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.
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。