Role of Merg1, a K+ Channel in the Onset of Skeletal Muscle Atrophy

描述（由申请人提供）： 广泛的，长期的目标是了解分子机制，调节骨骼肌（skm）萎缩的启动。本申请的目的是探索Merg 1a在皮肤萎缩起始中的作用。核心假设是Merg 1a激活泛素蛋白酶体途径（UPP），已知在萎缩组织中具有活性，并通过调节肌膜Ca++通道活性参与皮肤萎缩的发作。该假设基于显示Merg 1a存在于萎缩的肌肉中并且其功能可以诱导UPP活性和萎缩的数据。此外，数据显示E3连接酶（UPP的一部分）mRNA的转录响应Merg 1a的表达。已知两种途径诱导E3连接酶合成：IKK-2/I：B-1/NF-：B和PI 3 K/Akt/FOXO。因此，我们的前两个具体目标是确定：1）Merg 1a表达是否通过激活IKK-2/I：B-1/NF-：B通路启动UPP活性; 2）Merg 1a表达是否通过PI 3 K/Akt/FOXO通路启动UPP活性。数据还表明，Ca++水平和Merg 1a表达之间存在联系。因此，我们的第三个目的是确定Merg 1a表达对Ca++电流密度和细胞内Ca++浓度的影响，以及Ca++调节的变化是否是增加UPP活性所必需的。前两个目的将测量Merg 1a和已知参与导致E3连接酶产生的途径的蛋白质的组成型活性或显性负性形式的体内异位共表达的效果。第三个目的将涉及使用条件性表达Merg 1a的C2 C12克隆细胞系，以确定Merg 1a表达对Ca++电流密度、Ca++通道亚基表达、细胞内钙水平和钙蛋白酶活性的影响。最后，将使用Ca++处理的各种药理学调节剂来确定Ca++是否参与Merg 1a向UPP的信号传导。本案无关骨骼肌萎缩在许多病理条件下发生（例如，艾滋病、糖尿病、癌症恶病质等）并与残疾、发病率和死亡率的增加有关。这项研究意义重大，因为它将探索一种新的皮肤萎缩引发剂，并产生信息，允许开发更有效的治疗方法，从而减少这种衰弱状况的病例数。

DESCRIPTION (provided by applicant): The broad, long range objective is to understand molecular mechanisms that regulate initiation of skeletal muscle (skm) atrophy. The objective of this application is to explore the role of Merg1a in initiation of skm atrophy. The central hypothesis is that Merg1a activates the ubiquitin proteasome pathway (UPP), known to be active in atrophying tissue, and participates in the onset of skm atrophy by modulating sarcolemmal Ca++ channel activity. The hypothesis is based on data which show that Merg1a is present in atrophying muscle and that its function can induce UPP activity and atrophy. Addtionally, data show that E3 ligase (part of the UPP) mRNAs are transcribed in response to Merg1a expression. Two pathways are known to induce E3 ligase synthesis: IKK-2/I:B-1/NF-:B and PI3K/Akt/FOXO. Therefore, our first two specific aims are to determine if: 1) Merg1a expression initiates UPP activity by activating the IKK-2/I:B-1/NF-:B pathway; and 2) Merg1a expression initiates UPP activity by the PI3K/Akt/FOXO pathway. Data also show that a connection exists between Ca++ levels and Merg1a expression. Thus, our third aim is to determine the effect of Merg1a expression on Ca++ current density and intracellular Ca++ concentration and if changes in Ca++ modulation are necessary for increased UPP activity. The first two aims will measure the effect(s) of in vivo ectopic co- expression of Merg1a and either constitutively active or dominant negative forms of proteins known to participate in the pathways leading to E3 ligase production. The third aim will involve use of a C2C12 clonal cell line which conditionally expresses Merg1a to determine the effect(s) of Merg1a expression on Ca++ current density, Ca++ channel subunit expression, intracellular calcium levels, and calpain activity. Finally, various pharmacological modulators of Ca++ handling will be used to determine if Ca++ is involved in Merg1a signaling to the UPP. Relevance. Skeletal muscle atrophy occurs with many pathological conditions (e.g., AIDS, diabetes, cancer cachexia, etc.) and is related to increased disability, morbidity and mortality. This research is significant because it will explore a novel initiator of skm atrophy and produce information that will allow for development of more effective therapies and, thereby, reduce the number of cases of this debilitating condition.