Background: Catecholamine surges and resultant excessive β-adrenergic stimulation occur in a broad spectrum of diseases. Excessive β-adrenergic stimulation causes cardiomyocyte necrosis, but the underlying mechanism remains obscure. Necroptosis, a major form of regulated necrosis mediated by RIPK3-centered pathways, is implicated in heart failure; however, it remains unknown whether excessive β-adrenergic stimulation-induced cardiac injury involves necroptosis. Hence, we conducted the present study to address these critical gaps.

Methods and Results: Two consecutive daily injections of isoproterenol (ISO; 85 mg/kg, s.c.) or saline were administered to adult mixed-sex mice. At 24 h after the second ISO injection, cardiac area with Evans blue dye (EBD) uptake and myocardial protein levels of CD45, RIPK1, Ser166-phosphorylated RIPK1, RIPK3, and Ser345-phosphorylated MLKL (p-MLKL) were significantly greater, while Ser321-phosphorylated RIPK1 was significantly lower, in the ISO-treated than in saline-treated wild-type (WT) mice. The ISO-induced increase of EBD uptake was markedly less in RIPK3^−/− mice compared with WT mice (p = 0.016). Pretreatment with the RIPK1-selective inhibitor necrostatin-1 diminished ISO-induced increases in RIPK3 and p-MLKL in WT mice and significantly attenuated ISO-induced increases of EBD uptake in WT but not RIPK3^−/− mice.

Conclusions: A large proportion of cardiomyocyte necrosis induced by excessive β-adrenergic stimulation belongs to necroptosis and is mediated by a RIPK1–RIPK3-dependent pathway, identifying RIPK1 and RIPK3 as potential therapeutic targets for catecholamine surges.

背景：儿茶酚胺激增和由此产生的过度 β-肾上腺素能刺激发生在广泛的疾病中。过度的β-肾上腺素能刺激导致心肌细胞坏死，但其潜在机制仍不清楚。坏死性凋亡是由以 RIPK3 为中心的通路介导的调节性坏死的主要形式，与心力衰竭有关。然而，过度β-肾上腺素能刺激引起的心脏损伤是否涉及坏死性凋亡仍然未知。因此，我们进行了本研究以解决这些关键差距。

方法和结果：向成年混合性别小鼠连续每日两次注射异丙肾上腺素（ISO；85 mg/kg，sc）或生理盐水。在第二次 ISO 注射后 24 小时，伊文思蓝染料 (EBD) 摄取的心脏面积和 CD45、RIPK1、Ser166 磷酸化 RIPK1、RIPK3 和 Ser345 磷酸化 MLKL (p-MLKL) 的心肌蛋白水平显着增加，而在 ISO 处理的小鼠中，Ser321 磷酸化的 RIPK1 显着低于生理盐水处理的野生型 (WT) 小鼠。ISO 引起的 EBD 摄取增加在RIPK3^-/-小鼠与 WT 小鼠相比 (p= 0.016)。用 RIPK1 选择性抑制剂 necrostatin-1 预处理减少了 ISO 诱导的 WT 小鼠中 RIPK3 和 p-MLKL 的增加，并显着减弱了 ISO 诱导的 WT 而非 RIPK3 ^-/-小鼠中 EBD 摄取的增加。

结论：过度 β-肾上腺素能刺激诱导的大部分心肌细胞坏死属于坏死性凋亡，由 RIPK1-RIPK3 依赖性途径介导，将 RIPK1 和 RIPK3 确定为儿茶酚胺激增的潜在治疗靶点。