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Background: Transcription factors HAND1 and HAND2 (HAND1/2) play significant roles in cardiac organogenesis. Abnormal expression and deficiency of HAND1/2 result in severe cardiac defects. However, the function and mechanism of HAND1/2 in regulating human early cardiac lineage commitment and differentiation are still unclear. Methods: With NKX2.5eGFP H9 human embryonic stem cells (hESCs), we established single and double knockout cell lines for HAND1 and HAND2, respectively, whose cardiomyocyte differentiation efficiency could be monitored by assessing NKX2.5-eGFP+ cells with flow cytometry. The expression of specific markers for heart fields and cardiomyocyte subtypes was examined by quantitative PCR, western blot and immunofluorescence staining. Microelectrode array and whole-cell patch clamp were performed to determine the electrophysiological characteristics of differentiated cardiomyocytes. The transcriptomic changes of HAND knockout cells were revealed by RNA sequencing. The HAND1/2 target genes were identified and validated experimentally by integrating with HAND1/2 chromatin immunoprecipitation sequencing data. Results: Either HAND1 or HAND2 knockout did not affect the cardiomyocyte differentiation kinetics, whereas depletion of HAND1/2 resulted in delayed differentiation onset. HAND1 knockout biased cardiac mesoderm toward second heart field progenitors at the expense of first heart field progenitors, leading to increased expression of atrial and outflow tract cardiomyocyte markers, which was further confirmed by the appearance of atrial-like action potentials. By contrast, HAND2 knockout cardiomyocytes had reduced expression of atrial cardiomyocyte markers and displayed ventricular-like action potentials. HAND1/2-deficient hESCs were more inclined to second heart field lineage and its derived cardiomyocytes with atrial-like action potentials than HAND1 single knockout during differentiation. Further mechanistic investigations suggested TBX5 as one of the downstream targets of HAND1/2, whose overexpression partially restored the abnormal cardiomyocyte differentiation in HAND1/2-deficient hESCs. Conclusions: HAND1/2 have specific and redundant roles in cardiac lineage commitment and differentiation. These findings not only reveal the essential function of HAND1/2 in cardiac organogenesis, but also provide important information on the pathogenesis of HAND1/2 deficiency-related congenital heart diseases, which could potentially lead to new therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Cardiomyocyte growth and differentiation rely on precise gene expression regulation, with epigenetic modifications emerging as key players in this intricate process. Among these modifications, N6-methyladenosine (m6A) stands out as one of the most prevalent modifications on mRNA, exerting influence over mRNA metabolism and gene expression. However, the specific function of m6A in cardiomyocyte differentiation remains poorly understood. Results: We investigated the relationship between m6A modification and cardiomyocyte differentiation by conducting a comprehensive profiling of m6A dynamics during the transition from pluripotent stem cells to cardiomyocytes. Our findings reveal that while the overall m6A modification level remains relatively stable, the m6A levels of individual genes undergo significant changes throughout cardiomyocyte differentiation. We discovered the correlation between alterations in chromatin accessibility and the binding capabilities of m6A writers, erasers, and readers. The changes in chromatin accessibility influence the recruitment and activity of m6A regulatory proteins, thereby impacting the levels of m6A modification on specific mRNA transcripts. Conclusion: Our data demonstrate that the coordinated dynamics of m6A modification and chromatin accessibility are prominent during the cardiomyocyte differentiation. [ABSTRACT FROM AUTHOR]

Background and Aim: Generation of functional cardiomyocytes from human pluripotent stem cells (hPSCs) offers promising applications for cardiac regenerative medicine. Proper control of pluripotency and differentiation is vital for generating high-quality cardiomyocytes and repairing damaged myocardium. Cathepsin K, a lysosomal cysteine protease, is a potential target for cardiovascular disease treatment; however, its role in cardiomyocyte differentiation and regeneration is unclear. This study aims to investigate the effects and mechanisms of cathepsin K inhibition on the differentiation of human embryonic stem cell-induced cardiomyocytes (hESC-CMs) and myocardial generation., Methods: We cultured H9-hESCs in CDM3 medium to induce myocardial differentiation, adding cathepsin K inhibitor II (1 μM) on days 2, 5 and 8, respectively. Cells were observed and collected 48 h after each treatment. The morphology and contractile clusters of H9-hESCs were tracked with microscopy and video recording. Pluripotency and cardiac markers were assessed at each stage of differentiation. We also examined glucose and lipid metabolism, mitochondrion-related markers, apoptosis and autophagy., Results: CDM3 medium effectively differentiated high-density H9-hESCs into mature, spontaneously contracting cardiomyocytes. Cathepsin K inhibition accelerates the differentiation of H9-hESCs into cardiac mesoderm and cardiac precursor cells (CPCs) by reducing apoptosis, decreasing glycolysis and fatty acid metabolism at the early and middle stages, and subsequently facilitate the development and differentiation of cardiomyocytes by enhancing glucolipid metabolism and oxidative phosphorylation at the late stage. Meanwhile, cathepsin K inhibition enhanced mitochondrial function and lysosome-related gene transcription during the differentiation process., Conclusion: Our study highlights the potential of cathepsin K inhibition for renewable cardiomyocytes and suggests exploring metabolic pathways and signaling to improve cardiac regeneration and organoid development., Competing Interests: Declarations. Ethics approval and consent to participate: H9-hESCs were generously obtained from the Research Center for Eco-Environmental Sciences under the Chinese Academy of Sciences. The protocol was approved by the Hebei University Animal Welfare and Ethical Committee (Project: Role of Cathepsin K in the Development of Diabetic Atherosclerosis by regulating SERCA2-SUMOylation. No. IACUC-2021XG032, approved on Mar. 11, 2021). Competing interests: The authors declare no competing financial interests., (© 2025. The Author(s).)

Background: Bone marrow derived stem cells (BMSCs) have the potential to differentiate into cardiomyocytes, but the rate of differentiation is low and the mechanism of differentiation is unclear completely. Here, we aimed to investigate the role of miR1-2 in differentiation of mouse BMSCs into cardiomyocyte-like cells and reveal the involved signaling pathways in the procedure. Methods: Mouse BMSCs were treated with miR1-2 and 5-azacytine (5-aza). The expression of cardiac cell markers: NKx2.5, cTnI and GATA4 in BMSCs were examined by qPCR. The apoptosis rate was detected by flow cytometry and the activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the upstream protein of this signaling pathway. Results: After over-expression of miR1-2 in mouse BMSCs, the apoptosis rate was significantly lower than the 5-aza group, while the expressions of cardiac-specific genes: such as Nkx2.5, cTnI and GATA4 were significantly increased compared to the control group and the 5-aza group. Meanwhile, over-expression of miR1-2 in mouse BMSCs enhanced the expression of wnt11, JNK, β-catenin and TCF in the Wnt/β-catenin signaling pathway. Use of LGK-974, an inhibitor of Wnt/β-catenin signaling pathway, significantly reduced the expression of cardiac-specific genes and partially blocked the role of the miR1-2. Conclusion: Over-expression of miR1-2 in mouse BMSCs can induce them toward promoted cardiomyocyte differentiation via the activation of the Wnt/β-catenin signaling pathway. Compared to 5-aza, miR1-2 can induce differentiation of BMSCs into cardiomyocytes more effectively with a less cytotoxicity. [ABSTRACT FROM AUTHOR]

Background: Environmental factors are important for stem cell lineage specification, and increasing evidence indicates that the nanoscale geometry/topography of the extracellular matrix (ECM) directs stem cell fate. Recently, many three-dimensional (3D) biomimetic nanofibrous scaffolds resembling many characteristics of the native ECM have been used in stem cell-based myocardial tissue engineering. However, the biophysical role and underlying mechanism of 3D nanofibrous scaffolds in cardiomyocyte differentiation of induced pluripotent stem cells (iPSCs) remain unclear. Results: Here, we fabricated a 3D poly-(ε-caprolactone) (PCL) nanofibrous scaffold using the electrospinning method and verified its nanotopography and porous structure by scanning electron microscopy. We seeded murine iPSCs (miPSCs) directly on the 3D PCL nanofibrous scaffold and initiated non-directed, spontaneous differentiation using the monolayer method. After the 3D PCL nanofibrous scaffold was gelatin coated, it was suitable for monolayer miPSC cultivation and cardiomyocyte differentiation. At day 15 of differentiation, miPSCs differentiated into functional cardiomyocytes on the 3D PCL nanofibrous scaffold as evidenced by positive immunostaining of cardiac-specific proteins including cardiac troponin T (cTnT) and myosin light chain 2a (MLC2a). In addition, flow cytometric analysis of cTnT-positive cells and cardiac-specific gene and protein expression of cTnT and sarcomeric alpha actinin (α-actinin) demonstrated that the cardiomyocyte differentiation of miPSCs was more efficient on the 3D PCL nanofibrous scaffold than on normal tissue culture plates (TCPs). Furthermore, early inhibition of Wnt/β-catenin signaling by the selective antagonist Dickkopf-1 significantly reduced the activity of Wnt/β-catenin signaling and decreased the cardiomyocyte differentiation of miPSCs cultured on the 3D PCL nanofibrous scaffold, while the early activation of Wnt/β-catenin signaling by CHIR99021 further increased the cardiomyocyte differentiation of miPSCs. Conclusion: These results indicated that the electrospun 3D PCL nanofibrous scaffolds directly promoted the cardiomyocyte differentiation of miPSCs, which was mediated by the activation of the Wnt/β-catenin signaling during the early period of differentiation. These findings highlighted the biophysical role of 3D nanofibrous scaffolds during the cardiomyocyte differentiation of miPSCs and revealed its underlying mechanism involving Wnt/β-catenin signaling, which will be helpful in guiding future stem cell- and scaffold-based myocardium bioengineering. [ABSTRACT FROM AUTHOR]