BACKGROUND:Heart failure (HF), which is the terminal stage of many cardiovascular diseases, is associated with low survival rates and a severe financial burden. The mechanisms, especially the molecular mechanism combined with new theories, underlying the pathogenesis of HF remain elusive. We demonstrate that phosphorylation-regulated dynamic liquid–liquid phase separation of HIP-55 (hematopoietic progenitor kinase 1–interacting protein of 55 kDa) protects against HF.METHODS:Fluorescence recovery after photobleaching assay, differential interference contrast analysis, pull-down assay, immunofluorescence, and immunohistochemical analysis were used to investigate the liquid–liquid phase separation capacity of HIP-55 and its dynamic regulation in vivo and in vitro. Mice with genetic deletion of HIP-55 and mice with cardiac-specific overexpression of HIP-55 were used to examine the role of HIP-55 on β-adrenergic receptor hyperactivation-induced HF. Mutation analysis and mice with specific phospho-resistant site mutagenesis were used to identify the role of phosphorylation-regulated dynamic liquid–liquid phase separation of HIP-55 in HF.RESULTS:Genetic deletion of HIP-55 aggravated HF, whereas cardiac-specific overexpression of HIP-55 significantly alleviated HF in vivo. HIP-55 possesses a strong capacity for phase separation. Phase separation of HIP-55 is dynamically regulated by AKT-mediated phosphorylation at S269 and T291 sites, failure of which leads to impairment of HIP-55 dynamic phase separation by formation of abnormal aggregation. Prolonged sympathetic hyperactivation stress induced decreased phosphorylation of HIP-55 S269 and T291, dysregulated phase separation, and subsequent aggregate formation of HIP55. Moreover, we demonstrated the important role of dynamic phase separation of HIP-55 in inhibiting hyperactivation of the β-adrenergic receptor–mediated P38/MAPK (mitogen-activated protein kinase) signaling pathway. A phosphorylation-deficient HIP-55 mutation, which undergoes massive phase separation and forms insoluble aggregates, loses the protective activity against HF.CONCLUSIONS:Our work reveals that the phosphorylation-regulated dynamic phase separation of HIP-55 protects against sympathetic/adrenergic system–mediated heart failure.

中文翻译：

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HIP-55 磷酸化调节的动态相分离可预防心力衰竭

背景：心力衰竭（HF）是许多心血管疾病的晚期阶段，与低存活率和严重的经济负担相关。心力衰竭发病机制，特别是分子机制与新理论相结合，目前仍不清楚。我们证明，HIP-55（55 kDa 的造血祖细胞激酶 1 相互作用蛋白）的磷酸化调节的动态液-液相分离可防止 HF。 方法：光漂白测定、微分干涉对比分析、下拉测定后的荧光恢复，采用免疫荧光和免疫组织化学分析研究HIP-55的液-液相分离能力及其体内外动态调控。使用 HIP-55 基因缺失的小鼠和 HIP-55 心脏特异性过度表达的小鼠来研究 HIP-55 对 β-肾上腺素能受体过度激活诱导的心力衰竭的作用。采用突变分析和小鼠特异性磷酸抗性位点诱变来鉴定磷酸化调节的 HIP-55 动态液-液相分离在心力衰竭中的作用。 结果：HIP-55 基因缺失加重心力衰竭，而心脏特异性过表达则加重心力衰竭。 HIP-55 显着减轻体内心力衰竭。HIP-55具有很强的相分离能力。HIP-55 的相分离由 AKT 介导的 S269 和 T291 位点磷酸化动态调节，磷酸化失败会因形成异常聚集而导致 HIP-55 动态相分离受损。长时间的交感神经过度激活应激会导致 HIP-55 S269 和 T291 磷酸化降低、相分离失调以及随后 HIP55 聚集体的形成。此外，我们证明了 HIP-55 的动态相分离在抑制 β-肾上腺素能受体介导的 P38/MAPK（丝裂原激活蛋白激酶）信号通路过度激活中的重要作用。磷酸化缺陷的 HIP-55 突变会经历大量相分离并形成不溶性聚集体，从而失去对 HF 的保护活性。 结论：我们的工作表明，HIP-55 的磷酸化调节的动态相分离可防止交感神经/肾上腺素能系统–介导的心力衰竭。