From the Goethe University, Frankfurt am Main, Germany (F. Spallotta, C.C., S.A., S.Z., D.S., F. Schnütgen, H.v.M., A.F., I.F., A.M.Z., C.G.); University of Turin, Torino, Italy (D.G., M. Cocco, R.M., A.D.S., M.A., M. Collino, M. Bertinaria); Istituto Italiano di Tecnologia CLNS@Sapienza Rome, Italy (M.M.); Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (C.K., S.G., T.B.); Università Cattolica del Sacro Cuore, Rome, Italy (S.N.); Karolinska Institutet, Huddinge, Sweden (V.A.); University of Mainz, Germany (A.K., A.B.-F.); NXT-Dx, Ghent, Belgium (M. Braspenning); Ghent University, Belgium (W.v.C.); Baker IDI Heart and Diabetes Institute, Melbourne VIC, Australia (M.J.D.B., R.H.R.); IRCCS Policlinico San Donato, Milan, Italy (G.Z., F.M.); National Research Council, Rome, Italy (A.F., C.C.); and Sapienza University, Rome, Italy (B.B.).
Circ Res. 2018 Jan 5;122(1):31-46. doi: 10.1161/CIRCRESAHA.117.311300. Epub 2017 Nov 20.
Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes.
To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs.
Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response.
Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.
人心肌间充质细胞（CMSCs）是一种具有治疗相关性的原代细胞群体。由于代谢改变和稳定的表观遗传变化的掺入，糖尿病会损害 CMSC 的功能。
研究α-酮戊二酸（αKG）在 CMSCs 中 DNA 去甲基化的代谢控制中的作用。
定量全基因组分析、甲基化和羟甲基化 DNA 测序以及基因特异性 GC 甲基化检测显示，从糖尿病供体分离的人心肌间充质细胞的基因组 DNA 中 5-甲基胞嘧啶、5-羟甲基胞嘧啶和 5-甲酰基胞嘧啶积累。全心脏基因组 DNA 分析显示，高脂饮食（HFD）、链脲佐菌素注射或两者联合（链脲佐菌素/HFD）暴露的小鼠心脏中反复发生氧化胞嘧啶修饰积累。在这种情况下，非靶向和靶向代谢组学表明，糖尿病 CMSCs 和 HFD 小鼠心脏中的细胞内 αKG 合成减少。这一观察结果与 TDG（胸腺嘧啶 DNA 糖基化酶）和 TET1（十-十一易位蛋白 1）与 TET1 脱离核的关联和功能受损相平行。分子动力学和突变分析表明，αKG 在精氨酸 275 上结合 TDG，提供酶的变构激活。因此，该酶显著增加了其去除 G/T 核苷酸错配或 5-甲酰基胞嘧啶的能力。因此，αKG 的外源性来源通过促进 TDG 功能、TET1 核定位和 TET/TDG 关联，恢复了 DNA 去甲基化循环。CRISPR/Cas9 敲除或 TET/TDG siRNA 敲低导致 TDG 失活，导致 5-甲酰基胞嘧啶积累，从而在非糖尿病起源的细胞中部分模拟糖尿病表观遗传景观。鉴定为 αKG 脱氢酶抑制剂的新型化合物（S）-2-[(2,6-二氯苯甲酰)氨基]琥珀酸（AA6）增加了糖尿病 CMSCs 和 HFD 和链脲佐菌素小鼠心脏中的 αKG 水平，在 HFD 中引起 DNA 去甲基化、葡萄糖摄取和胰岛素反应。
恢复 DNA 去甲基化循环的代谢控制有望对受环境代谢变化影响的细胞产生有益影响。
