Small heat shock proteins (sHsps) protect the heart from chemotherapeutics-induced heart failure, by inhibiting p53-dependant apoptosis. However, mechanism of such protection has not been elucidated yet. Here we test a hypothesis that serine phosphorylation of sHsps is essential to inhibit the Doxorubicin-induced p53-dependent apoptotic pathway. Three transgenic mice (TG) lines with cardiomyocyte specific overexpression of human heat shock protein 27 (hHsp27), namely, wild type (MHC-hHsp27), S82A single mutant (MHC-mut-hHsp27(S82A) and tri-mutant (MHC-mut-hHsp27(S15A/S78A/S82A)) were generated. TG mice were treated with Dox (6mg/kg body weight; once in a week; 4 weeks) along with age-matched non-transgenic (Non-TG) controls. The Dox-treated MHC-hHsp27 mice showed improved survival and cardiac function (both MRI and echocardiography), in terms of contractility (%EF) and left ventricular inner diameter (LVID), compared to the Dox-treated Non-TG mice. However, both MHC-mut-hHsp27(S82A) and MHC-mut-hHsp27(S82A/S15A/S76A) mutants overexpressing TG mice did not show such a cardioprotection. Furthermore, transactivation of p53 was found to be attenuated only in Dox-treated MHC-hHsp27 mice-derived cardiomyocytes in vitro, as low p53 was detected in the nuclei, not in mutant hHsp27 overexpressing cardiomyocytes. Similarly, only in MHC-hHsp27 overexpressing cardiomyocytes, low Bax, higher mTOR phosphorylation and low apoptotic PARP-1 cleavage (89kDa fragment) were detected. Pharmacological inhibition of p53 was more effective in mutant-TG mice, compared to MHC-hHsp27 mice. We conclude that phosphorylation of overexpressed Hsp27 at S82 and its association with p53 is essential for the overall cardioprotective effect of Hsp27 against Dox-induced dilated cardiomyopathy. Only phosphorylated Hsp27 protect the heart by inhibiting p53 transactivation.

中文翻译：

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Hsp27 中的丝氨酸突变消除了其抑制 p53 依赖性细胞凋亡和阿霉素诱导的小鼠心力衰竭的能力

小热休克蛋白 (sHsps) 通过抑制 p53 依赖性细胞凋亡来保护心脏免受化疗药物引起的心力衰竭。然而，这种保护机制尚未阐明。在这里，我们测试了一个假设，即 sHsps 的丝氨酸磷酸化对于抑制多柔比星诱导的 p53 依赖性细胞凋亡途径至关重要。三种具有心肌细胞特异性过表达人热休克蛋白 27 (hHsp27) 的转基因小鼠 (TG) 系，即野生型 (MHC-hHsp27)、S82A 单突变体 (MHC-mut-hHsp27(S82A) 和三突变体 (MHC- mut-hHsp27(S15A/S78A/S82A)) 用 Dox（6mg/kg 体重；每周一次；4 周）和年龄匹配的非转基因（非 TG）对照处理 TG 小鼠。 Dox 处理的 MHC-hHsp27 小鼠显示出改善的存活率和心脏功能（MRI 和超声心动图），在收缩力 (%EF) 和左心室内径 (LVID) 方面，与 Dox 处理的非 TG 小鼠相比。然而，过表达 TG 小鼠的 MHC-mut-hHsp27(S82A) 和 MHC-mut-hHsp27(S82A/S15A/S76A) 突变体都没有表现出这样的心脏保护作用。此外，发现 p53 的反式激活仅在体外 Dox 处理的 MHC-hHsp27 小鼠衍生的心肌细胞中减弱，因为在细胞核中检测到低 p53，而不是在过表达 hHsp27 的突变体心肌细胞中。类似地，仅在 MHC-hHsp27 过表达的心肌细胞中，检测到低 Bax、更高的 mTOR 磷酸化和低凋亡 PARP-1 切割（89kDa 片段）。与 MHC-hHsp27 小鼠相比，p53 的药理学抑制在突变型 TG 小鼠中更有效。我们得出结论，S82 处过度表达的 Hsp27 的磷酸化及其与 p53 的关联对于 Hsp27 对 Dox 诱导的扩张型心肌病的整体心脏保护作用至关重要。只有磷酸化的 Hsp27 通过抑制 p53 反式激活来保护心脏。