Regulation of myocardial GPCRs by USP20 in normal and hypertrophied heart

项目概要 慢性压力超负荷导致左心室肥厚 (LVH) 和不良心脏重塑 是心力衰竭的主要原因之一，影响着数百万美国人。除了病理性的 损伤和生物力学因素、涉及通过 β1 协调信号传导的神经激素途径和 β2 肾上腺素能受体 (AR) 在心肌适应压力超负荷方面发挥着重要作用。因素 心肌对压力超负荷的反应从适应性 LVH 转变为适应不良的 LVH 仍然在很大程度上 晦涩难懂。我们的研究重点是 βAR 运输和信号传导的调节 泛素化/去泛素化机制。我们最近发现β1AR和β1AR的差异调节 β2AR 内吞转运可以通过称为泛素特异性蛋白酶 20 的去泛素酶 (DUB) 来实现 (USP20)，我们发现它使两种 βAR 去泛素化。响应 βAR 激动剂 (-)异丙肾上腺素 (Iso)， USP20 在 Ser333 上被蛋白激酶 A (PKA) 磷酸化；这种磷酸化会抑制 USP20 DUB 对 β2AR 的活性并调节 β2AR 向自噬体的运输。 USP20磷酸化 在体内也会发生：(1) 当人类心脏衰竭时，USP20 磷酸化显着升高 与非衰竭心脏相比，以及 (2) 通过主动脉横向缩窄 (TAC) 实现的压力超载 触发 WT 心肌细胞中的 USP20 磷酸化，但不触发 β1AR KO 小鼠。因此，在 LVH 期间， USP20 磷酸化需要心脏中的 β1AR 激活，并且可能在心脏病理重塑中发挥作用 左心室肥厚和/或心力衰竭。我们假设“USP20 及其磷酸化状态微调 βAR 信号转导、内吞运输和自噬，从而影响 LVH 的心脏重塑。” 我们将通过解决以下具体目标来检验我们的假设：（1）确定 心肌细胞-USP20在心功能障碍发展中的作用，（2）确定 通过 USP20 Ser333 磷酸化来调节心肌 βAR 信号传导，以及 (3) 确定协调的 β1AR和USP20在调节自噬蛋白Beclin1及其泛素化状态中的作用 对心肌细胞自噬的影响。

PROJECT SUMMARY Chronic pressure overload that leads to left ventricular hypertrophy (LVH) and adverse cardiac remodeling is one of the leading causes of heart failure and affects millions of Americans. In addition to pathological insults and biomechanical factors, neurohormonal pathways that involve coordinated signaling through β1 and β2 adrenergic receptors (ARs) can play a major role in myocardial adaptation to pressure overload. The factors that shift the myocardial response to pressure overload from adaptive to maladaptive LVH remain largely obscure. Our research has focused on the regulation of βAR trafficking and signaling by ubiquitination/deubiquitination mechanisms. We recently discovered that differential regulation of β1AR and β2AR endocytic trafficking can be achieved by the deubiquitinase (DUB) called ubiquitin-specific protease-20 (USP20), which we found deubiquitinates both βARs. In response to the βAR agonist (-)isoproterenol (Iso), USP20 is phosphorylated on Ser333 by protein kinase A (PKA); this phosphorylation inhibits USP20 DUB activity toward the β2AR and regulates trafficking of the β2AR to autophagosomes. USP20 phosphorylation occurs in vivo, as well: (1) USP20 phosphorylation is significantly elevated in failing human hearts when compared with non-failing hearts and (2) pressure overload achieved by transverse aortic constriction (TAC) triggers USP20 phosphorylation in cardiomyocytes of WT, but not β1AR KO mice. Accordingly, during LVH, USP20 phosphorylation requires β1AR activation in the heart and may play a role in pathologic remodeling in LVH and/or heart failure. We hypothesize that “USP20 and its phosphorylation status fine-tune βAR signal transduction, endocytic trafficking and autophagy, thus impacting cardiac remodeling in LVH.” We will test our hypotheses by addressing the following specific aims: (1) To determine the role of cardiomyocyte-USP20 in the development of cardiac dysfunction, (2) To determine the regulation of myocardial βAR signaling by USP20 Ser333 phosphorylation and (3) To determine the coordinated roles of β1AR and USP20 in regulating ubiquitination status of the autophagy protein Beclin1 and its effect in cardiomyocyte autophagy.