Regulation of myocardial GPCRs by USP20 in normal and hypertrophied heart

项目摘要 慢性压力超负荷导致左心室肥大（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 损伤和生物力学因素，涉及通过β 1和β 2的协调信号传导的神经激素途径， β 2肾上腺素能受体（AR）在心肌对压力超负荷的适应中起重要作用。的因素 使心肌对压力超负荷的反应从适应性LVH转变为不适应性LVH， 晦涩难懂。我们的研究集中在β AR运输和信号转导的调节， 泛素化/去泛素化机制。我们最近发现β 1 AR和β 2 AR的差异调节， β 2 AR的内吞运输可以通过称为泛素特异性蛋白酶-20的去泛素化酶（DUB）来实现 （USP 20），我们发现它可以使两种β AR去泛素化。对β AR激动剂（-）异丙肾上腺素（Iso）的反应， USP 20通过蛋白激酶A（PKA）在Ser333上磷酸化;这种磷酸化抑制USP 20 DUB 对β 2AR的活性，并调节β 2AR向自噬体的运输。USP20磷酸化 在体内也发生：（1）当发生以下情况时，USP 20磷酸化在衰竭的人类心脏中显著升高： 与非衰竭心脏相比;（2）通过横向主动脉缩窄（TAC）实现的压力超负荷 在WT而不是β 1AR KO小鼠的心肌细胞中触发USP20磷酸化。因此，在LVH期间， USP20磷酸化需要心脏中的β 1 AR活化，并可能在心脏病的病理性重塑中发挥作用。 LVH和/或心力衰竭。我们假设"USP20及其磷酸化状态微调β AR， 信号转导、内吞运输和自噬，从而影响LVH中的心脏重塑。 我们将通过以下具体目标来检验我们的假设：（1）确定 心肌细胞-USP20在心功能不全的发展中的作用，（2）确定 通过USP20 Ser333磷酸化的心肌β AR信号传导和（3）为了确定协调的 β 1 AR和USP 20在调节自噬蛋白Beclin 1泛素化状态中的作用及其意义 在心肌细胞自噬中的作用。

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.