Investigating Novel Regulatory Mechanisms of the Cardiac Calcium Pump by Inhibitory and Stimulatory Micropeptides.

项目概要/摘要 这两个目标项目的目标是探索调节心脏钙转运蛋白的动态机制， 塞卡。 SERCA 在心动周期中发挥着核心作用；因此，它的调节对于生存和适应至关重要 以满足不断变化的生理需求。这种调节主要由两种跨膜微肽介导： 抑制肽 Phospholamban (PLB) 和刺激肽矮开放阅读框 (DWORF)。这些 监管者竞争结合 SERCA 并控制其功能。我们发现细胞内 Ca2+ 升高会驱动 心肌收缩也会导致 SERCA 与 PLB 和 DWORF 的结合平衡发生动态变化。 具体来说，Ca2+升高同时降低SERCA对PLB的亲和力并增加SERCA对PLB的亲和力。 双狼。预计这会在 Ca2 瞬变峰值期间降低 SERCA 的抑制并增加刺激。 此外，我们的初步结果表明，PLB 单体的动态部分在 SERCA 过程中脱离 Ca2+升高被动态地隔离在PLB五聚体中。 PLB 五聚体缓慢解开导致 PLB 在快速心脏起搏过程中以五聚体形式积累，将 PLB 与 SERCA 隔离，以降低运动时的抑制 心率。目标 1 将探索 PLB 五聚体的这种频率依赖性积累如何介导关键的 PLB 在鲍迪奇效应中的作用，鲍迪奇效应是一种积极的力-频率关系，其中更快的心率会产生更大的力 心脏收缩。这种现象是调整运动心输出量的关键机制，重要的是， 缺乏心力衰竭。因此，实验将检验这种新机制在生理过程中是如何改变的。 心脏对肾上腺素的反应以及与心力衰竭相关的 PLB 突变的病理学反应。这些见解可能揭示 为什么携带这些突变的患者更容易出现心律失常/心力衰竭。另外，我们的初步数据 揭示 PLB 和 DWORF 亲和力发生 Ca2 依赖性变化是因为这些调节剂更喜欢结合不同的 SERCA 酶促循环的中间构象。目标 2 将研究能量学的明显变化 SERCA 酶促循环期间 SERCA-微肽结合的变化可能是不同的抑制和刺激作用的基础 分别是 PLB 和 DWORF 的影响。具体来说，实验将探索 PLB 与 ATP- 的结合有多紧密。 SERCA 的结合状态阻止 Ca2 结合以介导 PLB 抑制。另一方面，DWORF 更喜欢绑定到状态 当泵由于速率限制步骤而循环时，SERCA 的影响占主导地位。我们将确定 DWORF 是否稳定 酶循环的高能中间状态，可降低能量势垒并增加 SERCA 酶周转率。 拟议的实验将改变 SERCA 微肽调节的经典范式，并为以下领域的发展提供信息： 小分子治疗心力衰竭。

PROJECT SUMMARY/ABSTRACT The goal of this two aim project is to explore dynamic mechanisms that regulate the cardiac calcium transporter, SERCA. SERCA plays a central role in the cardiac cycle; therefore, its regulation is critical for both survival and adapting to changing physiological demands. This regulation is primarily mediated by two transmembrane micropeptides: the inhibitory peptide, Phospholamban (PLB), and the stimulatory peptide, dwarf open reading frame (DWORF). These regulators compete to bind SERCA and control its function. We have found that intracellular Ca2+ elevations that drive contractions in cardiac muscle also cause dynamic shifts the binding equilibria of SERCA with PLB and DWORF. Specifically, Ca2+ elevations simultaneously lower the affinity of SERCA for PLB and increase SERCA affinity for DWORF. This is expected to lower inhibition and increase stimulation of SERCA during the peak of Ca2+ transients. Additionally, our preliminary results revealed that a dynamic fraction of PLB monomers that unbind from SERCA during Ca2+ elevations are dynamically sequestered in PLB pentamers. Slow unbinding of PLB pentamers causes PLB to accumulate in pentamers during rapid cardiac pacing, sequestering PLB away from SERCA to lower inhibition at exercising heart rates. Aim 1 will explore how this frequency-dependent accumulation of the PLB pentamer may mediate a critical role for PLB in the Bowditch effect, a positive force-frequency relationship in which a faster heart rate causes more forceful contractions of the heart. This phenomenon is a critical mechanism that adjusts cardiac output for exercise and, importantly, it is lacking in heart failure. Thus, experiments will examine how this novel mechanism is altered physiologically during the heart’s response to adrenaline and pathologically by PLB mutations linked to heart failure. These insights may reveal why patients with these mutations are more susceptible to arrythmias/heart failure. Additionally, our preliminary data revealed that Ca2+-dependent changes in PLB and DWORF affinity occur because these regulators prefer to bind different intermediate conformations of the SERCA enzymatic cycle. Aim 2 will investigate how distinct changes in the energetics of SERCA-micropeptide binding during the SERCA enzymatic cycle may underly the distinct inhibitory and stimulatory effects of PLB and DWORF, respectively. Specifically, experiments will explore how tight binding of PLB to the ATP- bound state of SERCA deters Ca2+ binding to mediate PLB inhibition. On the other hand, DWORF prefers to bind to states of SERCA that predominate when the pump is cycling due to rate-limiting steps. We will determine if DWORF stabilizes high energy intermediate states of the enzymatic cycle to lower an energy barrier and increase SERCA enzyme turnover. The proposed experiments will shift classic paradigms of SERCA-micropeptide regulation and inform the development of small molecules to treat heart failure.