To explore the potential of UCH-L1 as a novel therapeutic and diagnostic target in heart failure

心力衰竭（HF）是退伍军人（VA）卫生系统中患者入院的头号原因， 消耗了退伍军人事务部很大一部分医疗资源然而，HF的分子机制很差 HF的治疗仍然停留在控制症状和减少危险因素的水平上 无法治愈因此，进一步研究开发针对HF的致病机制特异性新疗法 这是一个迫切的需要。我们的初步研究表明，去泛素化酶，泛素羧基- 末端水解酶L1（UCH-L1）在小鼠和人类衰竭心脏的心肌细胞中上调。在 此外，心肌细胞限制性（CR）UCH-L1转基因过表达（CR-UCH-L1 Tg） 压力超负荷（PO）诱导的小鼠模型中的心脏病理性重塑和功能障碍 心肌病和HF，CR-UCH-L1 TG诱导的不良表型可以通过 用UCH-L1的可逆、竞争性、act-site定向抑制剂LDN-57444治疗。此外，UCH-L1 能够抑制PO-心脏中的自噬，这是保护 对抗PO诱导的心肌病和HF。在分子水平上，UCH-L1最有可能促进 mTORC 1（雷帕霉素复合物1的机制靶点）依赖性ULK 1（非配位-51样）失活 激酶1）介导的自噬诱导和DAP 1（死亡相关蛋白1）介导的活化 抑制心肌细胞中的自噬流。有趣的是，外泌体UCH-L1的循环水平是 通过自噬抑制（AI）诱导的外泌体负载增加的机制， PO-心脏的心肌细胞中UCH-L1的分泌。总的来说，我们的研究结果有力地支持了 假设靶向UCH-L1是治疗HF和循环外泌体UCH的新方法， L1是HF的新生物标志物。这一假设将通过小鼠模型中的三个特定目标进行测试， 以及VA HF患者如下：目的1是确定靶向UCH-L1在HF中的治疗潜力 对小鼠CR-UCH-L1敲除（KO）的影响和UCH-L1抑制剂LDN-57444对PO-1的疗效。 将研究诱发的心肌病和HF。目的2是确定分子机制， UCH-L1介导HF，检验了UCH-L1控制mTORC 1组装的假设， 增加mTOR与ULK 1的接触，使ULK 1在S757处磷酸化，同时降低mTOR与ULK 1的相关性。 mTOR与DAP 1的结合用于DAP 1的去磷酸化以增强心肌细胞中的AI，从而夸大了 心脏病理性重塑和功能障碍。我们将确定CR-UCH-L1 TG诱导的不良反应是否 通过自噬的CR过表达，通过额外增强心脏自噬来挽救表型 相关基因（Atg）7或自噬激活剂、雷帕霉素。此外，我们将剖析信号机制， UCH-L1抑制自噬，最初集中于mTORC 1组装的调节，从而抑制自噬。 ULK 1介导的自噬诱导，同时增强DAP 1介导的自噬通量抑制， 心肌细胞目的3是确定PO诱导的心脏UCH-L1的上调和释放，重点是 外泌体释放UCH-L1进入循环的分子机制及其诊断 和/或VA HF患者中循环外泌体UCH-L1的预后价值。虽然人工智能在驾驶中的关键作用 热休克蛋白70（HSC 70）介导的UCH-L1外泌体负载和分泌载体的同源物 心肌细胞中细胞膜蛋白5（SCAMP 5）介导的UCH-L1外泌体释放 UCH-L1将被解剖，循环外泌体UCH-L1是一种新的生物标志物的概念，用于评估 将检测VA患者的HF。该结果将为靶向UCH-L1的转化研究铺平道路， 治疗和管理心脏病和HF。因此，除了建立一个 与VA医学研究使命相关的独特和独立的研究计划，导致 用于降低VA患者人群中HF负担的基于机制的治疗方法。

Heart failure (HF) is the number one reason for admission among patients in the Veteran (VA) health system, consuming a significant portion of VA medical resources. However, the molecular mechanism of HF is poorly understood and the treatment of HF still remains at the level of controlling symptom and reducing risk factors without a cure. Hence, further research into developing pathogenic mechanism-specific novel therapies for HF is an urgent need. Our pilot studies have demonstrated that a deubiquitinating enzyme, ubiquitin carboxyl- terminal hydrolase L1 (UCH-L1) is upregulated in the cardiomyocytes of mouse and human failing hearts. In addition, cardiomyocyte-restricted (CR) transgenic overexpression of UCH-L1 (CR-UCH-L1 Tg) exaggerates cardiac pathological remodeling and dysfunction in a mouse model of pressure overload (PO)-induced cardiomyopathy and HF, and the CR-UCH-L1 Tg-induced adverse phenotypes could be rescued by the treatment with a reversible, competitive, act-site directed inhibitor of UCH-L1, LDN-57444. Moreover, UCH-L1 is capable of suppressing autophagy in PO-hearts, which serves as crucial adaptive mechanism to protect against PO-induced cardiomyopathy and HF. At the molecular level, it is most likely that UCH-L1 facilitates mTORC1 (mechanistic target of rapamycin complex 1) dependent inactivation of ULK1 (uncoordinated-51-like kinase 1)-mediated autophagy induction and the activation of DAP1 (death-associated protein 1)-mediated inhibition of autophagy flux in cardiomyocytes. Interestingly, the circulating level of exosomal UCH-L1 is elevated conceivably via a mechanism of autophagy inhibition (AI)-induced increases in exosomal loading and secretion of UCH-L1 in cardiomyocytes of PO-hearts. Collectively, our findings compellingly support the hypothesis that targeting UCH-L1 is a novel approach for the treatment of HF and circulating exosomal UCH- L1 serves as novel biomarker of HF. This hypothesis will be tested by three specific aims in mouse models as well as in VA HF patients as follows: Aim 1 is to determine the therapeutic potential of targeting UCH-L1 in HF in mice. The impact of CR-UCH-L1 knockout (KO) and the efficacy of UCH-L1 inhibitor, LDN-57444 on PO- induced cardiomyopathy and HF will be investigated. Aim 2 is to determine the molecular mechanism by which UCH-L1 mediates HF, testing the hypothesis that UCH-L1 controls the assembly of mTORC1 in favor of increasing the access of mTOR to ULK1 for phosphorylation of ULK1 at S757 while decreasing the association of mTOR with DAP1 for dephosphorylation of DAP1 to enhance AI in cardiomyocytes, thereby exaggerating cardiac pathological remodeling and dysfunction. We will determine whether CR-UCH-L1 Tg-induced adverse phenotypes are rescued by additional enhancement of cardiac autophagy via CR overexpression of autophagy related gene (Atg)7 or autophagy activator, rapamycin. Also, we will dissect the signaling mechanism by which UCH-L1 inhibits autophagy with an initial focus on the regulation of mTORC1 assembly thereby inhibiting ULK1-mediated autophagy induction while enhancing DAP1-mediated suppression of autophagy flux in cardiomyocytes. Aim 3 is to define PO-induced upregulation and release of cardiac UCH-L1 with a focus on the molecular mechanism for exosomal release of UCH-L1 into circulation in animal models and the diagnostic and/or prognostic value of circulating exosomal UCH-L1 in VA HF patients. While a critical role of AI in driving cognate of heat shock protein 70 (HSC70)-mediated exosomal loading of UCH-L1 and secretory carrier membrane protein 5 (SCAMP5)-mediated exosomal release of UCH-L1 in cardiomyocytes with accumulated UCH-L1 will be dissected, the concept that circulating exosomal UCH-L1 is a novel biomarker for evaluating HF in VA patients will be tested. The outcome will pave the way for translational studies of targeting UCH-L1 to treat and manage cardiac disease and HF. Thus, this proposal outcome will, in addition to establishing a unique and independent research program relevant to the VA medical research mission, result in a mechanistically based therapeutic approach for reducing the HF burden within the VA patient population.