Department of Pharmacology, Chonnam National University Medical School, Hwasun, Republic of Korea.
Medical Research Center for Gene Regulation, Chonnam National University Medical School, Hwasun, Republic of Korea.
Cardiovasc Res. 2019 Nov 1;115(13):1850-1860. doi: 10.1093/cvr/cvy317.
Previously, we reported that phosphorylation of histone deacetylase 2 (HDAC2) and the resulting activation causes cardiac hypertrophy. Through further study of the specific binding partners of phosphorylated HDAC2 and their mechanism of regulation, we can better understand how cardiac hypertrophy develops. Thus, in the present study, we aimed to elucidate the function of one such binding partner, heat shock protein 70 (HSP70).
Primary cultures of rat neonatal ventricular cardiomyocytes and H9c2 cardiomyoblasts were used for in vitro cellular experiments. HSP70 knockout (KO) mice and transgenic (Tg) mice that overexpress HSP70 in the heart were used for in vivo analysis. Peptide-precipitation and immunoprecipitation assay revealed that HSP70 preferentially binds to phosphorylated HDAC2 S394. Forced expression of HSP70 increased phosphorylation of HDAC2 S394 and its activation, but not that of S422/424, whereas knocking down of HSP70 reduced it. However, HSP70 failed to phosphorylate HDAC2 in the cell-free condition. Phosphorylation of HDAC2 S394 by casein kinase 2α1 enhanced the binding of HSP70 to HDAC2, whereas dephosphorylation induced by the catalytic subunit of protein phosphatase 2A (PP2CA) had the opposite effect. HSP70 prevented HDAC2 dephosphorylation by reducing the binding of HDAC2 to PP2CA. HSP70 KO mouse hearts failed to phosphorylate S394 HDAC2 in response to isoproterenol infusion, whereas Tg overexpression of HSP70 increased the phosphorylation and activation of HDAC2. 2-Phenylethynesulfonamide (PES), an HSP70 inhibitor, attenuated cardiac hypertrophy induced either by phenylephrine in neonatal ventricular cardiomyocytes or by aortic banding in mice. PES reduced HDAC2 S394 phosphorylation and its activation by interfering with the binding of HSP70 to HDAC2.
These results demonstrate that HSP70 specifically binds to S394-phosphorylated HDAC2 and maintains its phosphorylation status, which results in HDAC2 activation and the development of cardiac hypertrophy. Inhibition of HSP70 has possible application as a therapeutic.
我们之前报道过组蛋白去乙酰化酶 2（HDAC2）的磷酸化及其导致的激活可引起心肌肥厚。通过进一步研究磷酸化 HDAC2 的特定结合伴侣及其调控机制，我们可以更好地了解心肌肥厚的发生机制。因此，本研究旨在阐明其结合伴侣之一热休克蛋白 70（HSP70）的功能。
本研究使用原代培养的新生大鼠心室肌细胞和 H9c2 心肌细胞进行体外细胞实验，使用 HSP70 敲除（KO）小鼠和心脏过表达 HSP70 的转基因（Tg）小鼠进行体内分析。肽沉淀和免疫沉淀实验显示 HSP70 优先与磷酸化的 HDAC2 S394 结合。强制表达 HSP70 增加了 HDAC2 S394 的磷酸化及其激活，但不增加 S422/424 的磷酸化，而 HSP70 敲低则降低了其磷酸化。然而，HSP70 在无细胞条件下不能使 HDAC2 磷酸化。酪蛋白激酶 2α1 磷酸化 HDAC2 S394 增强了 HSP70 与 HDAC2 的结合，而蛋白磷酸酶 2A（PP2CA）催化亚基诱导的去磷酸化则产生相反的效果。HSP70 通过减少 HDAC2 与 PP2CA 的结合来阻止 HDAC2 的去磷酸化。异丙肾上腺素输注不能使 HSP70 KO 小鼠心脏的 HDAC2 S394 发生磷酸化，而过表达 HSP70 则增加了 HDAC2 的磷酸化和激活。HSP70 抑制剂 2-苯乙磺酰胺（PES）可减轻去甲肾上腺素诱导的新生大鼠心室肌细胞肥大和主动脉缩窄诱导的小鼠心肌肥大。PES 通过干扰 HSP70 与 HDAC2 的结合来减少 HDAC2 S394 的磷酸化及其激活。
这些结果表明，HSP70 特异性地与 S394 磷酸化的 HDAC2 结合并维持其磷酸化状态，从而导致 HDAC2 的激活和心肌肥厚的发生。抑制 HSP70 可能具有治疗应用价值。
