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

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
P R R Van Gorp ◽  
J Zhang ◽  
J Liu ◽  
R Tsonaka ◽  
H Mei ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Heart Development ◽  
Troponin I ◽  
Specific Role ◽  
Neonatal Rat ◽  
Cardiomyocyte Differentiation ◽  
Atrial Myocytes ◽  
Uncharacterized Protein ◽  
Cardiomyogenic Differentiation ◽  
Cardiac Sarcomeres

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. FUNDunding 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.

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

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
J Liu ◽  
R Tsonaka ◽  
H Mei ◽  
B Akerboom ◽  
M Schalij ◽  
...  
Keyword(s):  
Stem Cells ◽  
Rna Sequencing ◽  
Cardiac Contraction ◽  
Neonatal Rat ◽  
Cardiomyocyte Differentiation ◽  
Atrial Myocytes ◽  
Contractile Phenotype ◽  
Cardiomyogenic Differentiation ◽  
Differentiation And Proliferation ◽  
Differentiation And Dedifferentiation

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

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
P R R Van Gorp ◽  
J Liu ◽  
S O Dekker ◽  
J Zhang ◽  
M J Schalij ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Mrna Levels ◽  
Atrial Myocytes ◽  
Rat Cardiomyocytes ◽  
Cardiomyogenic Differentiation ◽  
Whole Transcriptome Analysis ◽  
Whole Transcriptome ◽  
Six Genes

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

Application potential of three-dimensional silk fibroin scaffold using mesenchymal stem cells for cardiac regeneration

2021 ◽  
pp. 088532822110185
Author(s):  
Yuksel Cetin ◽  
Merve G Sahin ◽  
Fatma N Kok
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Silk Fibroin ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Cardiac Tissue Engineering ◽  
Swelling Ratio ◽  
Differentiation Potential ◽  
Cardiomyogenic Differentiation

Cardiac tissue engineering focusing on biomaterial scaffolds incorporating cells from different sources has been explored to regenerate or repair damaged area as a lifesaving approach.The aim of this study was to evaluate the cardiomyocyte differentiation potential of human adipose mesenchymal stem cells (hAD-MSCs) as an alternative cell source on silk fibroin (SF) scaffolds for cardiac tissue engineering. The change in surface morphology of SF scaffolds depending on SF concentration (1–6%, w/v) and increase in their porosity upon application of unidirectional freezing were visualized by scanning electron microscopy (SEM). Swelling ratio was found to increase 2.4 fold when SF amount was decreased from 4% to 2%. To avoid excessive swelling, 4% SF scaffold with swelling ratio of 10% (w/w) was chosen for further studies.Biodegradation rate of SF scaffolds depended on enzymatic activity was found to be 75% weight loss of SF scaffolds at the day 14. The phenotype of hAD-MSCs and their multi-linage potential into chondrocytes, osteocytes, and adipocytes were shown by flow cytometry and immunohistochemical staining, respectively.The viability of hAD-MSCs on 3D SF scaffolds was determined as 90%, 118%, and 138% after 1, 7, and 14 days, respectively. The use of 3D SF scaffolds was associated with increased production of cardiomyogenic biomarkers: α-actinin, troponin I, connexin 43, and myosin heavy chain. The fabricated 3D SF scaffolds were proved to sustain hAD-MSCs proliferation and cardiomyogenic differentiation therefore, hAD-MSCs on 3D SF scaffolds may useful tool to regenerate or repair damaged area using cardiac tissue engineering techniques.

Abstract 124: Function of the Cytoskeletal Proteins Alpha-catenins in Myocardial Growth Control

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Jifen Li ◽  
Sarah Carrante ◽  
Roslyn Yi ◽  
Frans van Roy ◽  
Glenn L. Radice
Keyword(s):  
Heart Development ◽  
Growth Control ◽  
Cytoskeletal Proteins ◽  
Neonatal Rat ◽  
Nuclear Accumulation ◽  
Mouse Heart ◽  
Cardiomyocyte Proliferation ◽  
Rat Cardiomyocytes ◽  
Neonatal Cardiomyocytes

Introduction: Mammalian heart possesses regenerative potential immediately after birth and lost by one week of age. The mechanisms that govern neonatal cardiomyocyte proliferation and regenerative capacity are poorly understood. Recent reports indicate that Yap-Tead transcriptional complex is necessary and sufficient for cardiomyocyte proliferation. During postnatal development, N-cadherin/catenin adhesion complex becomes concentrated at termini of cardiomyocytes facilitating maturation of a specialized intercellular junction structure, the intercalated disc (ICD). This process coincides with the time cardiomyocytes exit cell cycle soon after birth. Hypothesis: We hypothesize that coincident with maturation of ICD α-catenins sequester transcriptional coactivator Yap in cytosol thus preventing activation of genes critical for cardiomyocyte proliferation. Methods: We deleted αE-catenin / αT-catenin genes (α-cat DKO) in perinatal mouse heart and knockdown (KD) α-catenins in neonatal rat cardiomyocytes to study functional impact of α-catenins ablation on ICD maturation. Results: We previously demonstrated that adult α-cat DKO mice exhibited decrease in scar size and improved function post myocardial infarction. In present study, we investigated function of α-catenins during postnatal heart development. We found increase in the number of Yap-positive nuclei (58.7% in DKO vs. 35.8 % in WT, n=13, p<0.001) and PCNA (53.9% in DKO vs. 47.8%, n=8, p<0.05) at postnatal day 1 and day 7 of α-cat DKO heart, respectively. Loss of α-catenins resulted in reduction in N-cadherin at ICD at day 14. We observed an increase number of mononucleated myocytes and decrease number of binucleated myocytes in α-cat DKO compared to controls. Using siRNA KD, we were able to replicate α-cat DKO proliferative phenotype in vitro. The number of BrdU-positive cells was decreased in α-cat KD after interfering with Yap expression (2.91% in α-cat KD vs. 2.02% in α-cat/Yap KD, n>2500 cells, p<0.05), suggesting α-catenins regulate cell proliferation through Yap in neonatal cardiomyocytes. Conclusion: Our results suggest that maturation of ICD regulates α-catenin-Yap interactions in cytosol, thus preventing Yap nuclear accumulation and cardiomyocyte proliferation.

Abstract 1451: Hyperpolarization Induces Differentiation Of Human Cardiomyocyte Progenitor Cells

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Piet van Vliet ◽  
Teun P de Boer ◽  
Marcel A van der Heyden ◽  
Joost P Sluijter ◽  
Pieter A Doevendans ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Molecular Mechanisms ◽  
Troponin I ◽  
Lucifer Yellow ◽  
Myogenic Differentiation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Cardiomyogenic Differentiation ◽  
Low Potassium ◽  
Calcineurin Signaling

Background: Recently, we have isolated cardiomyocyte progenitor cells (hCMPCs) from human fetal and adult hearts. These cells differentiate into spontaneously beating cardiomyocytes when stimulated with 5-azacytidine. Subsequent stimulation by TGFβ enhances differentiation efficiency to nearly 100%. The underlying molecular mechanisms mediating this cardiomyogenic differentiation are not understood. In skeletal myoblasts, hyperpolarization-mediated activation of calcineurin signaling is crucial for myogenic differentiation. In hCMPCs, whole-cell patch clamp recordings showed a hyperpolarized membrane potential after stimulation with TGFβ or BMP. We hypothesized that hyperpolarization and calcineurin signaling regulate cardiomyogenic differentiation of hCMPCs after TGFβ stimulation. Methods & Results: To test whether hyperpolarization initiates cardiomyogenic differentiation, hyperpolarization was induced by 1) co-culture of hCMPCs with HEK 293 cells overexpressing a Kir2.1GFP fusion protein (KWGF cells) or 2) culture of hCMPCs overnight in medium containing low potassium concentrations. During co-culture, Lucifer Yellow dye injection in KWGF cells spread to neighboring hCMPCs, indicating cellular coupling. This resulted in stable hyperpolarization in hCMPCs, which could be blocked by addition of the gap junction inhibitor halothane. After two weeks, qPCR analysis revealed increased expression of cardiac sarcomeric genes in the hCMPCs in a dose-dependent manner. Induction of hyperpolarization by culturing hCMPCs with low potassium concentrations also resulted in increased expression of cardiac genes and the formation of spontaneously beating cells. Immunofluorescence staining revealed striated patterns of troponin I and α-actinin. Interestingly, hyperpolarization also increased intracellular calcium levels in hCMPCs, as measured by ratiometric imaging of indo-1 fluorescence, and, subsequently, a time-dependent increase in NFAT-Luciferase reporter activity, indicating activation of the calcineurin pathway. Conclusion: TGFβ and/or BMP-mediated hyperpolarization of hCMPCs induces calcineurin-mediated cardiomyogenic differentiation.

Developmental changes in passive stiffness and myofilament Ca2+ sensitivity due to titin and troponin-I isoform switching are not critically triggered by birth

2006 ◽  
Vol 291 (2) ◽  
pp. H496-H506 ◽  
Author(s):  
Martina Krüger ◽  
Thomas Kohl ◽  
Wolfgang A. Linke
Keyword(s):  
Guinea Pig ◽  
Heart Development ◽  
Troponin I ◽  
Collagen Matrix ◽  
Passive Stiffness ◽  
Adult Rats ◽  
Functional Changes ◽  
Fetal Rat ◽  
Isoform Switching ◽  
Species Specific

The giant protein titin, a major contributor to myocardial mechanics, is expressed in two main cardiac isoforms: stiff N2B (3.0 MDa) and more compliant N2BA (>3.2 MDa). Fetal hearts of mice, rats, and pigs express a unique N2BA isoform (∼3.7 MDa) but no N2B. Around birth the fetal N2BA titin is replaced by smaller-size N2BA isoforms and N2B, which predominates in adult hearts, stiffening their sarcomeres. Here we show that perinatal titin-isoform switching and corresponding passive stiffness (STp) changes do not occur in the hearts of guinea pig and sheep. In these species the shift toward “adult” proportions of N2B isoform is almost completed by midgestation. The relative contributions of titin and collagen to STp were estimated in force measurements on skinned cardiac muscle strips by selective titin proteolysis, leaving the collagen matrix unaffected. Titin-based STp contributed between 42% and 58% to total STp in late-fetal and adult sheep/guinea pigs and adult rats. However, only ∼20% of total STp was titin based in late-fetal rat. Titin-borne passive tension and the proportion of titin-based STp generally scaled with the N2B isoform percentage. The titin isoform transitions were correlated to a switch in troponin-I (TnI) isoform expression. In rats, fetal slow skeletal TnI (ssTnI) was replaced by adult carciac TnI (cTnI) shortly after birth, thereby reducing the Ca2+ sensitivity of force development. In contrast, guinea pig and sheep coexpressed ssTnI and cTnI in fetal hearts, and skinned fibers from guinea pig showed almost no perinatal shift in Ca2+ sensitivity. We conclude that TnI-isoform and titin-isoform switching and corresponding functional changes during heart development are not initiated by birth but are genetically programmed, species-specific regulated events.

scholarly journals Notch Signaling Commits Mesoderm to the Cardiac Lineage

2020 ◽  
Author(s):  
Evan S. Bardot ◽  
Bharati Jadhav ◽  
Nadeera Wickramasinghe ◽  
Amélie Rezza ◽  
Michael Rendl ◽  
...  
Keyword(s):  
Notch Signaling ◽  
Heart Development ◽  
Cardiomyocyte Differentiation ◽  
Mouse Development ◽  
Mesoderm Specification ◽  
Cardiac Lineage ◽  
Underlying Mechanisms ◽  
Early Mouse ◽  
Differentiation Efficiency ◽  
Cardiac Mesoderm

AbstractDuring development multiple progenitor populations contribute to the formation of the four-chambered heart and its diverse lineages. However, the underlying mechanisms that result in the specification of these progenitor populations are not yet fully understood. We have previously identified a population of cells that gives rise selectively to the heart ventricles but not the atria. Here, we have used this knowledge to transcriptionally profile subsets of cardiac mesoderm from the mouse embryo and have identified an enrichment for Notch signaling components in ventricular progenitors. Using directed differentiation of human pluripotent stem cells, we next investigated the role of Notch in cardiac mesoderm specification in a temporally controlled manner. We show that transient Notch induction in mesoderm increases cardiomyocyte differentiation efficiency, while maintaining cardiomyocytes in an immature state. Finally, our data suggest that Notch interacts with WNT to enhance commitment to the cardiac lineage. Overall, our findings support the notion that key signaling events during early heart development are critical for proper lineage specification and provide evidence for early roles of Notch and WNT during mouse and human heart development.Summary statementEarly fate decisions are dictated by the embryonic signaling environment. We show that Notch signaling is active during early mouse development and that activating Notch in human cardiac mesoderm enhances cardiomyocyte differentiation efficiency.

Abstract 1509: Inducible TBX3 Overexpression As A Tool For Biopacemaker Engineering

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Gerard J Boink ◽  
Martijn L Bakker ◽  
Arie O Verkerk ◽  
Diane Bakker ◽  
Jacques M de Bakker ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Heart Development ◽  
Single Gene ◽  
Neonatal Rat ◽  
Pcr Analysis ◽  
Patch Clamp Technique ◽  
Impulse Propagation ◽  
Transfer Strategies ◽  
Impulse Formation

Introduction: Currently constructed biopacemakers based on single gene transfer strategies function suboptimally, with periods of slow heart rates and instabilities during rest. In the sinoatrial node (SAN), the dominant native pacemaker, multiple genes are required for proper impulse formation and impulse propagation. TBX3 is an important regulator of the SAN gene program during heart development. We examined the effects of inducible TBX3 overexpression in adult hearts and in vitro we explored whether lentiviral TBX3 overexpression may be used in biopacemaker engineering. Methods: In vivo atrial and ventricular expression levels of the connexin isoforms Cx43 and Cx40 (impulse propagation) and SCN5A were studied in mice with tamoxifen inducible overexpression of TBX3 using quantitative PCR analysis. Single neonatal rat cardiac myocytes were transduced with TBX3 expressing lentivirus to analyze the effects of TBX3 on action potentials and membrane currents (impulse formation) using the perforated patch-clamp technique. Results: In vivo, Cx43, Cx40 and SCN5A, which are not or only moderately expressed in the native SAN, were severely down-regulated to 20%, 15%, and 40%, respectively, by TBX3 (n=12; p<0.01). Single neonatal cardiac myocytes overexpressing TBX3 exhibited faster spontaneous beating rates, along with decreased maximum diastolic potential, inward rectifier potassium current (I K1 ), and fast sodium current (I Na ). These properties are typical of SAN pacemaker cells. Conclusions: TBX3 can act as a strong repressor of the working myocardium gene program in the adult heart. Overexpression of TBX3 might be a useful tool in biopacemaker gene and cell therapy.

Abstract 241: Retinoic Acid Promotes Metabolic Maturation of Human Embryonic Stem Cell-derived Cardiomyocytes

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Shumei Miao ◽  
Xing Fang ◽  
Xiaoxiao Wang ◽  
Lingqun Ye ◽  
Jingsi Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Heart Development ◽  
Drug Testing ◽  
Disease Modeling ◽  
Time Window ◽  
Embryonic Stem ◽  
Early Time ◽  
Control Group ◽  
Cardiomyocyte Differentiation

Cardiomyocytes differentiated from human embryonic stem cells (hESCs) represent a promising cell source for heart repair, disease modeling and drug testing. However, improving the differentiation efficiency and maturation of hESC-derived cardiomyocytes (hESC-CMs) is still a major concern. Retinoic acid (RA) signaling plays multiple roles in heart development, and studies on RA can provide clues for understanding cardiomyocyte differentiation and maturation. In this study, we studied the roles of RA during cardiomyocyte differentiation and maturation, systematically. After adding RA at different stages of cardiomyocyte differentiation, we compared the efficiency of differentiation by quantitative real-time PCR and flow cytometry. We found that RA treatment at the lateral mesoderm stage (days 2-4) significantly improved cardiomyocyte differentiation, as evidenced by the upregulation of TNNT2, NKX2.5 and MYH6 on day 10 of differentiation. In addition, flow cytometry showed that the proportion of differentiated cardiomyocytes in the RA-treated group was significantly higher than that in control group. Furthermore, RA was added at different time intervals after purification to induce cardiomyocyte maturation. Our results demonstrated that RA treatment on days 15-20 increased cardiomyocyte area, sarcomere length, multinucleation and mitochondrial copy number, and promoted RNA splicing switch. Importantly, RA-treated cardiomyocytes showed decreased glycolysis and enhanced mitochondrial oxidative phosphorylation, with the increased utilization of fatty acid and exogenous pyruvate but not glutamine. In conclusion, our data indicated that RA treatment at an early time window (days 2-4) promotes the efficiency of cardiomyocyte differentiation and that RA treatment post beating (days 15-20) promotes cardiomyocyte metabolic maturation. The biphasic effects of RA provide new insights for improving cardiomyocyte differentiation and quality.

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