Early Stage Of Cardiomyocyte Differentiation Research Articles

A long non-coding RNA GATA6-AS1 adjacent to GATA6 is required for cardiomyocyte differentiation from human pluripotent stem cells.

Long noncoding RNAs (lncRNAs) are crucial in many cellular processes, yet relatively few have been shown to regulate human cardiomyocyte differentiation. Here, we demonstrate an essential role of GATA6 antisense RNA 1 (GATA6-AS1) in cardiomyocyte differentiation from human pluripotent stem cells (hPSCs). GATA6-AS1 is adjacent to cardiac transcription factor GATA6. We found that GATA6-AS1 was nuclear-localized and transiently upregulated along with GATA6 during the early stage of cardiomyocyte differentiation. The knockdown of GATA6-AS1 did not affect undifferentiated cell pluripotency but inhibited cardiomyocyte differentiation, as indicated by no or few beating cardiomyocytes and reduced expression of cardiomyocyte-specific proteins. Upon cardiac induction, the knockdown of GATA6-AS1 decreased GATA6 expression, altered Wnt-signaling gene expression, and reduced mesoderm development. Further characterization of the intergenic region between genomic regions of GATA6-AS1 and GATA6 indicated that the expression of GATA6-AS1 and GATA6 were regulated by a bidirectional promoter within the intergenic region. Consistently, GATA6-AS1 and GATA6 were co-expressed in several human tissues including the heart, similar to the mirror expression pattern of GATA6-AS1 and GATA6 during cardiomyocyte differentiation. Overall, these findings reveal a previously unrecognized and functional role of lncRNA GATA6-AS1 in controlling human cardiomyocyte differentiation.

MiR-25 Promotes Cardiomyocyte Proliferation by Targeting FBXW7

Induction of endogenous cardiomyocyte (CM) proliferation is one of the key strategies for heart regeneration. Increasing evidence points to the potential role of microRNAs (miRNAs) in the regulation of CM proliferation. Here, we used human embryonic stem cell (hESC)-derived CMs (hESC-CMs) as a tool to identify miRNAs that promote CM proliferation. We profiled miRNA expression at an early stage of CM differentiation and identified a list of highly expressed miRNAs. Among these miRNAs, miR-25 was enriched in early-stage hESC-CMs, but its expression decreased over time. Overexpression of miR-25 promoted CM proliferation. RNA sequencing (RNA-seq) analysis revealed that genes related to cell-cycle signal were strongly influenced by miR-25 overexpression. We further showed that miR-25 promoted CM proliferation by targeting FBXW7. Finally, the function of miR-25 in the regulation of CM proliferation was demonstrated in zebrafish. Our study suggested that miR-25 is a promising molecule for heart regeneration.

Downregulation of LGR5 Expression Inhibits Cardiomyocyte Differentiation and Potentiates Endothelial Differentiation from Human Pluripotent Stem Cells.

SummaryUnderstanding molecules involved in differentiation of human pluripotent stem cells (hPSCs) into cardiomyocytes and endothelial cells is important in advancing hPSCs for cell therapy and drug testing. Here, we report that LGR5, a leucine-rich repeat-containing G-protein-coupled receptor, plays a critical role in hPSC differentiation into cardiomyocytes and endothelial cells. LGR5 expression was transiently upregulated during the early stage of cardiomyocyte differentiation, and knockdown of LGR5 resulted in reduced expression of cardiomyocyte-associated markers and poor cardiac differentiation. In contrast, knockdown of LGR5 promoted differentiation of endothelial-like cells with increased expression of endothelial cell markers and appropriate functional characteristics, including the ability to form tube-like structures and to take up acetylated low-density lipoproteins. Furthermore, knockdown of LGR5 significantly reduced the proliferation of differentiated cells and increased the nuclear translocation of β-catenin and expression of Wnt signaling-related genes. Therefore, regulation of LGR5 may facilitate efficient generation of cardiomyocytes or endothelial cells from hPSCs.

Genome-Wide Temporal Profiling of Transcriptome and Open Chromatin of Early Cardiomyocyte Differentiation Derived From hiPSCs and hESCs.

Recent advances have improved our ability to generate cardiomyocytes from human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). However, our understanding of the transcriptional regulatory networks underlying early stages (ie, from mesoderm to cardiac mesoderm) of cardiomyocyte differentiation remains limited. To characterize transcriptome and chromatin accessibility during early cardiomyocyte differentiation from hiPSCs and hESCs. We profiled the temporal changes in transcriptome and chromatin accessibility at genome-wide levels during cardiomyocyte differentiation derived from 2 hiPSC lines and 2 hESC lines at 4 stages: pluripotent stem cells, mesoderm, cardiac mesoderm, and differentiated cardiomyocytes. Overall, RNA sequencing analysis revealed that transcriptomes during early cardiomyocyte differentiation were highly concordant between hiPSCs and hESCs, and clustering of 4 cell lines within each time point demonstrated that changes in genome-wide chromatin accessibility were similar across hiPSC and hESC cell lines. Weighted gene co-expression network analysis (WGCNA) identified several modules that were strongly correlated with different stages of cardiomyocyte differentiation. Several novel genes were identified with high weighted connectivity within modules and exhibited coexpression patterns with other genes, including noncoding RNA LINC01124 and uncharacterized RNA AK127400 in the module related to the mesoderm stage; E-box-binding homeobox 1 (ZEB1) in the module correlated with postcardiac mesoderm. We further demonstrated that ZEB1 is required for early cardiomyocyte differentiation. In addition, based on integrative analysis of both WGCNA and transcription factor motif enrichment analysis, we determined numerous transcription factors likely to play important roles at different stages during cardiomyocyte differentiation, such as T and eomesodermin (EOMES; mesoderm), lymphoid enhancer-binding factor 1 (LEF1) and mesoderm posterior BHLH transcription factor 1 (MESP1; from mesoderm to cardiac mesoderm), meis homeobox 1 (MEIS1) and GATA-binding protein 4 (GATA4) (postcardiac mesoderm), JUN and FOS families, and MEIS2 (cardiomyocyte). Both hiPSCs and hESCs share similar transcriptional regulatory mechanisms underlying early cardiac differentiation, and our results have revealed transcriptional regulatory networks and new factors (eg, ZEB1) controlling early stages of cardiomyocyte differentiation.

Expression and reconstitution of the bioluminescent Ca(2+) reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes.

In order to develop a novel method of visualizing possible Ca(2+) signaling during the early differentiation of hESCs into cardiomyocytes and avoid some of the inherent problems associated with using fluorescent reporters, we expressed the bioluminescent Ca(2+) reporter, apo-aequorin, in HES2 cells and then reconstituted active holo-aequorin by incubation with f-coelenterazine. The temporal nature of the Ca(2+) signals generated by the holo-f-aequorin-expressing HES2 cells during the earliest stages of differentiation into cardiomyocytes was then investigated. Our data show that no endogenous Ca(2+) transients (generated by release from intracellular stores) were detected in 1-12-day-old cardiospheres but transients were generated in cardiospheres following stimulation with KCl or CaCl2, indicating that holo-f-aequorin was functional in these cells. Furthermore, following the addition of exogenous ATP, an inositol trisphosphate receptor (IP3R) agonist, small Ca(2+) transients were generated from day 1 onward. That ATP was inducing Ca(2+) release from functional IP3Rs was demonstrated by treatment with 2-APB, a known IP3R antagonist. In contrast, following treatment with caffeine, a ryanodine receptor (RyR) agonist, a minimal Ca(2+) response was observed at day 8 of differentiation only. Thus, our data indicate that unlike RyRs, IP3Rs are present and continually functional at these early stages of cardiomyocyte differentiation.

Wnt-promoted Isl1 expression through a novel TCF/LEF1 binding site and H3K9 acetylation in early stages of cardiomyocyte differentiation of P19CL6 cells

Islet 1 (ISL1), a marker of second heart field progenitors, plays a crucial role in cardiomyocyte differentiation and proliferation. However, little is known about transcriptional regulating mechanisms on Isl1 gene expression. Recent studies have demonstrated that Wnt/β-catenin signaling regulates Isl1 expression during heart development. However, the detailed mechanisms still remain unclear. In the present study performed during differentiation of P19CL6 into cardiomyocytes, we explored the underlying regulating mechanisms on Wnt/β-catenin-mediated Isl1 expression after we first confirmed that Wnt/β-catenin signaling promoted cardiomyocyte differentiation partly through Isl1 activation. We found a novel TCF/LEF1 binding site that was located 2300 bp upstream of the Isl1 ATG. Furthermore, Wnt/β-catenin signaling upregulated histone H3K9 acetylation on TCF/LEF1 binding sites on the Isl1 promoter, resulting in upregulation of Isl1 expression. This Wnt-mediated H3K9 acetylation on the Isl1 promoter was modulated by the acetyltransferase CREB-binding protein (CBP), instead of p300, through interaction with β-catenin. Collectively, these results suggest that in early stages of cardiomyocyte differentiation Wnt/β-catenin signaling promotes Isl1 expression via two ways: a novel TCF/LEF1 binding site and H3K9 acetylation conducted by CBP on the Isl1 promoter. To our knowledge, this is the first study reporting Wnt/β-catenin-regulated H3K9 acetylation on promoters of its target genes. And this study gives new insights into transcriptional regulating mechanisms of Wnt-mediated Isl1 expression during cardiomyocyte differentiation.

Eosinophil cationic protein enhances stabilization of β-catenin during cardiomyocyte differentiation in P19CL6 embryonal carcinoma cells

Prior to gastrulation, the Wnt signaling pathway through stabilized β-catenin enhances the differentiation of mouse ES cell into cardiomyocytes. We have recently shown that cardiomyocyte differentiation is enhanced by eosinophil cationic protein (ECP) through accelerated expression of marker genes of early cardiac differentiation. Furthermore, ECP enhanced the expression of Wnt3a in P19CL6 cells which were stimulated to differentiate into cardiomyocytes by DMSO. Following these findings, we evaluated in this study the potential of ECP to activate the Wnt/β-catenin signaling pathway during cardiomyocyte differentiation. Analysis by real time qPCR revealed that ECP increased the expression of Frizzled genes such as Frizzled-1, -2, -4 and -10 in P19CL6 cells in the presence of DMSO. The increased expression of those Wnt receptors was found to inhibit the phosphorylation of β-catenin resulting in the stabilization and translocation of β-catenin into the nucleus of P19CL6 cells during the early stages of cardiomyocyte differentiation. When assessed for β-catenin/TCF transcriptional activity with a TCF-luciferase (TOP/FOP) assay, ECP enhanced luciferase activity in P19CL6 cells during 48 h after transfection with TOP/FOP flash reporter in a stoichiometric manner. Collectively, this suggests that ECP can activate a canonical Wnt/β-catenin signaling pathway by enhancing the stabilization of β-catenin during cardiomyocyte differentiation.

Interaction of Wnt/β‐catenin and notch signaling in the early stage of cardiac differentiation of P19CL6 cells

Notch and Wnt/β-catenin signaling both play essential roles and interact closely in cardiomyocyte differentiation but the mechanism of interaction is largely unknown. Here we show that activation of Notch signaling in undifferentiated P19CL6 cells promoted cardiac differentiation, indicated by upregulated expression of early cardiac markers and activated the canonical Wnt pathway, suggested by augmented nuclear translocation of β-catenin. Further activation of the Notch pathway in early differentiating cells (at day 3) inhibited expression of a specific cardiac progenitor marker Islet1 but had no influence on β-catenin translocation. Notch signaling thus played biphasic roles in the early stage of cardiomyocyte differentiation and Wnt/β-catenin signaling. Unlike Notch signaling, Wnt signaling promoted cardiomyocyte differentiation and activated the Notch pathway in either undifferentiated or early differentiating cells. Additionally, β-catenin, recombination signal sequence binding protein-Jkappa (RBP-Jκ), and Notch1 intracellular domain (NICD-1) formed a transcriptional complex which was recruited to the Hes1 promoter region, indicating direct transcriptional regulation of Hes1. We thus document a specific reciprocal interaction between these two signaling pathways during early stage cardiac differentiation of P19CL6 cells.

Vitamin B12 Promotes Cx40 and HCN4 Gene Expression at an Early Stage of Cardiomyocyte Differentiation

Vitamin B(12) (VB(12)) was investigated for its ability to influence the expression of genes required for cardiac cell differentiation from mouse embryonic stem cells. When VB(12) (0.5 mM) was added to the medium on day 3 of culture, the levels of Cx40 and HCN4 expression increased over those in the control, 2 to 3 days before the start of cardiomyocyte beating. In contrast, the expression levels of alpha-MHC, MLC-2a, and MLC-2v were almost the same as those of the control throughout the culture period. Our results suggest that VB(12) is involved in the promotion of the intercellular conducting system rather than the generation of contractile proteins.

WNT signaling promotes Nkx2.5 expression and early cardiomyogenesis via downregulation of Hdac1

The cardiac transcription factor NKX2.5 plays a crucial role in cardiomyogenesis, but its mechanism of regulation is still unclear. Recently, epigenetic regulation has become increasingly recognized as important in differentiation and development. In this study, we used P19CL6 cells to investigate the regulation of Nkx2.5 expression by methylation and acetylation during cardiomyocyte differentiation. During the early stage of differentiation, Nkx2.5 expression was upregulated, but the methylation status of the Nkx2.5 promoter did not undergo significant change; while the acetylation levels of histones H3 and H4 were increased, accompanied by a significant reduction in Hdac1 expression. Suppression of Hdac1 activity stimulated cardiac differentiation accompanied by increased expression of cardiac-specific genes and cell cycle arrest. Overexpression of Hdac1 inhibited cardiomyocyte formation and downregulated the expressions of Gata4 and Nkx2.5. Mimicking induction of the WNT pathway inhibited Hdac1 expression with upregulated Nkx2.5 expression. WNT3a and WNT3 downregulated the expression of Hdac1, contrary to the effect of SFRP2 and GSK3β. Cotransfection of β -catenin and Lef1 significantly downregulated the expression of Hdac1. Our data suggest that WNT signaling pathway plays important roles in the regulation of Hdac1 during the early stage of cardiomyocyte differentiation and that the downregulation of Hdac1 promotes cardiac differentiation.

Multiple roles for BMP signaling in cardiac development

The bone morphogenetic protein (BMP) family is the largest subfamily of the transforming growth factor-beta (TGF-β) superfamily. Some BMPs are expressed before cardioblast formation and throughout the late stages of heart development. BMPs have crucial roles in a broad range of biological events, including cellular proliferation, differentiation, migration and apoptosis during organ development. At least six BMPs (BMP2, 4, 5, 6, 7 and 10) are expressed in the heart, where they have both independent and redundant functions. Experiments on genetically modified mice have demonstrated the importance of BMP signaling for heart morphogenesis after the mid-gestation stage. Interestingly, BMPs play a dual role in heart development. Precise regulation of BMP inhibition and BMP stimulation is required for proper heart development during the early stage of cardiomyocyte differentiation. Knowledge of embryogenesis is frequently used in studies of stem cell biology. Through the application of BMP signal regulation in cardiac development, many systems for the differentiation of embryonic stem (ES) cells into cardiomyocytes are developed. These results reveal the crucial role of temporal and spatial regulation of BMPs and BMP antagonists in heart development.

HhLIM is involved in cardiomyogenesis of embryonic stem cells

Proteins of the LIM family play important roles in a variety of fundamental biological processes including cell lineage specification and organ development. Here we examined the function in cardiogenesis of a new member of the LIM family, hhLIM, by molecular analysis of early stages of cardiomyocyte differentiation in hhLIM-deficient P19 cell line and P19 cells stably overexpressing hhLIM. The results indicate that hhLIM is a potent transcriptional activator of several cardiac muscle-specific genes. Inhibition of hhLIM expression by antisense transcripts can interfere with expression of cardiac muscle genes and block development of beating cardiomyocytes in P19 embryonic stem cells. Overexpression of hhLIM in P19 cells can enhance expression of cardiac marker genes Nkx2.5 and GATA-4 and potentiate development of cardiomyocyte-like morphology. These findings suggest that, in addition to its role in activation of the cardiac genetic program, hhLIM may be the nuclear target of inductive factor for precardiac cells.

Phosphatidylinositol 3-Kinase–Akt Pathway Plays a Critical Role in Early Cardiomyogenesis by Regulating Canonical Wnt Signaling

We have recently reported that activation of phosphatidylinositol 3-kinase (PI3K) plays a critical role in the early stage of cardiomyocyte differentiation of P19CL6 cells. We here examined molecular mechanisms of how PI3K is involved in cardiomyocyte differentiation. DNA chip analysis revealed that expression levels of Wnt-3a were markedly increased and that the Wnt/beta-catenin pathway was activated temporally during the early stage of cardiomyocyte differentiation of P19CL6 cells. Activation of the Wnt/beta-catenin pathway during this period was required and sufficient for cardiomyocyte differentiation of P19CL6 cells. Inhibition of the PI3K/Akt pathway suppressed the Wnt/beta-catenin pathway by activation of glycogen synthase kinase-3beta (GSK-3beta) and degradation of beta-catenin. Suppression of cardiomyocyte differentiation by inhibiting the PI3K/Akt pathway was rescued by forced expression of a nonphosphorylated, constitutively active form of beta-catenin. These results suggest that the PI3K pathway regulates cardiomyocyte differentiation through suppressing the GSK-3beta activity and maintaining the Wnt/beta-catenin activity.

Early stage-specific inhibitions of cardiomyocyte differentiation and expression of Csx/Nkx-2.5 and GATA-4 by phosphatidylinositol 3-kinase inhibitor LY294002

Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) has been reported to block cardiomyocyte differentiation. However, at which stage PI3-kinase plays this important role and what its molecular targets are remain unknown. To answer these questions, we induced cardiomyocyte differentiation of P19CL6 mouse embryonal carcinoma cells and investigated the activation of PI3-kinase by analyzing phospho-Akt. We also treated P19CL6 cells with the PI3-kinase-specific inhibitor LY294002 either continuously or at various time points and monitored the expression of cardiac contractile proteins and transcription factors. Most cells differentiated into sarcomeric myosin heavy chain (MHC)-positive cardiomyocytes on day 16 after induction. An increase in phospho-Akt was observed after induction and was maintained throughout the differentiation. LY294002 treatment restricted to the phase from days 0 to 4 was sufficient to inhibit cardiomyocyte differentiation in a dose-dependent manner. In contrast, LY294002 treatment either from days 4 to 8 or from days 8 to 12 did not cause significant changes in sarcomeric MHC expression. LY294002 treatment from days 0 to 4 also suppressed Csx/Nkx-2.5 and GATA-4 expression. These results demonstrate that PI3-kinase becomes activated and plays a pivotal role at a very early stage of cardiomyocyte differentiation, possibly by modulating the expression of the cardiac transcription factors.

Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcription factor.

GATA-4 is a cardiac-specific member of the GATA family of zinc finger transcription factors. During embryogenesis, GATA-4 expression is detected very early in the cardiogenic area and persists later in the developing heart. Studies have shown that GATA-4 is a potent transcriptional activator of several cardiac muscle-specific genes and a key regulator of the cardiomyocyte gene program. Consistent with a role for GATA-4 in cardiomyocyte formation, inhibition of GATA-4 expression by antisense transcripts interferes with expression of cardiac muscle genes and blocks development of beating cardiomyocytes in P19 embryonic stem cells. In order to better define the function of GATA-4 in cardiogenesis, we have carried out molecular analysis of early stages of cardiomyocyte differentiation in GATA-4-deficient P19 cell lines and in P19 cells stably overexpressing GATA-4. The results indicate that GATA-4 is not required for either endodermal or mesodermal commitment or for initiation of the cardiac pathway. However, in the absence of GATA-4, differentiation is blocked at the precardiac (cardioblasts) stage and cells are lost through extensive apoptosis. In contrast, ectopic expression of GATA-4 in P19 cells accelerates cardiogenesis and markedly increases (over 10-fold) the number of terminally differentiated beating cardiomyocytes following cell aggregation. Together, these findings suggest that, in addition to its role in activation of the cardiac genetic program, GATA-4 may be the nuclear target of inductive and/or survival factors for precardiac cells.