Cardiogenic Genes Research Articles

Abstract 15721: CRISPR Activates Heterochromatic Cardiac Genes in Fibroblasts to Initiate Lineage Conversion Into Cardiac Progenitors

Introduction: Our previous studies have demonstrated that CRISPR activation (CRISPRa) of endogenous genes was sufficient to convert fibroblasts into induced cardiovascular progenitor cells (iCPCs) for myocardial regenerative therapy. However, reprogramming efficiency remains a challenge and little has been elucidated about the epigenetic mechanisms of cellular reprogramming, which will be important for methodological improvements or refinements. Hypothesis: CRISPRa complex can alter the chromatin accessibility and activity of heterochromatic cardiac genes in fibroblasts, directly initiating cardiac reprogramming. Methods: A fibroblast-specific CRISPRa mice model (Pdgfra-cre/ERT; ROSA-dCas9-SunTag) was generated. SgRNAs targeting the promoter region of Gata4, Isl1, Nkx2-5, Baf60c, or Tbx5 genes were transduced through an AAV system in cardiac fibroblast isolated from transgenic mice. iCPCs were confirmed via immunostaining of CPC markers and cardiac electrophysiological assays. iCPCs were collected for RNA-seq, ATAC-seq, and ChIP-seq after reprogramming induction to reveal the epigenetic landscape modulated by CRISPRa. Results: The CRISPRa system was activated in ~80% of fibroblasts after tamoxifen induction and AAV-sgRNA transfection. The reprogrammed iCPCs were characterized by immunostaining of proliferative markers (Ki67 and Pcna), progenitor surface markers (Flk1 and Ssea1), and markers of cardiac tri-lineage cells after induced differentiation. RNA-seq data showed the upregulation of genes associated with cell cycling (Cdkn1b, Aurkb, etc.) and heart development (Myh6, Irx4, etc.) in CRISPRa-induced cells. ATAC-seq showed increased chromatin accessibility at the loci of cardiogenic genes targeted by CRISPRa in fibroblasts. Furthermore, ChIP-seq of Gata4, Nkx2.5, and Tbx5 associated DNA shows that these factors can bind to their own promoter regions in fibroblasts after CRISPRa induction. Conclusions: CRISPRa complexes open the chromatin of the targeted genes in fibroblasts and establish an endogenous autoregulatory loop to ensure stable reprogramming into cardiac progenitors.

Modeling a variant of unknown significance in the Drosophila ortholog of the human cardiogenic gene NKX2.5.

Sequencing of human genome samples has unearthed genetic variants for which functional testing is necessary to validate their clinical significance. We used the Drosophila system to analyze a variant of unknown significance in the human congenital heart disease gene NKX2.5 (also known as NKX2-5). We generated an R321N allele of the NKX2.5 ortholog tinman (tin) to model a human K158N variant and tested its function in vitro and in vivo. The R321N Tin isoform bound poorly to DNA in vitro and was deficient in activating a Tin-dependent enhancer in tissue culture. Mutant Tin also showed a significantly reduced interaction with a Drosophila T-box cardiac factor named Dorsocross1. We generated a tinR321N allele using CRISPR/Cas9, for which homozygotes were viable and had normal heart specification, but showed defects in the differentiation of the adult heart that were exacerbated by further loss of tin function. We propose that the human K158N variant is pathogenic through causing a deficiency in DNA binding and a reduced ability to interact with a cardiac co-factor, and that cardiac defects might arise later in development or adult life.

CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression.

Haploinsufficiency of the chromo-domain protein CHD7 underlies most cases of CHARGE syndrome, a multisystem birth defect including congenital heart malformation. Context specific roles for CHD7 in various stem, progenitor, and differentiated cell lineages have been reported. Previously, we showed severe defects when Chd7 is absent from cardiopharyngeal mesoderm (CPM). Here, we investigate altered gene expression in the CPM and identify specific CHD7-bound target genes with known roles in the morphogenesis of affected structures. We generated conditional KO of Chd7 in CPM and analysed cardiac progenitor cells using transcriptomic and epigenomic analyses, in vivo expression analysis, and bioinformatic comparisons with existing datasets. We show CHD7 is required for correct expression of several genes established as major players in cardiac development, especially within the second heart field (SHF). We identified CHD7 binding sites in cardiac progenitor cells and found strong association with histone marks suggestive of dynamically regulated enhancers during the mesodermal to cardiac progenitor transition of mESC differentiation. Moreover, CHD7 shares a subset of its target sites with ISL1, a pioneer transcription factor in the cardiogenic gene regulatory network, including one enhancer modulating Fgf10 expression in SHF progenitor cells vs. differentiating cardiomyocytes. We show that CHD7 interacts with ISL1, binds ISL1-regulated cardiac enhancers, and modulates gene expression across the mesodermal heart fields during cardiac morphogenesis.

Essential role of MESP1-RING1A complex in cardiac differentiation.

Heart development is controlled by a complex transcriptional network composed of transcription factors and epigenetic regulators. Mutations in key developmental transcription factor MESP1 and chromatin factors, such as PRC1 and cohesin components, have been found in human congenital heart diseases (CHDs), although their functional mechanism during heart development remains elusive. Here, we find that MESP1 interacts with RING1A/RING1, the core component of PRC1. RING1A depletion impairs human cardiomyocyte differentiation, and cardiac abnormalities similar to those in patients with MESP1 mutations were observed in Ring1A knockout mice. Mechanistically, MESP1 associates with RING1A to activate cardiogenic genes through promoter-enhancer interactions regulated by cohesin and CTCF and histone acetylation mediated by p300. Importantly, CHD mutations of MESP1 significantly affect such mechanisms and impair target gene activation. Together, our results demonstrate the importance of MESP1-RING1A complex in heart development and provide insights into the pathogenic mechanisms of CHDs caused by mutations in MESP1, PRC1, and cohesin components.

Abstract P3101: Chromatin Remodeling BAF (SWI/SNF) Complex Controls Both Cardiogenesis And Neurogenesis From Human Pluripotent Stem Cells

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. Results: Here, we report ARID1A, a DNA-binding subunit of the BAF(SWI/SNF) epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Loss-of-ARID1A (ARID1A –/– ) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A –/– hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF. Conclusions: We uncover the key and opposite roles by the BAF(SWI/SNF) complex to govern early cardiac and neural development. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A-REST/NRSF interaction.

Abstract 9102: Cytosolic and Nuclear LncRNA HBL1 Crosstalk to Orchestrate Human Cardiogenesis via Interactions With MicroRNA-1 and PRC2 Epigenetic Complex

Introduction: Compared to rodents, human embryos exhibit unique dynamics of cardiogenesis, suggesting the existence of novel human-specific genetic programs underlying early-stage human heart development. Due to lack of in vivo model, human-specific mechanisms underlying cardiogenesis remain elusive. Hypothesis: Human Heart Brake LncRNA 1 (HBL1) functions as a new layer of regulator controlling cardiogenic gene transcription via coordinating with microRNA-1 and PRC2 epigenetic complex. Methods: Human pluripotent stem cells (hPSCs) were utilized to model human cardiogenesis. CRISPR/Cas-9 was utilized to knockout HBL1 and coding genes, and disrupted microRNA-1 binding sites on HBL1. HBL1 interacting proteins were pulled down by Co-IP-MS. EMSA and RNA-IP tested HBL1 interactions with PRC2 subunits. ChIP-seq uncovered the impact of HBL1 on genome-wide PRC2 occupancy. Luciferase assay confirmed JARID2 is a target of microRNA-1. Results: HBL1 expresses in cytoplasm and nucleus of hPSCs. Gain-of-HBL1 represses, whereas loss-of-HBL1 increases cardiomyocyte differentiation from hPSCs. Cytosolic HBL1 modulates cardiac development from hPSCs by counteracting microRNA-1. In hPSCs, disruption of microRNA-1 binding sites on HBL1 phenocopies HBL1 deficiency. Nuclear HBL1 interacts with two PRC2 subunits, JARID2 and EED. Loss-of-JARID2 or EED phenocopies HBL1 deficiency to enhance cardiac differentiation from hPSCs. HBL1 deficiency disrupts genome-wide PRC2 occupancy and H3K27me3 deposition on essential cardiogenic genes, and consequently enhances their transcription in hPSCs. Both HBL1 and JARID2 are required for PRC2 deposition to determine the epigenetic state of critical cardiogenic genes in hPSCs. Furthermore, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved “microRNA-1-JARID2” axis to modulate transcription of cardiogenic genes. Conclusions: Our findings delineate the indispensable role of human-specific HBL1 in early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to precisely control human cardiogenesis.

Resolution factor 15-epi-Lipoxin A4 modulates miRNA-499 induced differentiation of cardiosphere-derived stem cells through dual inhibition of Wnt/β-catenin and TGFβ/SMAD signalling axes

BackgroundCardiosphere derived stem cells (CDSCs) are signified as a valued source of stem cell therapeutics for treating a gamut of cardiovascular ailments. However, challenges in the cardiogenic differentiation process culminate in stem cell transplantation failure. Hence, it is imperative to understand the comprehensive mechanisms and the role of endogenous factors in cardiac differentiation. ObjectivesObjective of the current study is to check the effect of 15-epi-lipoxin A4 (15E-LXA4)—a stable analogue of lipoxin A4, a pro-resolving agonist—on miR-499 (a cardiac-specific microRNA)-induced differentiation of CDSCs. MethodsCDSCs were transfected with lentiviral vectors bearing miR-499 and subjected to 15E-LXA4 treatment. Then, the treatment effects on surface markers (c-kit and CD105), cardiogenic gene markers (NKX2.5, GATA4, and cTnI), Wnt/β-catenin signaling (Wnt3a and phosphorylated/dephosphorylated β-catenin ratio) and TGFβ/SMAD signaling (TGFβ1 and SMAD3) were evaluated. Results15E-LXA4 treatment repressed miR-499 over-expression induced cardiac differentiation, manifested by enhanced expression of surface markers, Wnt/β-catenin and TGFβ/SMAD signaling parameters and reduced expression cardiogenic gene markers, plausibly through activation of estrogen receptor (ERα). ConclusionsThese findings give reason to understand the role of endogenous resolution factors like 15E-LXA4, as it displayed reciprocal effect on miR-499 induced cardiac differentiation in CDSCs.

LncRNA HBL1 is required for genome-wide PRC2 occupancy and function in cardiogenesis from human pluripotent stem cells.

ABSTRACTPolycomb repressive complex 2 (PRC2) deposits H3K27me3 on chromatin to silence transcription. PRC2 broadly interacts with RNAs. Currently, the role of the RNA-PRC2 interaction in human cardiogenesis remains elusive. Here, we found that human-specific heart brake lncRNA 1 (HBL1) interacted with two PRC2 subunits, JARID2 and EED, in human pluripotent stem cells (hPSCs). Loss of JARID2, EED or HBL1 significantly enhanced cardiac differentiation from hPSCs. HBL1 depletion disrupted genome-wide PRC2 occupancy and H3K27me3 chromatin modification on essential cardiogenic genes, and broadly enhanced cardiogenic gene transcription in undifferentiated hPSCs and later-on differentiation. In addition, ChIP-seq revealed reduced EED occupancy on 62 overlapped cardiogenic genes in HBL1−/− and JARID2−/− hPSCs, indicating that the epigenetic state of cardiogenic genes was determined by HBL1 and JARID2 at pluripotency stage. Furthermore, after cardiac development occurs, the cytosolic and nuclear fractions of HBL1 could crosstalk via a conserved ‘microRNA-1-JARID2’ axis to modulate cardiogenic gene transcription. Overall, our findings delineate the indispensable role of HBL1 in guiding PRC2 function during early human cardiogenesis, and expand the mechanistic scope of lncRNA(s) that cytosolic and nuclear portions of HBL1 could coordinate to orchestrate human cardiogenesis.

Targeting the histone demethylase LSD1 prevents cardiomyopathy in a mouse model of laminopathy.

LMNA mutations in patients are responsible for a dilated cardiomyopathy. Molecular mechanisms underlying the origin and development of the pathology are unknown. Herein, using mouse pluripotent embryonic stem cells (ESCs) and a mouse model both harboring the p.H222P Lmna mutation, we found early defects in cardiac differentiation of mutated ESCs and dilatation of mutated embryonic hearts at E13.5, pointing to a developmental origin of the disease. Using mouse ESCs, we demonstrated that cardiac differentiation of LmnaH222P/+ was impaired at the mesodermal stage. Expression of Mesp1, a mesodermal cardiogenic gene involved in epithelial-to-mesenchymal transition of epiblast cells, as well as Snai1 and Twist expression, was decreased in LmnaH222P/+ cells compared with WT cells in the course of differentiation. In turn, cardiomyocyte differentiation was impaired. ChIP assay of H3K4me1 in differentiating cells revealed a specific decrease of this histone mark on regulatory regions of Mesp1 and Twist in LmnaH222P/+ cells. Downregulation or inhibition of LSD1 that specifically demethylated H3K4me1 rescued the epigenetic landscape of mesodermal LmnaH222P/+ cells and in turn contraction of cardiomyocytes. Inhibition of LSD1 in pregnant mice or neonatal mice prevented cardiomyopathy in E13.5 LmnaH222P/H222P offspring and adults, respectively. Thus, LSD1 appeared to be a therapeutic target to prevent or cure dilated cardiomyopathy associated with a laminopathy.

Brachyury engineers cardiac repair competent stem cells.

To optimize the regenerative proficiency of stem cells, a cardiopoietic protein‐based cocktail consisting of multiple growth factors has been developed and advanced into clinical trials for treatment of ischemic heart failure. Streamlining the inductors of cardiopoiesis would address the resource intensive nature of the current stem cell enhancement protocol. To this end, the microencapsulated‐modified‐mRNA (M3RNA) technique was here applied to introduce early cardiogenic genes into human adipose‐derived mesenchymal stem cells (AMSCs). A single mesodermal transcription factor, Brachyury, was sufficient to trigger high expression of cardiopoietic markers, Nkx2.5 and Mef2c. Engineered cardiopoietic stem cells (eCP) featured a transcriptome profile distinct from pre‐engineered AMSCs. In vitro, eCP demonstrated protective antioxidant capacity with enhanced superoxide dismutase expression and activity; a vasculogenic secretome driving angiogenic tube formation; and macrophage polarizing immunomodulatory properties. In vivo, in a murine model of myocardial infarction, intramyocardial delivery of eCP (600 000 cells per heart) improved cardiac performance and protected against decompensated heart failure. Thus, heart repair competent stem cells, armed with antioxidant, vasculogenic, and immunomodulatory traits, are here engineered through a protein‐independent single gene manipulation, expanding the available regenerative toolkit.

Using multiple-steps bioinformatic analysis to predict the potential microRNA targets by cardiogenic HoxA11.

In this study, our major aim is to using multiple-steps bioinformatic analysis to predict cardiogenic genes with targeting mRNA profiling for predicting cardiogenic HoxA11 gene. We first analyzed the microarray data with bioinformatic measurement, including combining with panel module 1 (mouse embryonic stem cells), panel module 2 (mouse induced pluripotent stem cells), and panel module 3 (gene list form literature of heart development). A literature-based comparison of the two microarrays and a software-based (Targetscan program, www.targetscan.org) comparative analysis of the two datasets. Furthermore, we select the common central pathways and potential candidate genes involved in the cardiomyocyte-lineaged differentiation and development. Schematic presentation of a putative miR181a target site in Hox-A11 3'UTR. The bioinformatic result showed that potential interacted cardiogenic targets of Tbx5, Tbx20, Mal2c, Nkx2.5, cTNT, Cx43, MHC, and MCK in different treatment groups of pluripotent stem cells by using a literature-based comparison of the two microarrays and a software-based gene-lineage system. Our findings support that mir181a is an up-stream regulating microRNA to target the 3'UTR of HoxA11 mRNA during the process of cardiomyocyte differentiation.

Abstract MP142: Genome-wide Studies Reveal the Essential and Opposite Roles of Arid1a in Controlling Human Cardiogenesis and Neurogenesis From Pluripotent Stem Cells

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene/complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. The evolutionarily conserved ATP-dependent SWI/SNF complex is one of the largest chromatin remodeling complexes, consisting of ~15 subunits, including SMARCA2 (also known as BRM) or SMARCA4 (also known as BRG1) as the ATPase catalytic subunit. Several BRG1-associated factors (BAFs), such as ARID1A (Baf250a), have DNA binding capacity and assemble with either BRM or BRG1 to form a functional chromatin-remodeling complex. A single amino acid mutation (Arid1aV 1068G/V1068G ), impaired Arid1a-DNA interactions and resulted in both cardiac neural defects. Mutations in 4 different SWI/SNF subunits including ARID1A/B were identified in human congenital syndromes that include both neural and cardiac defects. Results: Here, we report ARID1A, which is a DNA-binding-subunit of the SWI/SNF epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) via employing distinct mechanisms. CRISPR/Cas-9 knockout of ARID1A (ARID1A -/- ) led to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A -/- hESCs was prominently suppressed, whereas neural differentiation was significantly promoted. Whole genome-wide ChIP-seq and ATAC-seq analyses revealed that ARID1A was required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during neural development, transcription of most essential neurogenic genes was dependent on ARID1A and ARID1A could interact with REST, which is a known transcriptional repressor. Conclusions: We uncovered the key and opposite roles by ARID1A to govern both early human cardiac and neural development and characterized the mechanisms. We found global chromatin accessibility on cardiogenic genes was dependent on ARID1A, whereas transcriptional activity of neurogenic genes was regulated by ARID1A, possibly through ARID1A-REST interaction.

Genome-wide studies reveal the essential and opposite roles of ARID1A in controlling human cardiogenesis and neurogenesis from pluripotent stem cells

BackgroundEarly human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive.ResultsHere, we report ARID1A, a DNA-binding subunit of the SWI/SNF epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Knockout-of-ARID1A (ARID1A−/−) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A −/− hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF.ConclusionsWe uncover the opposite roles by ARID1A to govern both early cardiac and neural development from pluripotent stem cells. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A, possibly through ARID1A-REST/NRSF interaction.

Unique patterns of cardiogenic and fibrotic gene expression in rat cardiac fibroblasts.

Background and Aim:Cardiac fibroblasts are important for both normal and pathological states of the heart, but the knowledge in cell physiology and genomics is still poorly understood. The aims of the present study were; first, to investigate the expression of cardiac and fibrotic genes in rat cardiac fibroblasts compared to cardiomyocytes and other fibroblasts (skin and muscle fibroblasts), second, to examine the in vitro effect of serum concentration on fibroblast gene expression. The findings can potentially be applied in ischemia/reperfusion models.Materials and Methods:Rat cardiac fibroblasts were collected and cultured in different conditions, and their gene expression (21 cardiogenic genes and 16 fibrotic genes) was compared with cardiomyocytes and other fibroblasts using comparative quantitative polymerase chain reaction. We also mimicked myocardial ischemia/reperfusion by depleting and then adding a serum into the culture in conventional culture (10% serum).Results:Cardiac fibroblasts expressed most of the cardiogenic genes, but their expression levels were significantly lower than in cardiomyocytes, while almost all fibrotic genes in the cardiac fibroblasts were significantly more highly expressed than in cardiomyocytes, except matrix metallopeptidase 9 (Mmp9) which also had greater expression in other fibroblasts. After mimicking cardiac ischemia and reperfusion in vitro by starving and then adding a serum into the cardiac fibroblast culture, the results revealed that Mmp9 expression was significantly increased (>30 times) after increasing but not reducing the serum in the culture. The expression of most cardiogenic and fibrotic genes in cardiac fibroblasts tended to decrease after increasing the serum in the culture. These changes were specific to cardiac fibroblasts but no other fibroblasts.Conclusion:Cardiac fibroblasts have a distinct pattern of gene expression from other fibroblasts and cardiomyocytes. They are also sensitive to high serum concentration but not affected by serum depletion, suggesting that the process of developing cardiac fibrosis might be stimulated by reperfusion or overcirculation rather than ischemia. The cell starvation followed the adding of serum may serve as a useful model to study cardiac fibrosis cause by the change of blood flow.

Mechanistic basis of neonatal heart regeneration revealed by transcriptome and histone modification profiling

The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.

Role of Cdx factors in early mesodermal fate decisions.

Murine cardiac and hematopoietic progenitors are derived from Mesp1+ mesoderm. Cdx function impacts both yolk sac hematopoiesis and cardiogenesis in zebrafish, suggesting that Cdx family members regulate early mesoderm cell fate decisions. We found that Cdx2 occupies a number of transcription factor loci during embryogenesis, including key regulators of both cardiac and blood development, and that Cdx function is required for normal expression of the cardiogenic transcription factors Nkx2-5 and Tbx5 Furthermore, Cdx and Brg1, an ATPase subunit of the SWI/SNF chromatin remodeling complex, co-occupy a number of loci, suggesting that Cdx family members regulate target gene expression through alterations in chromatin architecture. Consistent with this, we demonstrate loss of Brg1 occupancy and altered chromatin structure at several cardiogenic genes in Cdx-null mutants. Finally, we provide evidence for an onset of Cdx2 expression at E6.5 coinciding with egression of cardiac progenitors from the primitive streak. Together, these findings suggest that Cdx functions in multi-potential mesoderm to direct early cell fate decisions through transcriptional regulation of several novel target genes, and provide further insight into a potential epigenetic mechanism by which Cdx influences target gene expression.

A Bioprinted Cardiac Patch Composed of Cardiac-Specific Extracellular Matrix and Progenitor Cells for Heart Repair.

Congenital heart defects are present in 8 of 1000 newborns and palliative surgical therapy has increased survival. Despite improved outcomes, many children develop reduced cardiac function and heart failure requiring transplantation. Human cardiac progenitor cell (hCPC) therapy has potential to repair the pediatric myocardium through release of reparative factors, but therapy suffers from limited hCPC retention and functionality. Decellularized cardiac extracellular matrix hydrogel (cECM) improves heart function in animals, and human trials are ongoing. In the present study, a 3D-bioprinted patch containing cECM for delivery of pediatric hCPCs is developed. Cardiac patches are printed with bioinks composed of cECM, hCPCs, and gelatin methacrylate (GelMA). GelMA-cECM bioinks print uniformly with a homogeneous distribution of cECM and hCPCs. hCPCs maintain >75% viability and incorporation of cECM within patches results in a 30-fold increase in cardiogenic gene expression of hCPCs compared to hCPCs grown in pure GelMA patches. Conditioned media from GelMA-cECM patches show increased angiogenic potential (>2-fold) over GelMA alone, as seen by improved endothelial cell tube formation. Finally, patches are retained on rat hearts and show vascularization over 14 d in vivo. This work shows the successful bioprinting and implementation of cECM-hCPC patches for potential use in repairing damaged myocardium.

Direct Cardiac Reprogramming: Progress and Promise.

The human adult heart lacks a robust endogenous repair mechanism to fully restore cardiac function after insult; thus, the ability to regenerate and repair the injured myocardium remains a top priority in treating heart failure. The ability to efficiently generate a large number of functioning cardiomyocytes capable of functional integration within the injured heart has been difficult. However, the ability to directly convert fibroblasts into cardiomyocyte-like cells both in vitro and in vivo offers great promise in overcoming this problem. In this review, we describe the insights and progress that have been gained from the investigation of direct cardiac reprogramming. We focus on the use of key transcription factors and cardiogenic genes as well as on the use of other biological molecules such as small molecules, cytokines, noncoding RNAs, and epigenetic modifiers to improve the efficiency of cardiac reprogramming. Finally, we discuss the development of safer reprogramming approaches for future clinical application.

Abstract 350: The Developmental Origin Of Cardiac Fibroblasts Influences the Efficiency of Direct Reprogramming to Cardiomyocytes

Direct conversion of fibroblasts to cardiomyocytes (CM) is advancing the field of cardiac regeneration. Despite advantages of direct reprogramming, the presence of residual epigenetic memory of the original cells may hinder clinical transition. Thus, choosing a starting cell with similar ontogeny to the desired reprogrammed cell may overcome some of the limitations. Expression of key cardiogenic genes shared between cardiac fibroblasts (CFb) and CM in addition to the abundance of these cells, suggest that CFb may be the optimal autologous cell source for therapeutic manipulation in treating heart disease. We hypothesized that progenitors, transiently expressing Mesp1 generate a sub-population of CFb which are more prone to direct reprogramming and adopt a cardiomyocyte gene profile due to their maintained epigenetic memory. We generated a Mesp1CremTmG mouse to label all cells expressing Mesp1 and their progeny and we observed that the majority of the cells in the heart including CFb are derived from Mesp1 cells. We showed that ~80% of resident CFb are derived from Mesp1 while a minor non-Mesp1 subset also exists. We compared the reprogramming efficiency of CFb of Mesp1 origin to CFb of other origin to induced CM (iCM) by overexpression of specific cardiac transcription factors. Results from immunostaining and gene analysis showed higher expression of cardiac muscle markers in CM induced from Mesp1 CFb. To further delineate potential differences between two subsets, we performed RNAseq and our results showed that the non Mesp1 CFb were enriched in neural crest related genes. We generated Pax3CremTmG mice to lineage trace neural crest-derived cells. Our results confirmed a minor contribution of Pax3 cells to CFb. We developed a modified CLARITY technique to transform the heart into an optically-transparent organ for light-sheet fluorescence imaging. We observed that Pax3 CFb were mainly located in the wall of aorta while Mesp1 CFb were distributed throughout the heart. Additionally, we are studying whether each CFb subset has the tendency to generate a specific subtype of iCM (ventricular, atrial, and nodal CM). These results can identify distinct sub-populations of CFb, which can generate functional cardiomyocytes for cardiac-regenerative therapies.

The Promise of Cardiac Regeneration by In Situ Lineage Conversion.

Although remarkable advances in acute coronary care have significantly improved outcomes following myocardial infarction (MI), survivors often experience progressive heart failure, a devastating condition with limited curative options. Given that the adult heart cannot regenerate heart muscle cells to restore contractile function, there is great demand to develop therapeutic strategies to build new heart muscle (Figure). Thus, many strategies have been conceived to induce de novo generation of cardiomyocytes for heart repair. Figure. Generation of new cardiomyocytes by direct reprogramming of cardiac fibroblasts in situ. Following a myocardial infarction (MI), a fundamental issue remains the irreversible loss of cardiomyocytes with subsequent replacement by fibroblast-derived scar tissue ( left ). Although current pharmacological treatments can slow the progression of heart failure, the ultimate therapeutic goal would be to completely restore normal cardiac function ( right ). Recent work has shown that activated fibroblasts in the MI border zone can be directly converted into functional cardiomyocytes by the addition of transcription factors (cardiogenic genes). In work described in this issue of Circulation , Mohamed et al4 show that specific small molecules can further augment the cardiac reprogramming process in situ. By far the most well-studied approach …