Role In Cardiomyocyte Differentiation Research Articles

Monoamine oxidase A-dependent ROS formation modulates human cardiomyocyte differentiation through AKT and WNT activation

During embryonic development, cardiomyocytes undergo differentiation and maturation, processes that are tightly regulated by tissue-specific signaling cascades. Although redox signaling pathways involved in cardiomyogenesis are established, the exact sources responsible for reactive oxygen species (ROS) formation remain elusive. The present study investigates whether ROS produced by the mitochondrial flavoenzyme monoamine oxidase A (MAO-A) play a role in cardiomyocyte differentiation from human induced pluripotent stem cells (hiPSCs). Wild type (WT) and MAO-A knock out (KO) hiPSCs were generated by CRISPR/Cas9 genome editing and subjected to cardiomyocyte differentiation. Mitochondrial ROS levels were lower in MAO-A KO compared to the WT cells throughout the differentiation process. MAO-A KO hiPSC-derived cardiomyocytes (hiPSC-CMs) displayed sarcomere disarray, reduced α- to β-myosin heavy chain ratio, GATA4 upregulation and lower macroautophagy levels. Functionally, genetic ablation of MAO-A negatively affected intracellular Ca2+ homeostasis in hiPSC-CMs. Mechanistically, MAO-A generated ROS contributed to the activation of AKT signaling that was considerably attenuated in KO cells. In addition, MAO-A ablation caused a reduction in WNT pathway gene expression consistent with its reported stimulation by ROS. As a result of WNT downregulation, expression of MESP1 and NKX2.5 was significantly decreased in MAO-A KO cells. Finally, MAO-A re-expression during differentiation rescued expression levels of cardiac transcription factors, contractile structure, and intracellular Ca2+ homeostasis. Taken together, these results suggest that MAO-A mediated ROS generation is necessary for the activation of AKT and WNT signaling pathways during cardiac lineage commitment and for the differentiation of fully functional human cardiomyocytes.

Abstract 12841: Overexpression of Scarna20 Promotes Small Nuclear RNA Pseudouridylation and Improves Cardiomyogenesis

Introduction: Non-coding RNAs, particularly small Cajal body-associated RNAs (scaRNAs), play an important role in alternative splicing and maturation of mRNAs through pseudouridylatory modification. Our small non-coding RNA sequencing analysis has identified that scaRNA20 plays a major role in cardiomyocyte differentiation. In this study, we aim to elucidate the role of scaRNA20 during cardiac differentiation using in vitro induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMC) subjected to 3% hypoxic conditions. Hypothesis: Overexpression (OE) of scaRNA20 in iPSCs leads to enhanced cardiomyogenesis and increases the endurance properties of cardiomyocytes by maintaining contractility under hypoxic conditions. Methods and results: We have used OE and knockdown (KD) assays to delineate the role of scaRNA20 during the process of iPSC to iCMC differentiation. The video recordings of scaRNA20-OE-iCMCs and scaRNA20-KD-iCMC contractilities were measured by a novel Particle Image Velocimetry (PIV) method. Our results show that the contractility was impaired in control iCMCs whereas in scaRNA20-OE-iCMCs it was well-maintained under the hypoxic conditions. In contrast, scaRNA20-KD-iCMCs resulted in poor differentiation, they were non-functional and had an impaired contractility. Furthermore, we investigated the pseudouridylation levels of spliceosomal RNA U12 which is a direct target for scaRNA20. We found an increased level of Ψ 28 in scaRNA20-OE iCMCs (Figure A-D) . In addition, the scaRNA20 overexpression in iCMCs resulted in the upregulation of DKC1, NHP2, and NOP10 and downregulation of GAR1, COIL, and WRAP53 and these effects regulate the activation of pTP53 and STAT3 and thus activating cardiac genes. Conclusion: Taken together, our results show for the first time that modulation of iCMCs with scaRNA20 helps maintain the endurance properties of cardiomyocytes while under hypoxic conditions, and it also improves cardiomyogenesis.

Sbk2, a Newly Discovered Atrium-Enriched Regulator of Sarcomere Integrity.

Heart development relies on tight spatiotemporal control of cardiac gene expression. Genes involved in this intricate process have been identified using animals and pluripotent stem cell-based models of cardio(myo)genesis. Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal line of conditionally immortalized neonatal rat atrial myocytes (NRAMs), which allows toggling between proliferative and differentiated (ie, excitable and contractile) phenotypes in a synchronized and homogenous manner. In this study, the unique properties of conditionally immortalized NRAMs (iAMs) were exploited to identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation. Transcriptome analysis of iAM-1 cells at different stages during one cycle of differentiation and subsequent dedifferentiation identified ≈13 000 transcripts, of which the dynamic changes in expression upon cardiomyogenic differentiation mostly opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many with known (lineage-specific) functions in cardiac muscle formation. Filtering for cardiac-enriched low-abundance transcripts, identified multiple genes with an uncharacterized role during cardio(myo)genesis including Sbk2 (SH3 domain binding kinase family member 2). Sbk2 encodes an evolutionarily conserved putative serine/threonine protein kinase, whose expression is strongly up- and downregulated during iAM-1 cell differentiation and dedifferentiation, respectively. In neonatal and adult rats, the protein is muscle-specific, highly atrium-enriched, and localized around the A-band of cardiac sarcomeres. Knockdown of Sbk2 expression caused loss of sarcomeric organization in NRAMs, iAMs and their human counterparts, consistent with a decrease in sarcomeric gene expression as evinced by transcriptome and proteome analyses. Interestingly, co-immunoprecipitation using Sbk2 as bait identified possible interaction partners with diverse cellular functions (translation, intracellular trafficking, cytoskeletal organization, chromatin modification, sarcomere formation). iAM-1 cells are a relevant and suitable model to identify (lowly expressed) genes with a hitherto unidentified role in cardiomyocyte differentiation as exemplified by Sbk2: a regulator of atrial sarcomerogenesis.

Changes in chromatin accessibility landscape and histone H3 core acetylation during valproic acid-induced differentiation of embryonic stem cells

Directed differentiation of mouse embryonic stem cells (mESCs) or induced pluripotent stem cells (iPSCs) provides powerful models to dissect the molecular mechanisms leading to the formation of specific cell lineages. Treatment with histone deacetylase inhibitors can significantly enhance the efficiency of directed differentiation. However, the mechanisms are not well understood. Here, we use CUT&RUN in combination with ATAC-seq to determine changes in both histone modifications and genome-wide chromatin accessibility following valproic acid (VPA) exposure. VPA induced a significant increase in global histone H3 acetylation (H3K56ac), a core histone modification affecting nucleosome stability, as well as enrichment at loci associated with cytoskeletal organization and cellular morphogenesis. In addition, VPA altered the levels of linker histone H1 subtypes and the total histone H1/nucleosome ratio indicative of initial differentiation events. Notably, ATAC-seq analysis revealed changes in chromatin accessibility of genes involved in regulation of CDK serine/threonine kinase activity and DNA duplex unwinding. Importantly, changes in chromatin accessibility were evident at several key genomic loci, such as the pluripotency factor Lefty, cardiac muscle troponin Tnnt2, and the homeodomain factor Hopx, which play critical roles in cardiomyocyte differentiation. Massive parallel transcription factor (TF) footprinting also indicates an increased occupancy of TFs involved in differentiation toward mesoderm and endoderm lineages and a loss of footprints of POU5F1/SOX2 pluripotency factors following VPA treatment. Our results provide the first genome-wide analysis of the chromatin landscape following VPA-induced differentiation in mESCs and provide new mechanistic insight into the intricate molecular processes that govern departure from pluripotency and early lineage commitment.

Auto/paracrine factors and early Wnt inhibition promote cardiomyocyte differentiation from human induced pluripotent stem cells at initial low cell density

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) have received increasing attention for their clinical use. Many protocols induce cardiomyocytes at an initial high cell density (confluence) to utilize cell density effects as hidden factors for cardiomyocyte differentiation. Previously, we established a protocol to induce hiPSC differentiation into cardiomyocytes using a defined culture medium and an initial low cell density (1% confluence) to minimize the hidden factors. Here, we investigated the key factors promoting cardiomyocyte differentiation at an initial low cell density to clarify the effects of cell density. Co-culture of hiPSCs at an initial low cell density with those at an initial high cell density showed that signals secreted from cells (auto/paracrine factors) and not cell–cell contact signals, played an important role in cardiomyocyte differentiation. Moreover, although cultures with initial low cell density showed higher expression of anti-cardiac mesoderm genes, earlier treatment with a Wnt production inhibitor efficiently suppressed the anti-cardiac mesoderm gene expression and promoted cardiomyocyte differentiation by up to 80% at an initial low cell density. These results suggest that the main effect of cell density on cardiomyocyte differentiation is inhibition of Wnt signaling at the early stage of induction, through auto/paracrine factors.

Transcriptome analysis of conditionally immortalized atrial myocytes: identification of a novel atrium-enriched protein involved in sarcomere assembly and maintenance

Abstract Background Heart development relies on the tight spatiotemporal control of cardiac gene expression. Genes involved in these processes have been identified using mainly (transgenic) animals models and pluripotent stem cell-derived cardiomyocytes (CMs). Recently, the repertoire of cardiomyocyte differentiation models has been expanded with iAM-1, a monoclonal cell line of conditionally immortalized neonatal rat atrial myocytes (NRAMs) which allows toggling between proliferative and differentiated (i.e. excitable and contractile) phenotypes in a synchronized and homogenous manner. Purpose To identify and characterize (lowly expressed) genes with an as-of-yet uncharacterized role in cardiomyocyte differentiation, dedifferentiation and proliferation by exploiting the unique properties of conditionally immortalized NRAMs (iAMs). Methods and results RNA sequencing was performed during a full cycle of iAM-1 differentiation and subsequent dedifferentiation, identifying ±13,000 transcripts, of which the dynamic expressional changes during cardiomyogenic differentiation in most cases opposed those during dedifferentiation. Among the genes whose expression increased during differentiation and decreased during dedifferentiation were many genes with a known (lineage-specific) role in cardiac muscle formation, thereby confirming the relevance of iAMs as cardiomyogenic differentiation model. Filtering for cardiomyocyte-enriched low abundancy transcripts, resulted in the identification of an uncharacterized protein, which is highly conserved among Nephrozoa and up- and downregulated during cardiomyocyte differentiation and dedifferentiation, respectively. In neonatal and adult rats, this protein is muscle-specific, highly atrium-enriched and localized around the C-zone of cardiac sarcomeres. Lentiviral shRNA-mediated knockdown resulted in loss of sarcomeric organization in both NRAMs and iAMs. Neither knockdown nor overexpression of this protein affected the electrophysiological properties of differentiated iAM monolayers. Conclusions iAM-1 cells offer a relevant model to identify and characterize novel (low abundancy) genes involved in cardiomyocyte (de)differentiation as exemplified by the identification a novel uncharacterized protein that is muscle-specific, highly atrium-enriched, localized around the C-zone of cardiac sarcomeres and plays a specific role in atrial sarcomerigenesis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Netherlands Organisation for Health Research and Development (ZonMw) Leiden Regenerative Medicine Platform Holding project with number (LRMPH) Figure 1. (A) Experimental setup. At the indicated timepoints iAM-1 cells were fixed for immunostaining and RNA extraction for transcriptome analysis. (B) Immunochemical staining of iAM-1 cells for the proliferation marker Ki-67 and the Z-line marker sarcomeric α-actinin. (C & D) Immunohistological double stainings of longitudinal sections of neonatal rat hearts for the uncharacterized protein (GOI 1) and the sarcomeric protein cardiac troponin I (TNNI3). LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle. Scale bar, 250 μm.

Pten Regulates Cardiomyocyte Differentiation by Modulating Non-CG Methylation via Dnmt3.

The regulation of cardiomyocyte differentiation is a fundamental aspect of cardiac development and regenerative medicine. PTEN plays important roles during embryonic development. However, its role in cardiomyocyte differentiation remains unknown. In this study, a low‐cost protocol for cardiomyocyte differentiation from mouse embryonic stem cells (ESCs) is presented and it is shown that Pten deletion potently suppresses cardiomyocyte differentiation. Transcriptome analysis shows that the expression of a series of cardiomyocyte marker genes is downregulated in Pten−/− cardiomyocytes. Pten ablation induces Dnmt3b expression via the AKT/FoxO3a pathway and regulates the expression of a series of imprinted genes, including Igf2. Double knockout of Dnmt3l and Dnmt3b rescues the deficiency of cardiomyocyte differentiation of Pten−/− ESCs. The DNA methylomes from wild‐type and Pten−/− embryoid bodies and cardiomyocytes are analyzed by whole‐genome bisulfite sequencing. Pten deletion significantly promotes the non‐CG (CHG and CHH) methylation levels of genomic DNA during cardiomyocyte differentiation, and the non‐CG methylation levels of cardiomyocyte genes and Igf2 are increased in Pten−/− cardiomyocytes. Igf2 or Igf1r deletion also suppresses cardiomyocyte differentiation through the MAPK/ERK signaling pathway, and IGF2 supplementation partially rescues the cardiomyocyte differentiation. Finally, Pten conditional knockout mice are generated and the role of PTEN in cardiomyocyte differentiation is verified in vivo.

Smad4 regulates the nuclear translocation of Nkx2-5 in cardiac differentiation

Bmp plays an important role in cardiomyocyte differentiation, but the function of Smad4 in Bmp signaling remains elusive. Here, we show that disruption of the Smad4 gene in cardiac progenitors expressing Sfrp5 led to embryonic lethality with hypoplastic heart formation. Although the expression of Nkx2-5 is regulated by Bmp signaling, expression of Nkx2-5 was weakly detected in the mutant heart. However, the nuclear translocation of Nkx2-5 was impaired. Expression of CK2 or PP1, which could alter the phosphorylation status of the NLS of Nkx2-5, was not affected, but Nkx2-5 was found to bind to Smad4 by co-immunoprecipitation experiments. Introduction of Smad4 into cells derived from Smad4 conditional knockout embryonic hearts restored the nuclear localization of Nkx2-5, and exogenous Nkx2-5 failed to translocate into the nucleus of Smad4-depleted fibroblasts. These results suggest that Smad4 plays an essential role in cardiomyocyte differentiation by controlling not only transcription but also the nuclear localization of Nkx2-5.

Nucleic‐Acid Mediated Expression of Cardiopoietic Markers in Human Umbilical Cord‐Derived Mesenchymal Stem Cells

The management of patients with heart failure focuses on halting the progression of disease. To date, no therapeutic approach has been able to restore cardiac function to its original baseline state. Cell therapies, such as cardiopoietic (CP) stem cells, have shown to be promising platforms to restore cardiac function. The CP platform enhances the cardioregenerative potency of mesenchymal stem cells (MSCs). CP cells are characterized by the robust expression and nuclear translocation of cardiac transcription factors such Mesoderm Posterior Basic Helix‐Loop‐Helix Transcription Factor 1 (MESP1), early cardiac transcription factor Nkx2.5, myocyte enhancer factor 2C (Mef2C), and zinc finger‐containing transcription factor GATA4. Clinical trials using CP cells showed signs of safety and efficacy in improving cardiac function in patients with heart failure. However, the costs, time, and complexities associated with the manufacturing process have precluded the adoption of the therapy as a large‐scale clinical practice. In response, we adopted a microencapsulated‐modified‐messenger RNA (M3RNA) platform as a cost‐effective and time‐saving alternative to induce high expression of cardiopoietic markers. We hypothesized that the transfection of human umbilical cord‐derived mesenchymal stem cells (hUC‐MSCs) with M3RNA encoding mesodermal and/or cardiac transcription factor(s) will induce the expression of cardiopoietic markers (MESP1, Nkx2.5, Mef2C, and GATA4). hUC‐MSCs were transfected with M3RNA encoding Brachyury (this protein plays a key role in cardiomyocyte differentiation) and MESP1, Nkx2.5, Mef2C, and GATA4 using the transfection reagent Lipofectamine STEM. Protein expression was measured and quantified using fluorescence microscopy and flow cytometry. hUC‐MSCs transfected with M3RNA encoding mesodermal and/or cardiac transcription factor(s) demonstrated rapid protein expression at 4 hours post‐transfection that reached peak levels between 24 hours and 48 hours post‐transfection. Immunocytochemistry demonstrated similar morphology between transfected and non‐transfected cells. Thus, hUC‐MSCs can be transfected with M3RNA with high efficiency to produce rapid and predictable protein expression in vitro. We here demonstrate a simple, cost‐effective, and time‐saving approach to induce the expression of cardiopoietic markers in UC‐MSCs using M3RNA, which can serve as a highly translatable platform to circumvent many burdens associated with current strategies and development of cell‐based therapies for future clinical trials and practice.Support or Funding InformationMayo Clinic Center for Regenerative Medicine, Van Cleve Cardiac Regenerative Medicine Program, Marriott Foundation, Michael S. and Mary Sue Shannon Family, NIH NHLBI HL134664, Mayo Clinic Regenerative Sciences Post‐Baccalaureate Training Program

P3503Transcriptome changes in atrial myocytes during the transition from a proliferative into a contractile phenotype and vice versa

Abstract Background iAM-1 cells are conditionally immortalized neonatal rat atrial myocytes allowing toggling between proliferative and contractile phenotypes by a single-component change in culture medium composition. In the absence of proliferation stimuli, the cells synchronously differentiate into functional cardiomyocytes. Following re-expression of the immortalization factor, the fully differentiated iAM-1 cells dedifferentiate and start to proliferate again. Purpose The aim of our study was to investigate the changes in gene expression profile in iAM-1 cells during one round of cardiac differentiation and dedifferentiation in order to identify potential (new) regulators of atrial myocyte differentiation and proliferation. Methods RNA sequencing was performed on iAM-1 cells at 9 time points during one cycle of cardiomyogenic differentiation and dedifferentiation (20 million 150-bp paired-end reads per sample, 4 samples per time point). The resulting sequence data were analysed by EdgeR. Hierarchical clustering and principle component analysis were performed in R. GO category enrichment was determined using DAVID. Results Approximately 13,000 genes were extracted from the RNA sequencing analysis. In general, dynamic changes in mRNA levels during the transition from a proliferative into a contractile phenotype opposed those that occurred when differentiated iAM-1 were re-exposed to proliferation stimuli. These inverse trends were most evident for genes involved in cell cycle progression, DNA replication, sarcomere formation and cardiac contraction. Moreover, the RNA-SEQ data allowed us to make a distinction between genes contributing to the early and late phases of cardiomyogenic differentiation and dedifferentiation and to identify similarities and differences in the transcriptional programs underlying the cardiomyogenic differentiation of iAM-1 cells versus those of embryonic stem cells and induced pluripotent stem cells. The transcriptome analysis also unveiled several genes with potentially important and previously unrecognized roles in cardiomyocyte differentiation and proliferation. iAM-1 differentiation and dedifferention Conclusions Due to their ability to homogenously and synchronously differentiate and dedifferentiate, iAM-1 cells offer unique new insights into the transcriptional regulation of cardiomyocyte differentiation and proliferation.

P5725Identification of novel cardiomyogenic factors by transcriptome analysis of conditionally immortalized atrial myocytes

Abstract Background Cardiac development involves the properly timed expression of cardiomyogenic differentiation factors (CDFs). CDFs have mainly been discovered using animal models and, more recently, pluripotent stem cell-derived cardiomyocytes (PSC-CMCs). These models are, however, laborious, time-consuming and costly. Also, cardiomyogenic differentiation of CMCs is heterogeneous and yields phenotypically immature CMCs. Recently, our research group generated a monoclonal line of conditionally immortalized atrial myocytes, called iAM-1. After removal of the proliferation stimulus these cells spontaneously and synchronously differentiate into mature atrial myocytes, making them ideally suited for transcriptome analysis and the discovery of novels factors involved in cardiomyogenic differentiation. Methods Whole transcriptome analysis of iAM-1 cells was performed at 9 different time points during cardiomyogenic differentiation and subsequent dedifferentiation by RNA sequencing. Six genes upregulated during cardiomyogenic differentiation were selected for knockdown studies in differentiating iAM-1 cells. Each of these genes was targeted by a bicistronic lentiviral vector (LV) driving expression of a specific short hairpin RNA (shRNA) and of enhanced green fluorescent protein (eGFP). Knockdown effects during cardiomyogenic differentiation were studied by immunocytology. The LVs were also used in primary neonatal atrial and ventricular rat cardiomyocytes to study the role of the selected genes in cardiomyocyte homeostasis. Results Whole transcriptome analysis of differentiating iAM-1 cells identified the dynamic expression levels of ± 13.000 genes, including the expected profile for genes known to play a role in atrial myocyte differentiation, like Nkx2–5, Tbx3, Tbx5 and Nppa. Six genes with an unknown role in cardiomyocyte differentiation and homeostasis were selected based on significant upregulation during iAM-1 differentiation, substantial mRNA levels and selective expression in cardiac tissue. Inhibiting gene expression by lentiviral RNA interference resulted for Nkx2–5 as well as for 3 out of 6 target genes in disturbed iAM-1 differentiation, as evinced by loss of sarcomeric cross-striations. Similar effects were observed in shRNA-expressing (i.e. eGFP-positive) primary atrial and ventricular neonatal rat myocytes. Taken together, these results highlight the importance of these novel genes during cardiomyogenic differentiation and homeostasis in atrial as well as ventricular myocytes. Conclusions Transcriptome analysis of cardiomyogenic differentiation in conditionally immortalized atrial myocytes combined with genetic knockdown experiments led to the identification of several novel factors involved in the differentiation and homeostasis of atrial and ventricular myocytes. These results highlight the suitability of iAM-1 as model for fundamental research of cardiomyogenic differentiation. Acknowledgement/Funding ZonMW

Down-Regulation of a Male-Specific H3K4 Demethylase, KDM5D, Impairs Cardiomyocyte Differentiation.

Despite the small number of Y chromosome genes, their adequate expression is required for regulation of transcription, translation, and protein stability in males, not just for sex determination. In addition to the role in male fertility, the Y chromosome has a significant role in the development and sexual dimorphism of healthy and disease phenotypes. We observed that KDM5D along with its X-counterpart, KDM5C, are up-regulated during the cardiac mesoderm stage of development. Down-regulation of KDM5D using siRNA resulted in accumulation of differentiating cells in the S-phase of the cell cycle and impaired progression to cardiomyocytes as reflected by an altered expression pattern of cardiac progenitor specific markers. Furthermore, while control cells started spontaneous beating at a normal physiological range on day 7 of differentiation induction, no spontaneous beating was observed in KDM5D down-regulated cells. Interestingly, the knockdown of KDM5D had no significant effect on the expression level of its X-counterpart, KDM5C. Thus, we suggest that KDM5D, in cooperation with its X homologue as a dose-sensitive gene, may have an important role in cardiomyocyte differentiation. Our study presents further evidence on the contribution of Y chromosome genes to sex-dependent development outside of sex determination.

Copy number variants in hypoplastic right heart syndrome.

Hypoplastic right heart syndrome (HRHS) is a rare congenital defect characterized by underdeveloped and malformed structures of the right heart. Familial recurrence of HRHS indicates genetic factors contribute to its etiology. Our study investigates the presence of copy number variants (CNVs) in HRHS cases. We genotyped 42 HRHS cases identified from live births throughout California (2003-2010) using the Illumina HumanOmni2.5-8 array. We identified 14 candidate CNVs in 14 HRHS cases (33%) based on the genes included in the CNVs and their functions. Duplications overlapping part of ERBB4 were identified in two unrelated cases. ERBB4 is a neuregulin receptor with a pivotal role in cardiomyocyte differentiation and heart development. We also described a 7.5 Mb duplication at 16q11-12. Multiple genes in the duplicated region have previously been linked to heart defects and cardiac development, including RPGRIP1L, RBL2, SALL1, and MYLK3. Of the 14 validated CNVs, we identified four CNVs in close proximity to genes linked to the Wnt signaling pathway. This study expands on our previous work supporting the role of genetics in HRHS. We identified CNVs affecting crucial genes and signaling pathways involved in right heart development. ERBB4 and duplication of the 16q11-12 region are important areas for future investigation.

β-Catenin stabilization promotes proliferation and increase in cardiomyocyte number in chick embryonic epicardial explant culture.

Cardiomyocyte (CM) differentiation from proepicardial organ- (PEO) and embryonic epicardium (eEpi)-derived cells or EPDCs in a developing heart emerges as a wide interest in purview of cardiac repair and regenerative medicine. eEpi originates from the precursor PEO and EPDCs, which contribute to several cardiac cell types including smooth muscle cells, fibroblasts, endothelial cells, and CMs during cardiogenesis. Here in this report, we have analyzed several cardiac lineage-specific marker gene expressions between PEO and eEpi cells. We have found that PEO-derived cells show increased level of CM lineage-specific marker gene expression compared to eEpi cells. Moreover, Wnt signaling activation results in increased level of CM-specific marker gene expression in both PEO and eEpi cells in culture. Interestingly, Wnt signaling activation also increases the number of proliferating and sarcomeric myosin (Mf20)-positive cells in eEpi explant culture. Together, this data suggests that eEpi cells as a source for CM differentiation and Wnt signaling mediator, β-catenin, might play an important role in CM differentiation from eEpi cells in culture.

5730Initiation of the first heart beat and its role in cardiomyocyte differentiation

5730Initiation of the first heart beat and its role in cardiomyocyte differentiation

MTORC1 and mTORC2 play different roles in regulating cardiomyocyte differentiation from embryonic stem cells.

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase and functions through two distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2), with their key components Raptor and Rictor, to play crucial roles in cellular survival and growth. However, the roles of mTORC1 and mTORC2 in regulating cardiomyocyte differentiation from mouse embryonic stem (mES) cells are not clear. In this study, we performed Raptor or Rictor knockdown experiments to investigate the roles of mTORC1 and mTORC2 in cardiomyocyte differentiation. Ablation of Raptor markedly increased the number of cardiomyocytes derived from mES cells with well-organized myofilaments. Expression levels of brachyury (mesoderm protein), Nkx2.5 (cardiac progenitor cell protein), and α-Actinin (cardiomyocyte marker) were increased in Raptor knockdown cells. In contrast, loss of Rictor prevented cardiomyocyte differentiation. The dual ablation of Raptor and Rictor also decreased the number of cardiomyocytes. The two complexes exerted a regulatory mechanism in such a manner that knockdown of Raptor/mTORC1 resulted in a decreased phosphorylation of Rictor (Thr1135), which subsequently activated Rictor/mTORC2 in the differentiation of mES cells into cardiomyocytes. In conclusion, mTORC1 and mTORC2 played different roles in cardiomyocyte differentiation from mES cells in vitro. The activation of Rictor/mTORC2 was critical for facilitating cardiomyocyte differentiation from mES cells. Thus, this complex may be a promising target for regulating myocardial differentiation from embryonic stem cells or induced pluripotent stem cells.

SMYD1, an SRF-Interacting Partner, Is Involved in Angiogenesis.

Previous studies have demonstrated that Smyd1 plays a critical role in cardiomyocyte differentiation, cardiac morphogenesis and myofibril organization. In this study, we uncovered a novel function of Smyd1 in the regulation of endothelial cells (ECs). Our data showed that Smyd1 is expressed in vascular endothelial cells, and knockdown of SMYD1 in endothelial cells impairs EC migration and tube formation. Furthermore, Co-IP and GST pull-down assays demonstrated that SMYD1 is associated with the Serum Response Factor (SRF). EMSA assays further showed that SMYD1 forms a complex with SRF and enhances SRF DNA binding activity. Our studies indicate that SMYD1 serves as an SRF-interacting protein, enhances SRF DNA binding activity, and is required for EC migration and tube formation to regulate angiogenesis.

MicroRNA-375 overexpression influences P19 cell proliferation, apoptosis and differentiation through the Notch signaling pathway.

Our previous study reported that microRNA-375 (miR-375) is significantly upregulated in ventricular septal myocardial tissues from 22-week-old fetuses with ventricular septal defect as compared with normal controls. In the present study, the specific effects of miR-375 on P19 cell differentiation into cardiomyocyte-like cells were investigated. Stable P19 cell lines overexpressing miR-375 or containing empty vector were established, which could be efficiently induced into cardiomyocyte-like cells in the presence of dimethyl sulfoxide in vitro. miR-375 overexpression was verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Cell proliferation was determined according to total cell counts; cell cycle distribution and apoptosis levels were examined using flow cytometry. Apoptosis-related morphological changes were observed using Hoechst staining and fluorescence microscopy. During P19 cell differentiation, the cardiomyogenesis-related mRNAs (cardiac troponin T, GATA binding protein 4, myocyte-specific enhancer factor 2C) and mRNAs involved in the Notch signaling pathway (Notch2, Delta-like 1 and hes family bHLH transcription factor 1) were detected at days 0, 4, 6 and 10. Their differential expression was examined using RT-qPCR; the apoptosis-related genes BAX and Bcl-2 were also detected using this method. The corresponding proteins were evaluated by western blotting. Compared with the control group, miR-375 overexpression inhibited proliferation but promoted apoptosis in P19 cells, and the associated mRNAs and proteins were decreased during differentiation. miR-375 has an important role in cardiomyocyte differentiation, and can disrupt this process via the Notch signaling pathway. The present findings contribute to the understanding of the mechanisms of congenital heart disease and facilitate the development of new gene therapies.

Sublethal caspase activation promotes generation of cardiomyocytes from embryonic stem cells.

Generation of new cardiomyocytes is critical for cardiac repair following myocardial injury, but which kind of stimuli is most important for cardiomyocyte regeneration is still unclear. Here we explore if apoptotic stimuli, manifested through caspase activation, influences cardiac progenitor up-regulation and cardiomyocyte differentiation. Using mouse embryonic stem cells as a cellular model, we show that sublethal activation of caspases increases the yield of cardiomyocytes while concurrently promoting the proliferation and differentiation of c-Kit+/α-actininlow cardiac progenitor cells. A broad-spectrum caspase inhibitor blocked these effects. In addition, the caspase inhibitor reversed the mRNA expression of genes expressed in cardiomyocytes and their precursors. Our study demonstrates that sublethal caspase-activation has an important role in cardiomyocyte differentiation and may have significant implications for promoting cardiac regeneration after myocardial injury involving exogenous or endogenous cell sources.

Dgcr8 is Indispensable for Cardiac Lineage Specification in Embryonic Stem Cells

Objective: microRNAs have been shown to play important roles in cellular behavior and lineage specification including cardiogenic differentiation. However, full understanding of their roles in cardiomyocyte differentiation has been impeded due to lack of proper cellular model. Here, we used an embryonic stem cell (ESC) that is lacking the important microprocessor Dgcr8 (or Pasha), which allows the introduction of individual miRNAs to study their role in cardiac differentiation and for more precise target selection. Methods: Dgcr8 KO ESC was cultured in LIF-supplemented ESC medium with mouse embryonic fibroblast feeders and cardiac differentiation was induced using an embryonic body-based differentiation protocol. Differentiation was monitored by measuring mRNA and protein levels of cardiogenic markers and heterochromatin changes using immunofluorescent staining and semi-quantitative PCR. Results and conclusion: We showed that Dgcr8 KO ESCs indeed are lacking a large population of small RNAs, including but not limited to mature microRNAs. The KO cells had a lower proliferation rate and were unable to differentiate into the cardiac lineage. To our surprise, in addition to a defect in microRNA processing, Dgcr8 KO embryonic stem cells are unable to form proper heterochromatin and to inactivate genotoxic centromeric repetitive elements. Our results argue that, in addition to controlling microRNA processing, Dgcr8 may serve a previously unrecognized role in heterochromatin silencing.