BACKGROUND Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease. Currently, there is no effective therapy for reducing cardiac I/R injury. Damage-associated molecular patterns are endogenous molecules released after cellular damage to exaggerate tissue inflammation and injury. RIPK3 (receptor-interacting protein kinase 3), a well-established intracellular mediator of cell necroptosis and inflammation, serves as a circulating biomarker of multiple diseases. However, whether extracellular RIPK3 also exerts biological functions in cardiac I/R injury remains totally unknown. METHODS Patients with acute myocardial infarction receiving percutaneous coronary intervention (PCI) were recruited independently in the discovery cohort (103 patients) and validation cohort (334 patients), and major adverse cardiovascular events were recorded. Plasma samples were collected before and after PCI (6 and 24 h) for RIPK3 concentration measurement. Cultured neonatal rat ventricular myocytes, macrophages and endothelial cells, and in vivo mouse models with myocardial injury induced by I/R (or hypoxia/reoxygenation) were used to investigate the role and mechanisms of extracellular RIPK3. Another cohort including patients with acute myocardial infarction receiving PCI and healthy volunteers was recruited to further explore the mechanisms of extracellular RIPK3. RESULTS In the discovery cohort, elevated plasma RIPK3 levels after PCI are associated with poorer short- and long-term outcomes in patients with acute myocardial infarction, as confirmed in the validation cohort. In both cultured cells and in vivo mouse models, recombinant RIPK3 protein exaggerated myocardial I/R (or hypoxia/reoxygenation) injury, which was alleviated by the RIPK3 antibody. Mechanistically, RIPK3 acted as a damage-associated molecular pattern and bound with RAGE (receptor of advanced glycation end-products), subsequently activating CaMKII (Ca2+/calmodulin-dependent kinase II) to elicit the detrimental effects. The positive correlation between plasma RIPK3 concentrations and CaMKII phosphorylation in human peripheral blood mononuclear cells was confirmed. CONCLUSIONS We identified the positive relationship between plasma RIPK3 concentrations and the risk of major adverse cardiovascular events in patients with acute myocardial infarction receiving PCI. As a damage-associated molecular pattern, extracellular RIPK3 plays a causal role in multiple pathological conditions during cardiac I/R injury through RAGE/CaMKII signaling. These findings expand our understanding of the physiological and pathological roles of RIPK3, and also provide a promising therapeutic target for myocardial I/R injury and the associated complications.

中文翻译：

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细胞外 RIPK3 作为一种危险相关的分子模式，夸大了心肌缺血/再灌注损伤。


背景 心肌缺血/再灌注 （I/R） 损伤已成为缺血性心脏病的重要治疗靶点。目前，尚无减少心脏 I/R 损伤的有效疗法。损伤相关分子模式是细胞损伤后释放的内源性分子，用于夸大组织炎症和损伤。RIPK3 （受体相互作用蛋白激酶 3） 是细胞坏死性凋亡和炎症的成熟细胞内介质，是多种疾病的循环生物标志物。然而，细胞外 RIPK3 是否也在心脏 I/R 损伤中发挥生物学功能仍然完全未知。方法 将接受经皮冠状动脉介入治疗 （PCI） 的急性心肌梗死患者独立纳入发现队列 （103 例） 和验证队列 （334 例） 中，记录主要不良心血管事件。在 PCI 前后 （6 小时和 24 小时） 收集血浆样品用于 RIPK3 浓度测量。培养的新生大鼠脑室肌细胞、巨噬细胞和内皮细胞以及由 I/R （或缺氧/复氧） 诱导的心肌损伤的体内小鼠模型用于研究细胞外 RIPK3 的作用和机制。招募了另一个队列，包括接受 PCI 的急性心肌梗死患者和健康志愿者，以进一步探索细胞外 RIPK3 的机制。结果在发现队列中，PCI 后血浆 RIPK3 水平升高与急性心肌梗死患者较差的短期和长期预后相关，如验证队列所证实的那样。 在培养细胞和体内小鼠模型中，重组 RIPK3 蛋白夸大了心肌 I/R（或缺氧/复氧）损伤，而 RIPK3 抗体减轻了这种损伤。从机制上讲，RIPK3 充当损伤相关分子模式并与 RAGE（晚期糖基化终产物受体）结合，随后激活 CaMKII （Ca2+/钙调蛋白依赖性激酶 II） 以引发有害影响。证实血浆 RIPK3 浓度与人外周血单核细胞 CaMKII 磷酸化呈正相关。结论 我们确定了血浆 RIPK3 浓度与接受 PCI 的急性心肌梗死患者发生主要不良心血管事件风险之间的正相关关系。作为一种损伤相关分子模式，细胞外 RIPK3 通过 RAGE/CaMKII 信号传导在心脏 I/R 损伤期间的多种病理状况中起因果作用。这些发现扩大了我们对 RIPK3 生理和病理作用的理解，也为心肌 I/R 损伤和相关并发症提供了有前途的治疗靶点。