CCND2 修饰 mRNA 激活心肌梗死后小鼠和猪心脏中的心肌细胞周期。
CCND2 Modified mRNA Activates Cell Cycle of Cardiomyocytes in Hearts With Myocardial Infarction in Mice and Pigs.
机构信息
Department of Biomedical Engineering, School of Medicine, School of Engineering (J.S., L.W., R.C.M., G.P.W., Y.-A.L., Y.W., Y.Z., J.Z.), University of Alabama at Birmingham.
Department of Medicine, Division of Cardiovascular Disease, School of Medicine (G.P.W., J.Z.), University of Alabama at Birmingham.
出版信息
Circ Res. 2023 Sep;133(6):484-504. doi: 10.1161/CIRCRESAHA.123.322929. Epub 2023 Aug 11.
BACKGROUND
Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte-specific overexpression of CCND2 (cyclin D2, in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our specific modified mRNA translation system (SMRTs) can induce CCND2 expression in cardiomyocytes and replicate the benefits observed in other studies of cardiomyocyte-specific CCND2 overexpression for myocardial repair.
METHODS
The CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) consists of 2 modRNA constructs: one codes for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the cardiomyocyte-specific expression of luciferase or green fluorescent protein (GFP), in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals.
RESULTS
CCND2 was abundantly expressed in cultured, postmitotic cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (eg, Ki67 and Aurora B kinase). When the GFP-cardiomyocyte SMRTs were intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in cardiomyocytes but no other cell type. In both MI models, cardiomyocyte proliferation (on day 7 and day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (days 7 and 28 in mice, days 10 and 28 in pigs) were significantly higher, and infarcts (day 28 in both species) were significantly smaller in animals treated with the CCND2-cardiomyocyte SMRTs than in any other group that underwent MI induction.
CONCLUSIONS
Intramyocardial injections of the CCND2-cardiomyocyte SMRTs promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with MI.
背景
在哺乳动物心脏损伤模型中的实验表明，心肌细胞特异性过表达 CCND2（人类中的细胞周期蛋白 D2）可改善心肌梗死（MI）后的恢复。本研究的主要目的是证明我们的特定修饰的 mRNA 翻译系统（SMRTs）可以在心肌细胞中诱导 CCND2 表达，并复制在其他心肌细胞特异性 CCND2 过表达的心肌修复研究中观察到的益处。
方法
CCND2-心肌细胞特异性修饰的 mRNA 翻译系统（心肌细胞 SMRTs）由 2 种 modRNA 构建体组成：一种编码 CCND2，包含 L7Ae 结合位点，另一种编码 L7Ae，包含心肌细胞特异性 microRNA miR-1 和 miR-208 的识别元件。因此，L7Ae 在非心肌细胞中抑制 CCND2 翻译，但在心肌细胞中被内源性 miR-1 和 miR-208 抑制，从而促进心肌细胞特异性 CCND2 表达。在 MI 的小鼠和猪模型中进行了实验，并在接受编码心肌细胞特异性表达荧光素酶或绿色荧光蛋白（GFP）的 SMRTs 治疗的动物、接受 L7Ae modRNA 单独或载体治疗的动物以及接受 Sham 手术的动物中进行了对照评估。
结果
在转染 CCND2-心肌细胞 SMRTs 后 2 天，培养的有丝分裂后心肌细胞中大量表达 CCND2，并且伴随着细胞周期激活和增殖标志物（例如 Ki67 和 Aurora B 激酶）的上调。当 GFP-心肌细胞 SMRTs 被心肌内注射到梗死的小鼠心脏中时，在心肌细胞中观察到 GFP 信号，但在其他细胞类型中没有。在两种 MI 模型中，与接受 MI 诱导的任何其他组相比，用 CCND2-心肌细胞 SMRTs 治疗的动物的心肌细胞增殖（在小鼠中的第 7 天和第 3 天，在猪中的第 10 天和第 28 天）显著增加，左心室射血分数（在小鼠中的第 7 天和第 28 天，在猪中的第 10 天和第 28 天）显著升高，梗死面积（在两种物种中均为第 28 天）显著减小。
结论
心肌内注射 CCND2-心肌细胞 SMRTs 可促进心肌细胞增殖，减少梗死面积，并改善小型和大型哺乳动物 MI 心脏的心脏功能。
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