons and serotonergic dorsal raphe (DR) neurons, promotes this state. Recently, this system has been proposed as a gamma wave generator, in part, because some neurons produce high-threshold, Ca(2+)-dependent oscillations at gamma frequencies. However, it is not known whether arousal-related inputs to these neurons generate such oscillations, or whether such oscillations are ever transmitted to neuronal targets. Since key arousal input arises from hypothalamic orexin (hypocretin) neurons, we investigated whether the unusually noisy, depolarizing orexin current could provide significant gamma input to cholinergic and serotonergic neurons, and whether such input could drive Ca(2+)-dependent oscillations. Whole-cell recordings in brain slices were obtained from mice expressing Cre-induced fluorescence in cholinergic LDT and PPT, and serotonergic DR neurons. After first quantifying reporter expression accuracy in cholinergic and serotonergic neurons, we found that the orexin current produced significant high frequency, including gamma, input to both cholinergic and serotonergic neurons. Then, by using a dynamic clamp, we found that adding a noisy orexin conductance to cholinergic neurons induced a Ca(2+)-dependent resonance that peaked in the theta and alpha frequency range (4-14 Hz) and extended up to 100 Hz. We propose that this orexin current noise and the Ca(2+) dependent resonance work synergistically to boost the encoding of high-frequency synaptic inputs into action potentials and to help ensure cholinergic neurons fire during EEG activation. This activity could reinforce thalamocortical states supporting arousal, REM sleep, and intracortical gamma.

An in vitro analysis of myocardial potential indicates that phenotypic plasticity is an innate property of early embryonic tissue.

Explants from gastrula-stage avian embryos have provided an important culture model for examining the formation of the vertebrate heart. Explants harvested from anterior regions containing the precardiac mesoderm faithfully recapitulate cardiogenesis and generate contractile tissue in culture. Posterior regions of the early embryo do not supply cellular material to the developing heart in situ, and thus have been commonly employed as negative control tissues for studying cardiogenic induction. To begin to understand the cellular mechanisms that account for the distinct cell fates of precardiac and posterior tissue within the embryo, we undertook a comprehensive investigation on the myocardial potential of presumptive noncardiac tissue. Myocardial differentiation was assayed by expression of the myocardium-associated transcription factor gene Nkx2.5 and positive immunostaining for sarcomeric myosin, muscle alpha-actinin, and smooth muscle alpha-actin. Our results demonstrate that regions of the early embryo that do not provide a cellular contribution to the myocardium in situ are capable of generating myocardial tissue when removed from their normal embryonic environment and placed in culture under nontreated conditions. Although treatment with the presumptive cardiac inducer Dickkopf-1 increased the frequency that cardiac tissue appeared within cultures of posterior tissue, no difference was observed in either the size or morphology of the myocardium-positive areas among treated and nontreated explants. These findings suggest that progenitor cells within the early embryo possess an innate phenotypic plasticity and that presumptive cardiac inducing signals do not induce cardiac differentiation but instead augment a pre-existing cardiac potential of embryonic tissue.

Adult stem cells and their cardiac potential.

Adult cardiac muscle is unable to repair itself following severe disease or injury. Because of this fundamental property of the myocardium, it was long believed that the adult myocardium is a postmitotic tissue. Yet, recent studies have indicated that new cardiac myocytes are generated throughout the life span of an adult and that extracardiac cells can contribute to the renewal of individual cells within the myocardium. In addition, investigations of the phenotypic capacity of adult stem cells have suggested that their potential is not solely restricted to the differentiated cell phenotypes of the source tissue. These observations have great implications for cardiac biology, as stem cells obtained from the bone marrow and other readily accessible adult tissues may serve as a source of replacement cardiac myocytes. In this review, we describe the evidence for these new findings and discuss their implications in context of the continuing controversy over stem cell plasticity.

Hematopoietic cells from bone marrow have the potential to differentiate into cardiomyocytes in vitro.

Recent studies have indicated that hematopoietic progenitor cells (HPCs) have the capacity to form cardiomyocytes. In the present study, we further examined the cardiac competence of HPCs by asking whether these cells by themselves can be provoked to undergo cardiac differentiation. Our data indicate that in response to growth factor treatment, HPCs from avian bone marrow (BM) can undergo cardiac differentiation, as indicated by their expression of multiple cardiac transcription factors and sarcomeric proteins. Furthermore, coculture experiments with adult mouse BM cells and embryonic heart tissue confirmed that HPCs are able to both integrate into cardiac tissue and differentiate into cardiomyocytes. In an additional set of experiments, we investigated whether other hematopoietic populations might possess cardiac potential by examining whether blood cells that normally are recruited to damaged tissue might act as a source of newly generated cardiomyocytes. Remarkably, macrophages cocultured with cardiac explants also demonstrated an ability to integrate into contractile heart tissue and undergo cardiac differentiation. Thus, our data suggest that the capacity of blood cells to transdifferentiate into cardiomyocytes is not limited to classically defined hematopoietic progenitors.

Stem cells and the formation of the myocardium in the vertebrate embryo.

A major goal in cardiovascular biology is to repair diseased or damaged hearts with newly generated myocardial tissue. Stem cells offer a potential source of replacement myocytes for restoring cardiac function. Yet little is known about the nature of the cells that are able to generate myocardium and the conditions they require to form heart tissue. A source of information that may be pertinent to addressing these issues is the study of how the myocardium arises from progenitor cells in the early vertebrate embryo. Accordingly, this review will examine the initial events of cardiac developmental biology for insights into the identity and characteristics of the stem cells that can be used to generate myocardial tissue for therapeutic purposes.