GATA4 is an early cardiac-specific transcription factor, and endogenous GATA4-positive cells play a critical role in cardioprotection after myocardial injury. As functional paracrine units of therapeutic cells, exosomes can partially reproduce the reparative properties of their parental cells. Here, we investigated the cardioprotective capabilities of exosomes derived from cardiac colony-forming unit fibroblasts (cCFU-Fs) overexpressing GATA4 (cCFU-FsGATA4) and the underlying mechanism through which these exosomes use microRNA (miRNA) delivery to regulate target proteins in myocardial infarction (MI). Exosomes were harvested from cCFU-Fs by ultracentrifugation. miRNA arrays were performed to determine differential miRNA expression between exosomes derived from cCFU-FsGATA4 (GATA4-Exo) and control cCFU-Fs (NC-Exo). A dual-luciferase reporter assay confirmed that miR221 directly targets the 3′ untranslated region (UTR) of the phosphatase and tensin homolog on chromosome ten (PTEN) gene. Cardiac function and myocardial infarct size were evaluated by echocardiography and Masson trichrome staining, respectively. Compared with NC-Exo, GATA4-Exo increased the survival and reduced the apoptosis of H9c2 cells. Direct intramyocardial transplantation of GATA4-Exo at the border of the ischemic region following ligation of the left anterior descending (LAD) coronary artery significantly restored cardiac contractile function and reduced infarct size. Microarray analysis revealed significantly increased miR221 expression in GATA4-Exo. qPCR confirmed higher miR221 levels in H9c2 cells treated with GATA4-Exo than in those treated with NC-Exo. miR221 mimic-transfected H9c2 cells demonstrated a significantly higher survival rate following exposure to hypoxic conditions than those transfected with miR221 inhibitor. A dual-luciferase reporter gene assay confirmed the PTEN gene as a target of miR221. Western blot analysis showed that H9c2 cells treated with GATA4-Exo exhibited lower PTEN protein expression and higher p-Akt expression. GATA4 overexpression enhances the protective effect of cCFU-F-derived exosomes on myocardial ischemic injury. In terms of the mechanism, it is at least partly due to the miR221 transferred by GATA4-Exo, which inhibits PTEN expression, activates the phosphatidylinositol 3 kinase (PI3K)/AKT signaling pathway, and subsequently alleviates apoptosis of myocardial cells (CMs).

中文翻译：

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GATA4的过表达通过miRNA221介导的PTEN / PI3K / AKT信号通路的靶向作用，增强了从心脏集落形成单位成纤维细胞分泌的外泌体的抗凋亡作用。

GATA4是一种早期的心脏特异性转录因子，内源性GATA4阳性细胞在心肌损伤后的心脏保护中起着至关重要的作用。作为治疗细胞的功能旁分泌单位，外泌体可以部分复制其亲本细胞的修复特性。在这里，我们研究了过表达GATA4（cCFU-FsGATA4）的心脏集落形成单位成纤维细胞（cCFU-Fs）衍生的外泌体的心脏保护能力，以及这些外泌体利用microRNA（miRNA）传递来调节心肌梗死中靶蛋白的潜在机制。 （MI）。通过超速离心从cCFU-Fs中收获外来体。进行miRNA阵列分析以确定ccFU-FsGATA4（GATA4-Exo）和对照cCFU-Fs（NC-Exo）衍生的外泌体之间的差异miRNA表达。双重荧光素酶报告基因测定证实miR221直接靶向10号染色体（PTEN）基因上的磷酸酶和张力蛋白同源物的3'非翻译区（UTR）。通过超声心动图和Masson三色染色分别评估心脏功能和心肌梗死面积。与NC-Exo相比，GATA4-Exo增加了H9c2细胞的存活率并降低了其凋亡。结扎左前降支（LAD）冠状动脉后，在缺血区域边界直接进行GATA4-Exo心肌内移植，可显着恢复心脏收缩功能并减少梗塞面积。微阵列分析显示，GATA4-Exo中miR221表达显着增加。qPCR证实，用GATA4-Exo处理的H9c2细胞中的miR221水平高于用NC-Exo处理的细胞。与低氧条件相比，miR221模拟转染的H9c2细胞暴露于低氧条件下的存活率明显更高。双重荧光素酶报告基因检测证实PTEN基因是miR221的靶标。蛋白质印迹分析表明，用GATA4-Exo处理的H9c2细胞表现出较低的PTEN蛋白表达和较高的p-Akt表达。GATA4的过表达增强了cCFU-F衍生的外来体对心肌缺血损伤的保护作用。就机理而言，这至少部分是由于由GATA4-Exo转移的miR221抑制了PTEN的表达，激活了磷脂酰肌醇3激酶（PI3K）/ AKT信号传导途径，并随后减轻了心肌细胞（CMs）的凋亡。