School of Basic Medicine, Qingdao University, Qingdao, 266071, China.
Department of Cardiology, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, 266003, China.
Cell Mol Biol Lett. 2024 Nov 12;29(1):140. doi: 10.1186/s11658-024-00649-8.
Circular RNAs (circRNAs) are differentially expressed in various cardiovascular diseases, including myocardial infarction (MI) injury. However, their functional role in necroptosis-induced loss of cardiomyocytes remains unclear. We identified a cardiac necroptosis-associated circRNA transcribed from the Cacna1c gene (circCacna1c) to investigate the involvement of circRNAs in cardiomyocyte necroptosis.
To investigate the role of circCacna1c during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with hydrogen peroxide (HO) to induce reactive oxygen species (ROS)-induced cardiomyocyte death. The N-methyladenosine (mA) modification level of circCacna1c was determined by methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) analysis. Additionally, an RNA pull-down assay was performed to identify interacting proteins of circCacna1c in cardiomyocytes, and the regulatory role of circCacna1c in target protein expression was tested using a western blotting assay. Furthermore, the MI mouse model was constructed to analyze the effect of circCacna1c on heart function and cardiomyocyte necroptosis.
The expression of circCacna1c was found to be reduced in cardiomyocytes exposed to oxidative stress and in mouse hearts injured by MI. Overexpression of circCacna1c inhibited necroptosis of cardiomyocytes induced by hydrogen peroxide and MI injury, resulting in a significant reduction in myocardial infarction size and improved cardiac function. Mechanistically, circCacna1c directly interacts with heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in the cytoplasm, preventing its nuclear translocation and leading to reduced Hnrnpf levels within the nucleus. This subsequently suppresses Hnrnpf-dependent receptor-interacting protein kinase 1 (RIPK1) expression. Furthermore, fat mass and obesity-associated protein (FTO) mediates demethylation of mA modification on circCacna1c during necrosis and facilitates degradation of circCacna1c.
Our study demonstrates that circCacna1c can improve cardiac function following MI-induced heart injury by inhibiting the Hnrnpf/RIPK1-mediated cardiomyocyte necroptosis. Therefore, the FTO/circCacna1c/Hnrnpf/RIPK1 axis holds great potential as an effective target for attenuating cardiac injury caused by necroptosis in ischemic heart disease.
环状 RNA（circRNAs）在多种心血管疾病中表达差异，包括心肌梗死（MI）损伤。然而，其在心肌细胞坏死性凋亡诱导的丢失中的功能作用尚不清楚。我们鉴定了一种心脏坏死性凋亡相关的环状 RNA，来源于 Cacna1c 基因（circCacna1c），以研究环状 RNA 在内皮细胞坏死性凋亡中的作用。
为了研究 circCacna1c 在氧化应激过程中的作用，我们用过氧化氢（H2O2）处理 H9c2 细胞和新生大鼠心肌细胞，以诱导活性氧（ROS）诱导的心肌细胞死亡。通过甲基化 RNA 免疫沉淀定量聚合酶链反应（MeRIP-qPCR）分析确定 circCacna1c 的 N6-甲基腺苷（m6A）修饰水平。此外，我们进行了 RNA 下拉实验以鉴定心肌细胞中 circCacna1c 的相互作用蛋白，并通过 Western blot 实验测试 circCacna1c 对靶蛋白表达的调节作用。此外，构建了 MI 小鼠模型，以分析 circCacna1c 对心脏功能和心肌细胞坏死性凋亡的影响。
我们发现，在氧化应激作用下的心肌细胞和 MI 损伤的小鼠心脏中，circCacna1c 的表达减少。过表达 circCacna1c 可抑制 H2O2 和 MI 损伤诱导的心肌细胞坏死性凋亡，导致心肌梗死面积减小，心脏功能改善。机制上，circCacna1c 可直接与细胞质中的异质核核糖核蛋白 F（Hnrnpf）相互作用，阻止其核转位，从而降低核内 Hnrnpf 水平。这继而抑制 Hnrnpf 依赖性受体相互作用蛋白激酶 1（RIPK1）的表达。此外，脂肪量和肥胖相关蛋白（FTO）介导了坏死过程中 circCacna1c 的 m6A 修饰去甲基化，并促进了 circCacna1c 的降解。
本研究表明，circCacna1c 可通过抑制 Hnrnpf/RIPK1 介导的心肌细胞坏死性凋亡来改善 MI 诱导的心肌损伤后的心脏功能。因此，FTO/circCacna1c/Hnrnpf/RIPK1 轴有望成为缺血性心脏病中抑制坏死性凋亡引起的心脏损伤的有效靶点。
