Targeting Immune-Responsive Gene 1 (Irg1) and Itaconate for Cardioprotection of the Donor Heart for Transplantation

心脏移植被认为是终末期心力衰竭的黄金标准治疗方法。然而，需求 由于多重挑战，目前远远供不应求。一个重要的限制是发生了初级 移植物功能障碍(PDG)在10%-20%的患者中存在，并极大地导致不利的临床结果和 资源利用率。当供心功能和输出量不足时，就会发生PGD。风险 供心保存时间大于4h时，PDG显著升高。丙戊酸(VPA)， 组蛋白去乙酰酶抑制剂，是美国食品和药物管理局(FDA)批准的药物，传统上用于 癫痫的治疗。我们现在令人信服地证明，添加VPA可以显著提高 与组氨酸-色氨酸-色氨酸保存相比，供心功能和改善缺血耐受性 单用酮戊二酸(HTK)保存液。在小鼠心脏再灌流模型中发现了这一点 体外灌流和移植的设置。此外，我们展示了VPA通过以下方式实现这一点的证据 上调三元酸循环酶IRG1，该酶产生抗炎代谢物“衣康酸”。 事实上，我们的心脏再灌注模型证实了IRG1在基线水平上的显著上调。 VPA治疗，伴随着抗氧化途径机制的强劲激活 NRF2转录因子。染色质免疫沉淀显示VPA处理使IRG1增强子增加 活性通过乙酰化H3K27组蛋白占有率的增加来指示。重要的是，VPA治疗 保存的人供体心脏也上调了IRG1的表达，降低了炎性细胞因子的表达 建议翻译与大型动物和人类临床环境相关的调解人。对于这项建议，我们 计划：(1)确定IRG1起作用的细胞类型，我们假设它最有可能通过 心肌细胞(CM)和内皮细胞(EC)。这是使用具有条件的转基因小鼠实现的 应用诱导型Cre-Lox技术检测这些细胞类型中IRG1的缺失。我们还将检查过度表达 用腺相关病毒介导的模型表达IRG1mRNA。(2)利用细胞培养，我们将确定 IRG1/衣康酸介导的Nrf2途径抗氧化蛋白的烷基化修饰是否影响其 功能。我们将用衣康酸处理细胞，然后鉴定和突变相关的烷基化修饰。 位于半胱氨酸残基的抗氧化蛋白质决定其重要性。(3)我们会决定 丙戊酸改善猪和人供心功能和缺血耐受性的效果。我们还将 在小鼠模型中证实丙戊酸介导的心脏保护机制。该项目具有关键的 临床意义，如降低PGD的发生率，允许供者心脏运输更长时间 促进器官分配的距离，并改善临床移植结果。减少 利用VPA和IRG1/衣康酸的心脏保护作用造成围手术期供心损伤是一种新的方法 并可能与肝脏和肾脏等其他器官的保存相关。

Heart transplantation is considered gold standard therapy for end-stage heart failure. However, demand currently far outstrips supply due to multiple challenges. An important limitation is the occurrence of primary graft dysfunction (PDG) in 10-20% of patients and contributes greatly to adverse clinical outcomes and resource utilization. PGD occurs when donor heart function and output is inadequate end organ perfusion. Risk for significant PDG increases when donor heart preservation time is greater than 4 hours. Valproic acid (VPA), a histone deacetylase inhibitor, is a “Food and Drug Administration (FDA)” approved drug traditionally used for the treatment of epilepsy. We now convincingly demonstrate that addition of VPA can dramatically improve donor heart function and improve ischemic tolerance compared to preservation using Histidine-Tryptophan- Ketoglutarate (HTK) preservation solution alone. This was seen in murine heart reperfusion models in the setting of ex-vivo perfusion and transplantation. Furthermore, we show evidence that VPA achieves this by upregulating tricarboxylic acid cycle enzyme Irg1 which produces the anti-inflammatory metabolite “itaconate”. Indeed, our cardiac reperfusion model confirms the impressive upregulation of Irg1 above baseline driven by VPA treatment, and this was accompanied by robust activation of antioxidant pathway mechanisms through Nrf2 transcription factor. Chromatin immunoprecipitation showed that VPA treatment increased Irg1 enhancer activity as indicated by increased occupancy by acetylated H3K27 histone. Importantly, VPA treatment of stored human donor hearts also upregulated Irg1 expression and decreased the expression of inflammatory mediators suggesting translational relevance for large animal and human clinical settings. For this proposal, we plan to: (1) Identify the cell type through which Irg1 acts and we hypothesize that it is most likely through cardiomyocytes (CM) and endothelial cells (EC). The is achieved using transgenic mice with conditional deficiency of Irg1 in these cell types using inducible Cre-Lox technology. We will also examine overexpression models using adeno-associated virus mediated expression Irg1 mRNA. (2) Using cell culture, we will determine whether Irg1/itaconate mediated alkylation modifications on Nrf2 pathway antioxidant proteins impacts their function. We will treat cells with itaconate and then identify as well as mutate relevant alkylation modifications sites at the cysteine residue of antioxidant proteins to determine their importance. (3) We will determine the efficacy of VPA for improving donor heart function and ischemic tolerance in pigs and humans. We will also corroborate mechanisms of VPA mediated cardioprotection identified in murine models. This project has critical clinical implications such as decreasing the PGD incidence, allow transport of donor hearts over longer distances to facilitate organ allocation, and improve clinical transplantation outcomes. Reduction in perioperative donor heart injury by harnessing the cardioprotective effect of VPA and Irg1/itaconate is novel and potentially relevant for preservation of other organs such as the livers and kidneys.