The Role of Striated Muscle Preferentially Expressed Protein Kinase in the Regulation of Ryanodine Receptor Type-2.

对于调节心肌肌浆细胞的机制， 内质网（SR）钙通过Ryanodine受体2型（RyR 2）释放。更全面地理解 需要调节RyR 2以更好地理解心血管疾病中钙失调的机制。 病理横纹肌前体表达蛋白激酶（SPEG）是RyR 2的结合伴侣， 在心力衰竭中下调的具有双激酶结构域的蛋白质。SPEG的心脏丧失导致 通过增加RyR 2的舒张期钙渗漏减少SR储存。虽然它的激酶活性没有 经过广泛的研究，有证据表明，内部激酶结构域，SPEG 1，是催化活性。作为 先前的磷酸化事件已经显示出改变RyR 2活性，因此SPEG可能作为一种抑制剂， RyR 2通过其激酶活性调节。此应用程序的总体目标是确定 SPEG的激酶活性对SR钙处理有影响，更具体地说，SPEG的激酶活性是否 在RyR 2的调节中起重要作用。申请人实验室生成的初步数据显示， 在心脏特异性SPEG敲除小鼠中RyR 2-S2367位点的磷酸化显著降低。 该位点位于RyR 2的关键钳夹区，其磷酸化在文献中尚未报道。 中心假设是RyR 2-S2367位点的磷酸化导致舒张期钙降低 通过RyR 2的稳定性泄漏。这项研究的基本原理是， 新的磷酸化位点和这种磷酸化事件的机制有可能导致新的 调节RyR 2活性的方法。假设将通过以下具体目标进行检验：1）确定 SPEG是否主要在S2367位点磷酸化RyR 2，2）检验S2367位点磷酸化RyR 2的假设， S2367位点抑制舒张期RyR 2开放，和3）确定SPEG 1激酶活性是否是治疗性的 心脏衰竭对于第一个目标，我们将使用两种方法来确定RyR 2-S2367是否被SPEG磷酸化。 体外激酶测定和用腺相关病毒过表达SPEG 1激酶结构域。下 目的2，我们将利用重组RyR 2和敲入RyR 2-S2367磷酸模拟物的小鼠模型， 磷酸化抗性突变，以研究这种磷酸化事件对钙释放的影响， 用分离的心肌细胞进行单通道RyR 2研究和钙成像。对于目标3，我们将确定 用腺相关病毒过表达SPEG 1激酶结构域是否能改善射血分数 和SR钙处理在心力衰竭的非缺血性小鼠模型中。这项研究的意义在于， 将促进对一个新的RyR 2磷酸化位点和SPEG的生理重要性的理解， 激酶活性这一知识有可能帮助确定新的治疗靶点， 治疗心血管疾病，如心力衰竭。

There is a fundamental gap in understanding of the mechanisms that regulate cardiac sarcoplasmic reticulum (SR) calcium release through Ryanodine Receptor Type-2 (RyR2). A fuller understanding of the regulation of RyR2 is needed to better comprehend mechanisms of calcium dysregulation in cardiovascular pathology. Striated Muscle Preferentially Expressed Protein Kinase (SPEG), a binding partner of RyR2, is a protein with dual kinase domains that is downregulated in heart failure. Cardiac loss of SPEG results in decreased SR stores through increased diastolic calcium leak from RyR2. Although its kinase activity has not been extensively studied, there is evidence that the internal kinase domain, SPEG1, is catalytically active. As previous phosphorylation events have been shown to modify RyR2 activity, it is possible that SPEG acts as a regulator of RyR2 through its kinase activity. The overall objective of this application is to determine what role SPEG’s kinase activity has on SR calcium handling and more specifically whether SPEG’s kinase activity is important in the regulation of RyR2. Preliminary data generated in the applicant’s laboratory revealed a significant decrease in phosphorylation at the RyR2-S2367 site in a cardiac specific SPEG knock-out mouse. Phosphorylation of this site, located in the critical clamp region of RyR2, has not been reported in the literature. The central hypothesis is that phosphorylation of the RyR2-S2367 site results in decreased diastolic calcium leak through stabilization of RyR2. The rationale for this research is that an understanding of the effects of this novel phosphorylation site and the mechanism of this phosphorylation event has the potential to lead to novel approaches to modulate RyR2 activity. The hypothesis will be tested with the following specific aims 1) Determine whether SPEG primarily phosphorylates RyR2 at the S2367 site, 2) Test the hypothesis that phosphorylation at the S2367 site inhibits diastolic RyR2 opening, and 3) Determine whether SPEG1 kinase activity is therapeutic in heart failure. For the first aim, we will determine whether RyR2-S2367 is phosphorylated by SPEG using both an in vitro kinase assay and overexpression of the SPEG1 kinase domain with adeno-associated virus. Under aim 2, we will utilize recombinant RyR2 and knock-in mouse models with RyR2-S2367 phospho-mimetic and phospho-resistant mutations to study the effects of this phosphorylation event on calcium release using both single channel RyR2 studies and calcium imaging with isolated cardiomyocytes. For aim 3, we will determine whether overexpression of the SPEG1 kinase domain with adeno-associated virus improves ejection fraction and SR calcium handling in a non-ischemic mouse model of heart failure. This research is significant in that it will advance understanding of a novel RyR2 phosphorylation site and the physiological importance of SPEG’s kinase activity. This knowledge has the potential to aid in the identification of novel therapeutic targets for the treatment of cardiovascular diseases such as heart failure.