Adult Heart Cells Research Articles

Modeling acute myocardial infarction and cardiac fibrosis using human induced pluripotent stem cell-derived multi-cellular heart organoids.

Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.

Recent Advances in Maturation of Induced pluripotent Stem Cells-derived Cardiomyocytes

Induced pluripotent stem cells -derived cardiomyocytes (iPSC-CMs) increasingly become an important tool for studying genetic heart diseases, drug screening and heart cell therapy. However, iPSC-CMs differentiated by the traditional ways are immature and the challenge of producing fully matured iPSC-CMs that functionally resemble adult cardiomyocytes persists. Immature iPSC-CMs exhibit characteristics typical of fetal-stage cells, including less developed contractility, electrophysiological properties, and metabolic processes. These limitations restrict their clinical applications, especially in regenerative medicine. Over the past decade, numerous strategies have been developed to enhance the maturation of iPSC-CMs. These include prolonged culture periods, biochemical induction using specific hormones and energy substrates, and the application of biophysical stimuli such as electrical and mechanical stimulation. In addition, advances in biomaterials, such as extracellular matrices and hydrogels, have been crucial in replicating the physiological environment needed to support iPSC-CM development. Together, these methods aim to produce cardiomyocytes that more closely mimic adult heart cells in structure and function. This review explores recent progress in optimizing the maturation of iPSC-CMs and highlights their potential impact on future applications in cell therapy and heart disease modeling.

Abstract 9865: Temporal Dynamics of T Cell Response After Cardiac Injury

Introduction: Neonatal heart can regenerate after myocardial injury unlike adult heart. Its capacity diminishes and disappears by post neonatal day 7 (P7). Recent studies have shown that immune cells especially T cells plays an important role to regulate inflammation, however, their role in cardiac regeneration/repair after injury is understudied. Therefore, in the present study we will characterize the temporal dynamics of T cells in neonatal and adult heart after injury and will study how temporal regulation is essential to maintain repair processes. Hypothesis: Regulatory T cells plays an important role in mediating reparative processes after ischemic cardiac injury. Methods and Results: Myocardial infarction (MI) was induced in neonatal mice at P2 (n=30) and in 8-week-old (n=10) C57BL/6 mice by ligating the left anterior descending artery (LAD). CD4+, CD8+ and Foxp3+ T cells were sorted by FACS at post MI day 2, 3, 4, 5 and 7 in neonatal mice and post MI day 7 in adult mice and analyzed for the differences in transcriptome by ultra-low input RNA sequencing. Flow cytometry analysis showed CD4+ T cells peaked at post MI day 4 compared to CD8+ T cells that peaked at post MI day 7. FoxP3+ T cells increased at post MI day 2 and diminished by post MI day 7 which coincides with the non-regenerative window of the heart. CD4+ and Foxp3+ T subsets showed increased expression of transcript in anatomical structure morphogenesis and regulation of response to stimulus. Our data showed 11.2%, 12.8% and 13.4% genes in CD4+, CD8+ and Foxp3+ T cells subsets were upregulated in neonatal mice versus adult animals. Enrichment analysis resulted that cellular component morphogenesis and cell development were activated in CD4+ T cells, and cellular component biogenesis and organelle organization were elevated in Foxp3+ T cells in neonatal mice. In addition, chromosome organization and cell cycle were upregulated in CD8+ T cells. Additionally, Foxp3+ T cells showed 198 genes uniquely expressed in neonatal hearts which are involved in cardiomyocytes proliferation and myofibroblasts phenotype transition. Conclusions: CD4+/CD8+/Foxp3+ regulate inflammation and mediate reparative processes in cardiac cells including myocytes and fibroblast after myocardial ischemic injury.

Resident stem cells in the heart.

Cardiovascular disease is the leading cause of mobility and morality worldwide, in which the ischemic heart disease is the most common type of the diseases. During last decade, a major progress in the study of the pathogenesis of heart disease has been achieved. For example, the discovery of adult stem/progenitor cells in the heart and vessel tissues may play a role in tissue regeneration. However, the issue of 31 retractions for cardiac stem cell work has caused a "storm of trust" in the heart stem cell field, in which both founders and scientists have become cautious and conservative in stem cell research of the heart. Despite that the existence of adult cardiac stem cells has been denied, recent studies confirmed that there are many other resident stem/progenitor cells in adult heart. Although these cells cannot differentiate into cardiomyocytes, the role they played in heart repair after injury should not be ignored. The purpose of this short article is to briefly review the current research progress in resident stem/progenitor cells in the heart, to discuss how they function during cardiac repair and to point out unanswered questions in the research field.

Endothelial Klf2-Foxp1-TGFβ signal mediates the inhibitory effects of simvastatin on maladaptive cardiac remodeling.

Aims: Pathological cardiac fibrosis and hypertrophy are common features of left ventricular remodeling that often progress to heart failure (HF). Endothelial cells (ECs) are the most abundant non-myocyte cells in adult mouse heart. Simvastatin, a strong inducer of Krüppel-like Factor 2 (Klf2) in ECs, ameliorates pressure overload induced maladaptive cardiac remodeling and dysfunction. This study aims to explore the detailed molecular mechanisms of the anti-remodeling effects of simvastatin.Methods and Results: RGD-magnetic-nanoparticles were used to endothelial specific delivery of siRNA and we found absence of simvastatin's protective effect on pressure overload induced maladaptive cardiac remodeling and dysfunction after in vivo inhibition of EC-Klf2. Mechanism studies showed that EC-Klf2 inhibition reversed the simvastatin-mediated reduction of fibroblast proliferation and myofibroblast formation, as well as cardiomyocyte size and cardiac hypertrophic genes, which suggested that EC-Klf2 might mediate the anti-fibrotic and anti-hypertrophy effects of simvastatin. Similar effects were observed after Klf2 inhibition in cultured ECs. Moreover, Klf2 regulated its direct target gene TGFβ1 in ECs and mediated the protective effects of simvastatin, and inhibition of EC-Klf2 increased the expression of EC-TGFβ1 leading to simvastatin losing its protective effects. Also, EC-Klf2 was found to regulate EC-Foxp1 and loss of EC-Foxp1 attenuated the protective effects of simvastatin similar to EC-Klf2 inhibition.Conclusions: We conclude that cardiac microvasculature ECs are important in the modulation of pressure overload induced maladaptive cardiac remodeling and dysfunction, and the endothelial Klf2-TGFβ1 or Klf2-Foxp1-TGFβ1 pathway mediates the preventive effects of simvastatin. This study demonstrates a novel mechanism of the non-cholesterol lowering effects of simvastatin for HF prevention.

Hydrogen peroxide diffusion and scavenging shapes mitochondrial network instability and failure by sensitizing ROS-induced ROS release

The mitochondrial network of cardiac cells is finely tuned for ATP delivery to sites of energy demand; however, emergent phenomena, such as mitochondrial transmembrane potential oscillations or propagating waves of depolarization have been observed under metabolic stress. While regenerative signaling by reactive oxygen species (ROS)-induced ROS release (RIRR) has been suggested as a potential trigger, it is unknown how it could lead to widespread responses. Here, we present a novel computational model of RIRR transmission that explains the mechanisms of this phenomenon. The results reveal that superoxide mediates neighbor-neighbor activation of energy-dissipating ion channels, while hydrogen peroxide distributes oxidative stress to sensitize the network to mitochondrial criticality. The findings demonstrate the feasibility of RIRR as a synchronizing factor across the dimensions of the adult heart cell and illustrate how a cascade of failures at the organellar level can scale to impact cell and organ level functions of the heart.

Fate Tracing of Isl1+Cells in Adult Mouse Hearts under Physiological and Exercise Conditions.

Myocardial damage due to dysfunctional myocardium has been increasing, and the prognosis of pharmacological and device-based therapies remain poor. Isl1-expressing cells were thought to be progenitor cells for cardiomyocyte proliferation after specific stimuli. However, the true origin of the proliferating myocardiac cells and the role of Isl1 in adult mammals remain unresolved. In this study, Isl1-CreERT2 knock-in mouse model was constructed using CRISPR/Cas9 technology. Using tamoxifen-inducible Isl1-CreERT/Rosa26R-LacZ system, Isl1+cells and their progeny were permanently marked by lacZ-expression. X-gal staining, immunostaining, and quantitative PCR were then used to reveal the fate of Isl1+cells under physiological and exercise conditions in mouse hearts from embryonic stage to adulthood. Isl1+cells were found to localize to the sinoatrial node, atrioventricular node, cardiac ganglia, aortic arch, and pulmonary roots in adult mice heart. However, they did not act as cardiac progenitor cells under physiological and exercise conditions. Although Isl1+cells showed progenitor cell properties in early mouse embryos (E7.5), this ability was lost by E9.5. Furthermore, although the proliferation and regeneration of heart cell was observed in response to exercise, the cells associated were not Isl1 positive.

Fibroblast Primary Cilia Are Required for Cardiac Fibrosis.

The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor β-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor β-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

Expression Pattern of Angiogenic Factors in Healthy Heart in Response to Physical Exercise Intensity.

Recently, many studies showing the regeneration potential of both cardiac and hematopoietic stem cells in adult heart following injury were definitively retracted by the literature. Therefore, stimulating myocardial angiogenesis becomes to be important for preventing cardiovascular diseases. Regular endurance exercise has been reported to induce capillary growth in healthy and diseased myocardium resulting in cardioprotective phenotype. Previously, we demonstrated a significantly increased capillary proliferation in mouse hearts following 30 and 45 days of endurance training. In the present study, we examined the localization and expression pattern of vascular endothelial growth factor receptors (VEGFR-1/Flt-1 and VEGFR-2/Flk-1), hypoxia-inducible factor-1α (HIF-1α), and inducible nitric oxide synthase (iNOS) in heart neocapillarization in response to a mild, moderate, and high intensity of endurance training. Sixty-three Swiss male mice were divided into four untrained control groups and three groups trained for 15 (T15), 30 (T30), and 45 (T45) days with a gradually increasing intensity on a treadmill. We observed the localization of studied proteins with immunostaining and their expression level with Western blot analyses. We found that VEGFR-2/Flk-1 expression progressively increased in trained groups compared with controls, while VEGFR-1/Flt-1 and HIF-1α were higher in T15 than in controls, T30, and T45 animals. Differently, iNOS levels enhanced after 15 and 30 days of exercise. The localization of these factors was not altered by exercise. The results showed that the expression of VEGFR-1/Flt-1, VEGFR-2/Flk-1, HIF-1α, and iNOS is differently regulated in cardiac angiogenesis according to the exercise intensity. VEGFR-1/Flt-1 and HIF-1α are upregulated by a mild intensity exercise, while VEGFR-2/Flk-1 progressively enhances with increasing workload. Differently, iNOS protein is modulated by a moderate intensity exercise. VEGF pathway appears to be involved in exercise-related angiogenesis in heart and VEGF might act in a paracrine and endocrine manner. Understanding this relationship is important for developing exercise strategies to protect the heart by insults.

Gene-Editing Technology Accelerates Cardiovascular Research: Clinical Applications Being Explored.

The emergence of an inexpensive and efficient tool for gene editing is accelerating the pace of cardiovascular research and beginning to show potential as a treatment tool. The new gene-editing technology leverages a bacterial defense mechanism against viral infections. Bacteria store sections of viral DNA in repeated sequences called CRISPR within their own genomes and use those sequences to identify and destroy viruses. Now, scientists are inserting genes they would like to target into CRISPR and pairing it with CAS9, an enzyme that cuts DNA. This process allows scientists to efficiently remove or replace a targeted gene. “It’s been a transformative technology,” said Kiran Musunuru, MD, PhD, MPH, an associate professor of cardiovascular medicine and genetics at the University of Pennsylvania’s Perelman School of Medicine. Gene editing has been a common tool in research for decades—particularly for producing model organisms with a particular genetic mutation. However, older techniques were more time-consuming, expensive, and more error-prone. CRISPR-CAS is allowing scientists to more easily and quickly produce model animals or human cell lines with specific genetic variants allowing experiments to proceed at an unprecedented pace or scale. Some preliminary studies have suggested that it also may be a useful tool for treating some forms of heart disease. The most immediate impact of CRISPR-CAS9 has been speeding the production …

Prokineticin receptor-1-dependent paracrine and autocrine pathways control cardiac tcf21+ fibroblast progenitor cell transformation into adipocytes and vascular cells

Cardiac fat tissue volume and vascular dysfunction are strongly associated, accounting for overall body mass. Despite its pathophysiological significance, the origin and autocrine/paracrine pathways that regulate cardiac fat tissue and vascular network formation are unclear. We hypothesize that adipocytes and vasculogenic cells in adult mice hearts may share a common cardiac cells that could transform into adipocytes or vascular lineages, depending on the paracrine and autocrine stimuli. In this study utilizing transgenic mice overexpressing prokineticin receptor (PKR1) in cardiomyocytes, and tcf21ERT-creTM-derived cardiac fibroblast progenitor (CFP)-specific PKR1 knockout mice (PKR1tcf−/−), as well as FACS-isolated CFPs, we showed that adipogenesis and vasculogenesis share a common CFPs originating from the tcf21+ epithelial lineage. We found that prokineticin-2 is a cardiomyocyte secretome that controls CFP transformation into adipocytes and vasculogenic cells in vivo and in vitro. Upon HFD exposure, PKR1tcf−/− mice displayed excessive fat deposition in the atrioventricular groove, perivascular area, and pericardium, which was accompanied by an impaired vascular network and cardiac dysfunction. This study contributes to the cardio-obesity field by demonstrating that PKR1 via autocrine/paracrine pathways controls CFP–vasculogenic- and CFP-adipocyte-transformation in adult heart. Our study may open up new possibilities for the treatment of metabolic cardiac diseases and atherosclerosis.

In This Issue

HomeCirculation ResearchVol. 121, No. 5In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published18 Aug 2017https://doi.org/10.1161/RES.0000000000000173Circulation Research. 2017;121:469Role of Hey2 in Transmural Electrical Patterning (p 537)Download figureDownload PowerPointVeerman et al determine the likely effect of a Brugada syndrome risk allele.Brugada syndrome is characterized by abnormal electrical signals in the heart and a heightened risk of sudden death. A genome-wide association study (GWAS) has identified a Brugada-linked single nucleotide polymorphism (SNP) at the genetic locus 6q22.31. This SNP lies close to the gene for transcription factor HEY2; however, whether HEY2 contributes to Brugada syndrome is unclear. Veerman and colleagues examined gene expression data from 190 human left ventricle samples and found that, of 8 genes in the 6q22.31 region, only HEY2 showed significant association—increased expression—with the Brugada risk allele. Further, genome-wide coexpression analyses showed that, of 15 617 genes examined, the expression of KCNIP2, which encodes an ion channel subunit, was most tightly correlated with the expression of HEY2. The team went on to show that myocardial expression of KCNIP2 was lower in HEY2-deficient mice than in wild-type animals, suggesting that HEY2 regulates KCNIP2. Indeed, these HEY2-deficient animals exhibited abnormal electrical patterning in the ventricle wall consistent with altered ion channel function. The results indicate that high HEY2 expression increases the risk of Brugada syndrome and highlight the utility of expression analyses for uncovering potential functions of GWAS identified loci.Notch and Sick Sinus Syndrome (p 549)Download figureDownload PowerPointTransient Notch activation causes long-term electrophysiological problems in the heart, say Qiao et al.Cell–cell signaling via transmembranous Notch proteins is essential for a variety of developmental processes, including the formation and function of several cardiovascular structures and systems. In adult mouse heart cells, Notch signaling is generally quiescent, but after an injury—such as a myocardial infarction—Notch is transiently reactivated, which in turn leads to altered ion channel expression and aberrant electrical properties. Qiao and colleagues have now examined the effects of such transient Notch reactivation in more detail. They found that in mice subjected to transient atrial Notch activation, there was significant heart rate slowing, sinus pausing, and dysregulation of sinus node and atrial conduction factors. Furthermore, these transcriptional and electrophysiological changes were sustained long after Notch signaling had returned to normal. Heart rate, for example, remained low for a year. Because cardiac injuries can predispose patients to atrial arrhythmias, the researchers suggest that a thorough understanding of the long-term cardiac effects of transient, injury-induced Notch activation may reveal pathways and factors that could be targeted for antiarrhythmic interventions.Inhibition of Meg3 Prevents Cardiac Remodeling (p 575)Download figureDownload PowerPointPiccoli and colleagues target a noncoding RNA to prevent cardiac remodeling.Fibroblasts contribute to myocardial remodeling during pressure overload-induced cardiac hypertrophy. These cells secrete factors that reorganize the extracellular matrix (ECM), creating fibrosis and heart dysfunction. To learn more about the remodeling actions of cardiac fibroblasts, Piccoli and colleagues investigated the array of long noncoding RNAs (lncRNAs) produced by these cells. LncRNAs regulate a variety of cellular processes and certain lncRNAs have been implicated in cardiovascular disease. However, the roles of cardiac fibroblast lncRNAs have not been thoroughly studied. In mice with pressure-induced hypertrophy, the team identified 3 lncRNAs that were specifically dysregulated in cardiac fibroblasts. Of these, one called Meg3 was the most abundant and has previously been implicated in fibrosis. Functional studies of Meg3 revealed that the RNA, by binding and activating transcription factor p53, promotes the expression of an ECM enzyme called MMP-2. The team also showed that antisense inhibition of Meg3 could prevent the pressure-induced increase in MMP-2 as well as the associated fibrosis and hypertrophy in mice. Inhibition of Meg3 may, therefore, be an effective way to prevent cardiac fibrosis and dysfunction during heart disease, say the authors. Previous Back to top Next FiguresReferencesRelatedDetails August 18, 2017Vol 121, Issue 5 Advertisement Article InformationMetrics © 2017 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000173 Originally publishedAugust 18, 2017 PDF download Advertisement

Characterization of Electrochemically Gated Graphene Field-Effect Transistor for Bioelectronic Applications

This work provides an experimental characterization and equivalent circuit modelling of electrochemically gated field effect transistors (EGFETs) using a graphene layer. The aim is to demonstrate the detection limit of these sensing devices for bioelectronics applications. Towards that, we present a detail study of the; (i) sources of intrinsic noise, (ii) the effect of ionic density in charge transfer resistance and (iii) the existence of parasitic conducting paths in the surrounding electrolyte. To investigate the liquid-based applications of graphene based EGFETs, quantifying the basic physical properties of graphene/solution interface is crucial. Towards that, we present a detail study of zero bias charge transfer resistance by using different type of ionic solutions and cell culture mediums. Measurements of the electrical noise were also performed to determine the EGFET detection limit. The performance of the EGFET to record the electrophysiological signals was evaluated using adult zebra fish heart and electrogenic cell cultures. The electrocardiogram of the zebrafish heart was monitored with signal-to-noise-ratio higher than 7. This contribution also discusses the application of graphene based EGFETS to measure electrophysiological signals in cell cultures. For instance, ultra-week extracellular signals resulting from cell metabolic activity or low frequency calcium waves. Keywords. Graphene, electrochemically gated field effect transistor, charge transport, intrinsic noise, extracellular signals. Figure 1

Cardiosphere-derived cells do not improve cardiac function in rats with cardiac failure

BackgroundHeart failure represents an important public health issue due to its high costs and growing incidence worldwide. Evidence showing the regenerative potential of postmitotic heart tissue has suggested the existence of endogenous cardiac stem cells in adult hearts. Cardiosphere-derived cells (CDC) constitute a candidate pool of such cardiac stem cells. Previous studies using acute myocardial infarction (MI) models in rodents demonstrated an improvement in cardiac function after cell therapy with CDC. We evaluated the therapeutic potential of CDC 60 days after MI in a rat model.MethodsCDC were obtained from human discarded myocardial tissue and rat hearts by enzymatic digestion with collagenase II. At 10–15 days after isolation, small, round, phase-bright cells (PBCs) appeared on top of the adherent fibroblast-like cells. The PBCs were collected and placed on a nonadherent plate for 2 days, where they formed cardiospheres which were then transferred to adherent plates, giving rise to CDC. These CDC were characterized by flow cytometry. Wistar rats were submitted to MI through permanent occlusion of the anterior descending coronary artery. After 60 days, they were immunosuppressed with cyclosporine A during 10 days. On the third day, infarcted animals were treated with 5 × 105 human CDC (hCDC) or placebo through intramyocardial injection guided by echocardiogram. Another group of animals was treated with rat CDC (rCDC) without immunosuppression. hCDC and rCDC were stably transduced with a viral construct expressing luciferase under control of a constitutive promoter. CDC were then used in a bioluminescence assay. Functional parameters were evaluated by echocardiogram 90 and 120 days after MI and by Langendorff at 120 days.ResultsCDC had a predominantly mesenchymal phenotype. Cell tracking by bioluminescence demonstrated over 85% decrease in signal at 5–7 days after cell therapy. Cardiac function evaluation by echocardiography showed no differences in ejection fraction, end-diastolic volume, or end-systolic volume between groups receiving human cells, rat cells, or placebo. Hemodynamic analyses and infarct area quantification confirmed that there was no improvement in cardiac remodeling after cell therapy with CDC.ConclusionOur study challenges the effectiveness of CDC in post-ischemic heart failure.

Abstract 4728: Apoptotic priming is regulated by a developmental program and predisposes children to therapy-induced toxicity

Abstract Pediatric cancer patients frequently suffer higher levels of treatment-induced toxicities than adults, limiting the use of potentially curative therapies. For example, brain irradiation contributes to the cure of medulloblastomas in 80% of children yet also causes cell death in healthy neurons, resulting in a permanent and devastating loss of IQ. Likewise, children commonly experience dose-limiting cardiotoxicity from doxorubicin treatment. These treatments are comparatively well tolerated in adults yet the basis for this dramatic contrast in sensitivity is unknown. Both radiation and cytotoxic chemotherapies can induce an apoptotic cell death, prompting us to hypothesize that apoptosis may be regulated in a fundamentally different manner in children versus adults. By testing the functional state of the apoptotic pathway in tissues with BH3 Profiling, we found that healthy brain, heart and kidney tissues from young mice are extremely sensitive to pro-apoptotic signals and are therefore “primed for apoptosis.” In stark contrast, these same tissues are completely insensitive to even saturating amounts of pro-apoptotic signals in adult mice. Apoptosis could be triggered in adult brain, heart or kidney cells only when complemented with exogenous BAX protein suggesting that these tissues lack sufficient levels of BAX or BAK for mitochondrial permeabilization and are thus “incompetent for apoptosis.” Immunoblotting revealed that BAX and BAK protein levels are strongly downregulated in these tissues during postnatal development and become nearly undetectable by adulthood, along with several other components of the apoptotic machinery. Parallel studies with spleen and bone marrow demonstrated high levels of apoptotic priming in young animals which continued into adulthood, highlighting the organ-specific nature of apoptosis regulation. In agreement with these findings, we observed apoptotic cell death in response to radiation damage in the brain, heart and kidneys of early postnatal but not adult mice. Likewise, the extent of doxorubicin-induced damage to cardiac tissue correlated strongly with changes in apoptotic priming and competence. Furthermore, we utilized BAX and/or BAK knockout mice to characterize the pivotal roles of these proteins in regulating apoptotic competence and damage responses in somatic tissues. We extended these findings to humans by BH3 profiling normal brain tissue and confirmed that brain tissue in young children is primed to undergo apoptosis while in adults it is apoptotically incompetent. Finally, we identified the epigenetic mechanisms that modulate apoptotic pathways during postnatal development. Our findings elucidate the molecular mechanisms responsible for the devastating vital organ sensitivity to damage in children and a strategy for its prevention. Citation Format: Kristopher A. Sarosiek, Michael Ziller, Cameron Fraser, Patrick Bhola, Jeremy Ryan, Jing Deng, Brian Jian, Marti Goldenberg, Joseph Madsen, Ruben Carrasco, Shenandoah Robinson, Javid Moslehi, Anthony Letai. Apoptotic priming is regulated by a developmental program and predisposes children to therapy-induced toxicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4728. doi:10.1158/1538-7445.AM2015-4728

PDGFRα demarcates the cardiogenic clonogenic Sca1+ stem/progenitor cell in adult murine myocardium.

Cardiac progenitor/stem cells in adult hearts represent an attractive therapeutic target for heart regeneration, though (inter)-relationships among reported cells remain obscure. Using single-cell qRT–PCR and clonal analyses, here we define four subpopulations of cardiac progenitor/stem cells in adult mouse myocardium all sharing stem cell antigen-1 (Sca1), based on side population (SP) phenotype, PECAM-1 (CD31) and platelet-derived growth factor receptor-α (PDGFRα) expression. SP status predicts clonogenicity and cardiogenic gene expression (Gata4/6, Hand2 and Tbx5/20), properties segregating more specifically to PDGFRα+ cells. Clonal progeny of single Sca1+ SP cells show cardiomyocyte, endothelial and smooth muscle lineage potential after cardiac grafting, augmenting cardiac function although durable engraftment is rare. PDGFRα− cells are characterized by Kdr/Flk1, Cdh5, CD31 and lack of clonogenicity. PDGFRα+/CD31− cells derive from cells formerly expressing Mesp1, Nkx2-5, Isl1, Gata5 and Wt1, distinct from PDGFRα−/CD31+ cells (Gata5 low; Flk1 and Tie2 high). Thus, PDGFRα demarcates the clonogenic cardiogenic Sca1+ stem/progenitor cell.

MZF1 is differentially expressed in human cardiac tissue of different age and c-KIT+ progenitor cells in the human adult heart

Objectives: The paradigm of a quiescent myocardium is obsolete since several resident progenitor cell populations have been identified within the heart by well-known stem cell surface markers like Sca1 or c-Kit. Recently we identified a potential role of the transcription factor myeloid zinc finger 1 (Mzf1) during murine embryonic heart development. Therefore, we investigated the role of MZF1 in human cardiac tissue of various age and in resident c-KIT+ progenitor cells obtained from the human adult heart.

Cardiac primitive cells in the adult human heart are influenced by Angiotensin II in chronic heart failure

Angiotensin II (AngII) levels are often increased in patients with cardiovascular disease and different AngII receptor (ATR) blockers are commonly used in heart failure therapy. An increase in number of cardiac primitive cells (CPCs) has been found in adult human pathological heart and these cells express AT1 and AT2 receptors (ATR). Hence, it is conceivable that AngII and ATR blockers influence CPC properties. In this study we investigated the effects of AngII and AT1R, AT2R blocking on human CPCs in vitro. CPCs were isolated from adult human hearts with ischemic cardiomyopathy (n=11) by tissue digestion and adherent CD117(+) cells immunomagnetic separation. Cells were cultured in the presence of AngII 0,1 and 1μM for 24 hr with and without pretreatment with 1μM valsartan - AT1R blocker (AT1RB), 1μM PD 123319 - AT2R blocker (AT2RB), 10μM telmisartan - AT1RB with PPAR activity, 1μM GW9662 - PPARγ blocker (PPARB). The expression of PPARγ was evaluated by electrophoresis and immunoblotting, while the proliferation of cells was evaluated by MTT assay. Administration of AngII stimulated proliferation of CPCs. The expression of PPAR γ increased 8,5 and 11,8-fold (p<0,05, n=3) in the presence of 0,1 and 1uM AngII, respectively. In the presence of AT1RB, proliferation of cells did not change significantly but it increased 1,5 and 2,1-fold (p<0,05, n=3) after addition of 0,1 and 1uM AngII, respectively, suggesting that this effect was modulated by stimulation of AT2R. In the presence of AT2RB, both basal and AngII-stimulated proliferation increased, particularly at lower AngII concentration. Interestingly, PPARγ antagonist reduced cell survival, but this effect was inversed by addition of AngII, probably due to the AngII-mediated increase of PPAR expression. Pretreatment of CPCs with AT1RB with PPAR activity increased basal cell proliferation, but was not beneficial in the presence of AngII when compared to pure AT1RB. The results indicate that AT1RB increases CPC proliferation mostly at higher AngII concentrations, while AT2RB has similar effect in the presence of low AngII levels. Hence, complex interactions between AngII and both AT1 and AT2 receptors take place in heart failure patients and influence the survival of CPCs in concentration dependant manner.

Cardioprotective SUR2A promotes stem cell properties of cardiomyocytes

Cardioprotective SUR2A promotes stem cell properties of cardiomyocytes

Left Atrial Appendages from Adult Hearts Contain a Reservoir of Diverse Cardiac Progenitor Cells

AimsThere is strong evidence supporting the claim that endogenous cardiac progenitor cells (CPCs) are key players in cardiac regeneration, but the anatomic source and phenotype of the master cardiac progenitors remains uncertain. Our aim was to investigate the different cardiac stem cell populations in the left atrial appendage (LAA) and their fates.Methods and ResultsWe investigated the CPC content and profile of adult murine LAAs using immunohistochemistry and flow cytometry. We demonstrate that the LAA contains a large number of CPCs relative to other areas of the heart, representing over 20% of the total cell number. We grew two distinct CPC populations from the LAA by varying the degree of proteolysis. These differed by their histological location, surface marker profiles and growth dynamics. Specifically, CD45pos cells grew with milder proteolysis, while CD45neg cells grew mainly with more intense proteolysis. Both cell types could be induced to differentiate into cells with cardiomyocyte markers and organelles, albeit by different protocols. Many CD45pos cells expressed CD45 initially and rapidly lost its expression while differentiating.ConclusionsOur results demonstrate that the left atrial appendage plays a role as a reservoir of multiple types of progenitor cells in murine adult hearts. Two different types of CPCs were isolated, differing in their epicardial-myocardial localization. Considering studies demonstrating layer-specific origins of different cardiac progenitor cells, our findings may shed light on possible pathways to study and utilize the diversity of endogenous progenitor cells in the adult heart.