PDF(Pubmed)
Abstract：
The prevalence of heart failure is constantly increasing, and the prognosis of patients remains poor. New treatment strategies to preserve cardiac function and limit cardiac hypertrophy are therefore urgently needed. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as an experimental platform for cardiac in vitro studies. However, in contrast to adult cardiomyocytes, hiPSC-CMs display immature morphology, contractility, gene expression and metabolism and hence express a naive phenotype that resembles more of a foetal cardiomyocyte.
A library of 14 novel compounds was synthesized in-house and screened for GATA4-NKX2-5 reporter activity and cellular toxicity. The most potent compound, 3i-1262, along with previously reported GATA4-acting compounds, were selected to investigate their effects on hypertrophy induced by endothelin-1 or mechanical stretch. Morphological changes and protein expression were characterized using immunofluorescence staining and high-content analysis. Changes in gene expression were studied using qPCR and RNA sequencing.
The prototype compound 3i-1262 inhibited GATA4-NKX2-5 synergy in a luciferase reporter assay. Additionally, the isoxazole compound 3i-1262 inhibited the hypertrophy biomarker B-type natriuretic peptide (BNP) by reducing BNP promoter activity and proBNP expression in neonatal rat ventricular myocytes and hiPSC-CMs, respectively. Treatment with 3i-1262 increased metabolic activity and cardiac troponin T expression in hiPSC-CMs without affecting GATA4 protein levels. RNA sequencing analysis revealed that 3i-1262 induces gene expression related to metabolic activity and cell cycle exit, indicating a change in the identity and maturity status of hiPSC-CMs. The biological processes that were enriched in upregulated genes in response to 3i-1262 were downregulated in response to mechanical stretch, and conversely, the downregulated processes in response to 3i-1262 were upregulated in response to mechanical stretch.
There is currently a lack of systematic understanding of the molecular modulation and control of hiPSC-CM maturation. In this study, we demonstrated that the GATA4-interfering compound 3i-1262 reorganizes the cardiac transcription factor network and converts hypertrophic signalling towards enhanced cardiomyocyte identity and maturity. This conceptually unique approach provides a novel structural scaffold for further development as a modality to promote cardiomyocyte specification and maturity.
A library of 14 novel compounds was synthesized in-house and screened for GATA4-NKX2-5 reporter activity and cellular toxicity. The most potent compound, 3i-1262, along with previously reported GATA4-acting compounds, were selected to investigate their effects on hypertrophy induced by endothelin-1 or mechanical stretch. Morphological changes and protein expression were characterized using immunofluorescence staining and high-content analysis. Changes in gene expression were studied using qPCR and RNA sequencing.
The prototype compound 3i-1262 inhibited GATA4-NKX2-5 synergy in a luciferase reporter assay. Additionally, the isoxazole compound 3i-1262 inhibited the hypertrophy biomarker B-type natriuretic peptide (BNP) by reducing BNP promoter activity and proBNP expression in neonatal rat ventricular myocytes and hiPSC-CMs, respectively. Treatment with 3i-1262 increased metabolic activity and cardiac troponin T expression in hiPSC-CMs without affecting GATA4 protein levels. RNA sequencing analysis revealed that 3i-1262 induces gene expression related to metabolic activity and cell cycle exit, indicating a change in the identity and maturity status of hiPSC-CMs. The biological processes that were enriched in upregulated genes in response to 3i-1262 were downregulated in response to mechanical stretch, and conversely, the downregulated processes in response to 3i-1262 were upregulated in response to mechanical stretch.
There is currently a lack of systematic understanding of the molecular modulation and control of hiPSC-CM maturation. In this study, we demonstrated that the GATA4-interfering compound 3i-1262 reorganizes the cardiac transcription factor network and converts hypertrophic signalling towards enhanced cardiomyocyte identity and maturity. This conceptually unique approach provides a novel structural scaffold for further development as a modality to promote cardiomyocyte specification and maturity.
摘要：
背景：心力衰竭的患病率不断增加，患者的预后仍然较差。因此，迫切需要新的治疗策略来保护心脏功能并限制心脏肥大。人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）越来越多地用作心脏体外研究的实验平台。然而,与成年心肌细胞相反，hiPSC-CM表现出未成熟的形态，收缩性，基因表达和代谢，因此表达更类似于胎儿心肌细胞的幼稚表型。
方法：内部合成了14种新化合物的文库，并筛选了GATA4-NKX2-5报告活性和细胞毒性。最有效的化合物,3i-1262，以及先前报道的GATA4作用化合物，选择它们来研究它们对内皮素-1或机械拉伸诱导的肥大的影响。使用免疫荧光染色和高含量分析来表征形态学变化和蛋白质表达。使用qPCR和RNA测序研究基因表达的变化。
结果:原型化合物3i-1262在荧光素酶报告基因测定中抑制GATA4-NKX2-5协同作用。此外,异恶唑化合物3i-1262通过降低新生大鼠心室肌细胞和hiPSC-CM中的BNP启动子活性和proBNP表达来抑制肥大生物标志物B型利钠肽（BNP），分别。用3i-1262处理增加了hiPSC-CM中的代谢活性和心肌肌钙蛋白T表达,而不影响GATA4蛋白水平。RNA测序分析显示，3i-1262诱导与代谢活性和细胞周期退出相关的基因表达，表明hiPSC-CM的身份和成熟状态的变化。响应3i-1262的上调基因富集的生物过程被下调，响应机械拉伸，反过来,响应3i-1262的下调过程响应机械拉伸而上调。
结论：目前对hiPSC-CM成熟的分子调节和控制缺乏系统的了解。在这项研究中,我们证明,干扰GATA4的化合物3i-1262重组了心脏转录因子网络,并将肥大信号转换为增强的心肌细胞同一性和成熟度.这种概念上独特的方法提供了一种新颖的结构支架，可进一步发展为促进心肌细胞规格和成熟的方式。
方法：内部合成了14种新化合物的文库，并筛选了GATA4-NKX2-5报告活性和细胞毒性。最有效的化合物,3i-1262，以及先前报道的GATA4作用化合物，选择它们来研究它们对内皮素-1或机械拉伸诱导的肥大的影响。使用免疫荧光染色和高含量分析来表征形态学变化和蛋白质表达。使用qPCR和RNA测序研究基因表达的变化。
结果:原型化合物3i-1262在荧光素酶报告基因测定中抑制GATA4-NKX2-5协同作用。此外,异恶唑化合物3i-1262通过降低新生大鼠心室肌细胞和hiPSC-CM中的BNP启动子活性和proBNP表达来抑制肥大生物标志物B型利钠肽（BNP），分别。用3i-1262处理增加了hiPSC-CM中的代谢活性和心肌肌钙蛋白T表达,而不影响GATA4蛋白水平。RNA测序分析显示，3i-1262诱导与代谢活性和细胞周期退出相关的基因表达，表明hiPSC-CM的身份和成熟状态的变化。响应3i-1262的上调基因富集的生物过程被下调，响应机械拉伸，反过来,响应3i-1262的下调过程响应机械拉伸而上调。
结论：目前对hiPSC-CM成熟的分子调节和控制缺乏系统的了解。在这项研究中,我们证明,干扰GATA4的化合物3i-1262重组了心脏转录因子网络,并将肥大信号转换为增强的心肌细胞同一性和成熟度.这种概念上独特的方法提供了一种新颖的结构支架，可进一步发展为促进心肌细胞规格和成熟的方式。
