Retinoid Metabolism in the Adult Heart and Heart Failure

项目摘要 本研究旨在探讨类维生素A代谢改变在心力衰竭中的作用 （HF）.我们最近发现，在特发性扩张型心肌病（IDCM）和实验性心脏 心力衰竭（HF）时，维生素A代谢物和强效激素的心脏水平下降高达40%， 全反式维甲酸（ATRA），尽管有足够的维生素A。我们还表明，直接管理 ATRA可预防心力衰竭，以应对压力过载。然而，对HF实施ATRA治疗可能面临 考虑到ATRA信号的多效性和ATRA在循环中的有限半衰期，可选择地， 通过靶向心脏类维生素A代谢的酶可能最好地实现治疗特异性。 该建议解决了我们对ATRA机制的理解中的3个关键知识缺口 代谢及其对出生后心脏的影响。1.负责新陈代谢的酶 在成年哺乳动物心脏中视黄醇到视黄酸的变化是未知的。我们已经开始确认 酶在人干细胞衍生的心肌细胞（hSC-CM）中的作用。我们发现，Dhrs 4调节 人干细胞衍生的心肌细胞hSC-CM中的视黄醇库。我们详细介绍了一个工作流程，以验证其他 候选人，利用先进的hSC-CM和人类工程心脏组织（hEHT）。2.的 临床相关的心脏ATRA功能不全的病理生理机制尚未得到解决。 细胞视黄醇结合蛋白1（Crbp 1）的敲除重现了在人类IDCM中观察到的ATRA下降。 在此，我们发现ATRA的下降足以引起舒张功能障碍和缓慢的肌原纤维松弛 动力学我们通过条件性心脏基因敲除，在小鼠中评估了视黄醇还原酶Dhrs 4的作用。 心我们验证了这样一个假设，即加强心肌细胞ATRA可以预防甚至挽救HF， 骑自行车过桥我们建议操纵DHRS 4和ATRA水平，以识别ATRA敏感的 小鼠心脏中的转录程序和hEHT。3. ATRA下降在HF病因中的普遍程度 尽管缺血性心肌病（ICM）和射血功能保留的HF的蛋白质组学生物特征尚不清楚， 分数（HFpEF）与ATRA下降一致。即便如此，另一项研究表明，心脏ATRA增加 在晚期冠心病的情况下。确定适合ATRA稳态治疗的队列 需要在HF病因学中量化HF中ATRA变化的幅度和方向。我们将 定量类维生素A和类维生素A相关蛋白多种病因，包括射血分数降低的HF （HFrEF）、HFpEF、ICM和肥厚型心肌病（HCM），使用最先进的靶向肿块 通过质谱分析（LC-MS 3，isPRM）测定它们的浓度，并使用MALDI-MS成像检查它们在心脏内的分布。 重申，这一建议从根本上解决了新的心脏生物学的转录主调节 利用人类心脏细胞工程的最新进展， 模型、先进的质谱技术和人类HF队列。

Project Summary This proposal examines the previously unaddressed role of altered retinoid metabolism in heart failure (HF). We have recently shown that in both idiopathic dilated cardiomyopathy (IDCM) and experimental heart failure (HF) there is up to a 40% decline in the cardiac levels of the vitamin A metabolite and potent hormone, all-trans retinoic acid (ATRA), despite adequate vitamin A. We have also shown that direct administration of ATRA prevents HF, in response to pressure-overload. However, implementing ATRA therapy for HF could face headwinds, given the pleiotropy of ATRA signaling and the limited half-life of ATRA in the circulation. Alternatively, therapeutic specificity might best be achieved by targeting the enzymes of cardiac retinoid metabolism. This proposal addresses 3 critical knowledge gaps in our understanding of the mechanism of ATRA metabolism and its impact on the post-natal heart. 1. The enzymes that are responsible for the metabolism of retinol to retinoic acid in the adult mammalian heart are unknown. We have begun to identify the pertinent enzymes in human stem cell-derived cardiomyocytes (hSC-CMs). We show that Dhrs4 regulates the retinaldehyde pool in human stem-cell-derived cardiomyocytes hSC-CMs. We detail a workflow to validate other candidates, leveraging advances hSC-CMs and human engineered heart tissue (hEHT). 2. The pathophysiological mechanisms of a clinically-pertinent cardiac ATRA insufficiency have not been addressed. Knockout of cellular retinol-binding protein 1 (Crbp1) recapitulates the ATRA decline seen in human IDCM. Herein, we show the ATRA decline is sufficient to cause diastolic dysfunction and slow myofibrillar relaxation kinetics. We evaluate the role of the retinaldehyde reductase Dhrs4 by conditional cardiac knockout in the mouse heart. We test the hypothesis that boosting cardiomyocyte ATRA can prevent or even rescue HF and ameliorate cross-bridge cycling. We propose to manipulate DHRS4 and ATRA levels to identify ATRA-sensitive transcriptional programs in mouse hearts, and hEHT. 3. How pervasive the ATRA decline is across HF etiologies is unknown, though proteomic biosignatures of ischemic cardiomyopathy (ICM) and HF with preserved ejection fraction (HFpEF) are consistent with ATRA decline. Even so, another study has shown increased cardiac ATRA in the setting of advanced coronary heart disease. Identifying suitable cohorts for ATRA homeostasis therapy requires that the magnitude and direction of ATRA changes in HF be quantified across HF etiologies. We will quantify retinoids and retinoid-associated protein multiple etiologies including HF with reduced ejection fraction (HFrEF), HFpEF, ICM, and hypertrophic cardiomyopathy (HCM), using state-of-the-art targeted mass spectrometry assays (LC-MS3, isPRM) and examine their distribution within the heart using MALDI-MS Imaging. Restated, this proposal addresses fundamentally novel cardiobiology of a transcriptional master regulator with translational potential, by leveraging the latest advances in human heart cell engineering, novel mouse models, advanced mass spectrometry techniques, and human HF cohorts.