From the Division of Cardiology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, MD (C.U.O., T.N., S.P., S.M., K.M.K.-S., B.L.L., A.C., G.Z., D.B., D.I.L., D.A.K., M.J.R.).
Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, Japan (T.N.).
Circ Res. 2020 Jul 31;127(4):522-533. doi: 10.1161/CIRCRESAHA.119.315714. Epub 2020 May 12.
Stimulated PKG1α (protein kinase G-1α) phosphorylates TSC2 (tuberous sclerosis complex 2) at serine 1365, potently suppressing mTORC1 (mechanistic [mammalian] target of rapamycin complex 1) activation by neurohormonal and hemodynamic stress. This reduces pathological hypertrophy and dysfunction and increases autophagy. PKG1α oxidation at cysteine-42 is also induced by these stressors, which blunts its cardioprotective effects.
We tested the dependence of mTORC1 activation on PKG1α C42 oxidation and its capacity to suppress such activation by soluble GC-1 (guanylyl cyclase 1) activation.
Cardiomyocytes expressing wild-type (WT) PKG1α (PKG1α) or cysteine-42 to serine mutation redox-dead (PKG1α) were exposed to ET-1 (endothelin 1). Cells expressing PKG1α exhibited substantial mTORC1 activation (p70 S6K [p70 S6 kinase], 4EBP1 [elF4E binding protein-1], and Ulk1 [Unc-51-like kinase 1] phosphorylation), reduced autophagy/autophagic flux, and abnormal protein aggregation; all were markedly reversed by PKG1α expression. Mice with global knock-in of PKG1α subjected to pressure overload (PO) also displayed markedly reduced mTORC1 activation, protein aggregation, hypertrophy, and ventricular dysfunction versus PO in PKG1α mice. Cardioprotection against PO was equalized between groups by co-treatment with the mTORC1 inhibitor everolimus. TSC2-S1365 phosphorylation increased in PKG1α more than PKG1α myocardium following PO. TSC2 (TSC2 S1365 phospho-null, created by a serine to alanine mutation) knock-in mice lack TSC2 phosphorylation by PKG1α, and when genetically crossed with PKG1α mice, protection against PO-induced mTORC1 activation, cardiodepression, and mortality in PKG1α mice was lost. Direct stimulation of GC-1 (BAY-602770) offset disparate mTORC1 activation between PKG1α and PKG1α after PO and blocked ET-1 stimulated mTORC1 in TSC2-expressing myocytes.
Oxidation of PKG1α at C42 reduces its phosphorylation of TSC2, resulting in amplified PO-stimulated mTORC1 activity and associated hypertrophy, dysfunction, and depressed autophagy. This is ameliorated by direct GC-1 stimulation.
受刺激的蛋白激酶 G-1α（protein kinase G-1α）可使 TSC2（结节性硬化复合物 2）丝氨酸 1365 磷酸化，从而强烈抑制神经激素和血液动力应激引起的 mTORC1（机械 [哺乳动物] 雷帕霉素靶蛋白复合物 1）激活。这减少了病理性肥大和功能障碍，并增加了自噬。这些应激源还诱导 PKG1α 半胱氨酸 42 上的氧化，从而削弱其心脏保护作用。
我们测试了 mTORC1 激活对 PKG1α C42 氧化的依赖性及其通过可溶性 GC-1（鸟苷酸环化酶 1）激活来抑制这种激活的能力。
表达野生型（WT）PKG1α（PKG1α）或半胱氨酸 42 至丝氨酸突变的氧化还原失活（PKG1α）的心肌细胞暴露于 ET-1（内皮素 1）。表达 PKG1α 的细胞表现出大量的 mTORC1 激活（p70 S6K[p70 S6 激酶]，4EBP1[eIF4E 结合蛋白-1]和 Ulk1[Unc-51 样激酶 1]磷酸化），减少自噬/自噬通量，并异常蛋白聚集；所有这些都被 PKG1α 的表达明显逆转。在压力超负荷（PO）下全身敲入 PKG1α 的小鼠也显示出明显减少的 mTORC1 激活，与 PO 相比，蛋白聚集，肥大和心室功能障碍。通过共同治疗 mTORC1 抑制剂 everolimus，两组之间的 PO 保护作用均等化。PKG1α 后 PO 时 PKG1α 心肌中的 TSC2-S1365 磷酸化增加。TSC2（TSC2 S1365 磷酸化无效，通过丝氨酸到丙氨酸突变创建）敲入小鼠缺乏 PKG1α 的 TSC2 磷酸化，并且当与 PKG1α 小鼠遗传交叉时，PO 诱导的 mTORC1 激活，心功能抑制和 PKG1α 小鼠的死亡率丧失。GC-1（BAY-602770）的直接刺激抵消了 PO 后 PKG1α 和 PKG1α 之间不同的 mTORC1 激活，并阻断了 TSC2 表达心肌细胞中 ET-1 刺激的 mTORC1。
PKG1α 半胱氨酸 42 上的氧化减少了其对 TSC2 的磷酸化，导致增强的 PO 刺激的 mTORC1 活性和相关的肥大，功能障碍和抑制自噬。这可以通过直接 GC-1 刺激来改善。
