Aims Circular RNAs (circRNAs) are important players involved in a variety of physiological and pathological processes. However, their functions and mechanisms during myocardial ischaemic injury and protection remain largely unknown. We recently found significant alterations of many circRNAs including circCHSY1 following myocardial ischaemia/reperfusion (I/R) injury, whereas their exact functions are unclear. Here, we investigated the roles of circCHSY1 in the acute myocardial I/R injury and the potential mechanisms involved. Methods and results The expression of circCHSY1 was detected in cardiomyocytes from mouse, rat, and human embryonic stem cells (hESC-CMs). It was further up-regulated in mouse I/R (30 min/24 h) hearts, oxygen glucose deprivation/reperfusion (OGD/R, 6 h/2 h) primary neonatal rat ventricular cardiomyocytes (NRCMs) and OGD/R (48 h/2 h) hESC-CMs. Adenovirus-mediated circCHSY1 overexpression significantly decreased infarct size and lactate dehydrogenase (LDH) release in mouse I/R hearts. Consistently, circCHSY1 overexpression reduced the LDH release in the OGD/R NRCMs and hESC-CMs, improved cell viability, and preserved mitochondrial function in the OGD/R NRCMs, whereas there were no significant differences in cell viability and LDH release between the OGD/R NRCMs with and without small interfering RNA (siRNA)-mediated circCHSY1 knockdown. Mechanistically, circCHSY1 was detected to bind with miR-24-3p analysed by dual-luciferase assay and RNA pull-down assays. CircCHSY1 overexpression-mediated protective effects on cells and mitochondria in OGD/R NRCMs were reversed by the miR-24-3p mimic. Furthermore, dual-luciferase assay showed that miR-24-3p was directly bound to heme oxygenase 1 (HO1) via its 3′UTR. The protein level of HO1 was down-regulated by miR-24-3p mimic in OGD/R NRCMs but enhanced by the circCHSY1 overexpression in vitro and in vivo. Functionally, the HO1 knockdown by adenovirus in vivo and by siRNA in vitro eliminated cardioprotective effects of circCHSY1 overexpression. Conclusion CircCHSY1 is up-regulated following myocardial I/R injury. The higher level of circCHSY1 protects I/R hearts and cardiomyocytes. The protection of circCHSY1 is mediated through enhancement of the HO1 level, resulting in preserving mitochondrial homoeostasis via targeting miR-24-3p in cardiomyocytes. These findings suggest circCHSY1 as a protective factor.

中文翻译：

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CircCHSY1 通过 miR-24-3p 增强血红素加氧酶 1 的表达，保护心脏免受缺血/再灌注损伤


Circular RNAs （circRNAs） 是参与多种生理和病理过程的重要参与者。然而，它们在心肌缺血性损伤和保护期间的功能和机制在很大程度上仍然未知。我们最近发现许多 circRNAs 在心肌缺血/再灌注 （I/R） 损伤后发生显着改变，包括 circCHSY1，而它们的确切功能尚不清楚。在这里，我们研究了 circCHSY1 在急性心肌 I/R 损伤中的作用以及所涉及的潜在机制。方法和结果 在小鼠、大鼠和人胚胎干细胞 （hESC-CMs） 的心肌细胞中检测到 circCHSY1 的表达。它在小鼠 I/R （30 min/24 h） 心脏、氧葡萄糖剥夺/再灌注 （OGD/R，6 h/2 h） 原代新生大鼠心室心肌细胞 （NRCMs） 和 OGD/R （48 h/2 h） hESC-CMs 中进一步上调。腺病毒介导的 circCHSY1 过表达显著降低小鼠 I/R 心脏梗死面积和乳酸脱氢酶 （LDH） 释放。一致地，circCHSY1 过表达降低了 OGD/R NRCM 和 hESC-CMs 中 LDH 的释放，提高了细胞活力，并保留了 OGD/R NRCM 中的线粒体功能，而 OGD/R NRCM 在有和没有小干扰 RNA （siRNA） 介导的 circCHSY1 敲低的 OGD/R NRCM 之间没有显着差异。从机制上讲，通过双荧光素酶测定和 RNA 下拉测定检测到 circCHSY1 与 miR-24-3p 结合。CircCHSY1 过表达介导的对 OGD/R NRCM 细胞和线粒体的保护作用被 miR-24-3p 模拟物逆转。此外，双荧光素酶测定显示 miR-24-3p 通过其 3′UTR 直接与血红素加氧酶 1 （HO1） 结合。 HO1 的蛋白水平在 OGD/R NRCMs 中被 miR-24-3p 模拟物下调，但在体外和体内被 circCHSY1 过表达增强。从功能上讲，腺病毒在体内和 siRNA 在体外敲低 HO1 消除了 circCHSY1 过表达的心脏保护作用。结论 CircCHSY1 在心肌 I/R 损伤后上调。较高水平的 circCHSY1 可保护 I/R 心脏和心肌细胞。circCHSY1 的保护是通过增强 HO1 水平介导的，导致通过靶向心肌细胞中的 miR-24-3p 来保持线粒体匀态。这些发现表明 circCHSY1 是一个保护因素。