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

在退伍军人(VA)医疗系统中，心力衰竭(HF)是患者入院的首要原因， 消耗了退伍军人管理局医疗资源的很大一部分。然而，目前对心衰的分子机制研究较少。 认识和治疗心衰仍停留在控制症状、降低危险因素的水平 没有治愈的方法。因此，进一步研究开发针对心力衰竭发病机制的新疗法 是一种迫切的需要。我们的初步研究表明，一种脱泛素酶泛素羧基- 末端水解酶L1(UCH-L1)在小鼠和人衰竭心脏的心肌细胞中表达上调。在……里面 此外，心肌细胞限制性(CR)转基因UCH-L1(CR-UCH-L1TG)的过度表达被夸大 压力超负荷(PO)诱导的小鼠心脏病理重塑和功能障碍 心肌病和心衰，以及CR-UCH-L1 TG诱导的不良表型可以通过 使用可逆的、竞争性的、作用位点定向的Uch-L1、LDn-57444抑制剂进行治疗。此外，UCH-L1 能够抑制PO心脏的自噬，这是保护心脏的关键适应机制 抗PO引起的心肌病和心力衰竭。在分子水平上，UCH-L1最有可能促进 MTORC1(雷帕霉素复合体1的机制靶点)依赖于ULK1(非配位-51样蛋白)的失活 激酶1)介导的自噬诱导和DAP1(死亡相关蛋白1)介导的激活 抑制心肌细胞的自噬通量。有趣的是，胞外体UCH-L1的循环水平是 可能是通过自噬抑制(AI)诱导的外体负荷增加和 PO-心脏心肌细胞分泌UCH-L1的研究总体而言，我们的发现令人信服地支持 假设靶向UCH-L1是治疗心力衰竭和循环外体UCH的新方法。 L1可作为心力衰竭的新生物标志物。这一假设将通过三个特定的目标在小鼠模型中进行验证 以及对VA心衰患者的治疗如下：目标1是确定靶向UCH-L1在心力衰竭中的治疗潜力 在老鼠身上。CR-UCH-L1基因敲除对PO的影响及UCH-L1抑制剂LDN-57444的作用 诱发性心肌病和心力衰竭将被研究。目标2是确定分子机制，通过它 Uch-L1介导HF，验证了Uch-L1控制mTORC1组装有利于 在S757增加mTOR对ULK1的访问以磷酸化ULK1，同时减少结合 MTOR与DAP1一起去磷酸化DAP1以增强心肌细胞AI，从而夸大 心脏病理性重构和功能障碍。我们将确定CR-UCH-L1 TG是否会导致不良反应 通过CR过度表达自噬进一步增强心脏自噬可挽救表型 相关基因(ATG)7或自噬激活剂雷帕霉素。此外，我们还将剖析信号机制，通过它 UCH-L1抑制自噬，最初集中在mTORC1组装的调节上，从而抑制 ULK1介导的自噬诱导和增强DAP1介导的自噬通量抑制 心肌细胞。目标3是定义PO诱导的心脏UCH-L1的上调和释放，重点是 UCH-L1体外释放进入循环动物模型的分子机制及诊断 和/或循环外体UCH-L1在VA HF患者中的预后价值。虽然人工智能在驾驶中的关键作用 热休克蛋白70(HSC70)介导的UCH-L1和分泌载体胞外负载的同源基因 心肌细胞膜蛋白5(SCAMP5)介导的UCH-L1胞外积聚释放 UCH-L1将被剖析，循环外体UCH-L1是一种新的生物标志物 将对VA患者的心衰进行检测。该结果将为以UCH-L1为靶点的翻译研究铺平道路 治疗和管理心脏病和心力衰竭。因此，这项提案的成果除了建立一个 与退伍军人管理局医学研究任务相关的独特和独立的研究计划，结果是 降低VA患者群体内心力衰竭负担的机械性治疗方法。

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.