背景:心肌梗塞(MI)是全球范围内发病率和死亡率高的主要疾病。然而,现有的治疗方法远不能令人满意,使得探索有效的分子靶标更加势在必行。先前已报道E3泛素连接酶RING指蛋白5(RNF5)通过调节泛素化介导的蛋白质降解而参与几种疾病。然而,很少有报道关注它在心血管疾病中的作用,包括MI。
方法:在本研究中,我们通过精确的CRISPR介导的基因组编辑建立了RNF5基因敲除小鼠,并在9-11周龄的雄性C57BL/6小鼠中使用了左冠状动脉前降支结扎.随后,进行血清生化分析和心脏组织病理学检查。此外,我们设计了调节RNF5表达的腺病毒,并对新生大鼠心肌细胞进行氧-葡萄糖剥夺(OGD)以模拟缺血条件,证明RNF5操作对细胞活力的影响。基因和蛋白质表达分析提供了对分子机制的见解。严格采用统计方法来评估实验结果的重要性。
结果:我们发现RNF5在接受OGD治疗的小鼠和NRCM的梗死心脏组织中下调。RNF5基因敲除导致小鼠心脏功能障碍加剧,更严重的炎症反应,MI手术后细胞凋亡增加。体外,RNF5敲低加剧了OGD诱导的细胞活性下降,细胞凋亡增加,而RNF5过表达则有相反的作用。机械上,已证明,由凋亡信号调节激酶1(ASK1)激活引发的激酶级联受RNF5的密切调节,并介导了RNF5在MI期间的保护功能。
结论:我们证明了RNF5对心肌梗死的保护作用,其功能依赖于抑制ASK1的激活,这为心肌梗死相关网络增加了新的调节成分,并有望实现新的治疗策略。
BACKGROUND: Myocardial infarction (MI) is a major disease with high morbidity and mortality worldwide. However, existing treatments are far from satisfactory, making the exploration of potent molecular targets more imperative. The E3 ubiquitin ligase RING finger protein 5 (
RNF5) has been previously reported to be involved in several diseases by regulating ubiquitination-mediated protein degradation. Nevertheless, few reports have focused on its function in cardiovascular diseases, including MI.
METHODS: In this study, we established
RNF5 knockout mice through precise CRISPR-mediated genome editing and utilized left anterior descending coronary artery ligation in 9-11-week-old male C57BL/6 mice. Subsequently, serum biochemical analysis and histopathological examination of heart tissues were performed. Furthermore, we engineered adenoviruses for modulating RNF5 expression and subjected neonatal rat cardiomyocytes to oxygen-glucose deprivation (OGD) to mimic ischemic conditions, demonstrating the impact of RNF5 manipulation on cellular viability. Gene and protein expression analysis provided insights into the molecular mechanisms. Statistical methods were rigorously employed to assess the significance of experimental findings.
RESULTS: We found RNF5 was downregulated in infarcted heart tissue of mice and NRCMs subjected to OGD treatment.
RNF5 knockout in mice resulted in exacerbated heart dysfunction, more severe inflammatory responses, and increased apoptosis after MI surgery. In vitro, RNF5 knockdown exacerbated the OGD-induced decline in cell activity, increased apoptosis, while
RNF5 overexpression had the opposite effect. Mechanistically, it was proven that the kinase cascade initiated by apoptosis signal-regulating kinase 1 (ASK1) activation was closely regulated by RNF5 and mediated
RNF5\'s protective function during MI.
CONCLUSIONS: We demonstrated the protective effect of RNF5 on myocardial infarction and its function was dependent on inhibiting the activation of ASK1, which adds a new regulatory component to the myocardial infarction associated network and promises to enable new therapeutic strategy.