Estrogen, Myosin Phosphorylation and Muscle Thermogenesis

我的研究计划的长期目标是进一步了解肌球蛋白的翻译后磷酸化修饰如何调节骨骼肌的收缩功能。在哺乳动物横纹肌中，磷酸部分从三磷酸腺苷（即ATP）转移到肌球蛋白调节轻链（RLC）上的丝氨酸残基是由骨骼肌球蛋白轻链激酶（skMLCK）催化的。这种反应是由钙和钙调素信号级联调控的，在收缩过程中被激活。反过来，skMLCK催化的肌球蛋白RLC的磷酸化增加了收缩蛋白的钙敏感性，从而增加了次极大力并产生所谓的“增强”。收缩诱导的RLC磷酸化和增强之间的强烈关联已经在各种啮齿动物快速抽搐骨骼肌模型中得到证实。相比之下，尽管啮齿动物的慢抽搐肌有有限的酶磷酸化RLC的能力，但它们没有表现出增强；因此，在慢肌表型中存在基础RLC磷酸化可能具有不同的目的，可能与快速肌表型共享，也可能不共享。因为调节肌肉收缩的钙信号也激活skMLCK活性，所以工作的骨骼肌通常处于肌球蛋白RLC磷酸化产生的增强状态。在这方面有趣的是，skMLCK活性和RLC磷酸化可能对激素影响敏感，这是一种连接系统和肌肉生理学的信号通路。因此，本应用程序的目的是研究雌激素等激素如何影响小鼠快速抽搐骨骼肌的肌凝蛋白RLC磷酸化（从而增强）（如Lai等人2016年提出的），以及这种影响是否涉及skMLCK信号传导。我们将研究系统生理学-肌肉生理学联系的另一种方法是检查肌球蛋白RLC磷酸化如何调节肌肉代谢率和产热，即产热。肌球蛋白RLC磷酸化调节肌肉产热的推测能力（McNamara et al. 2015）可能会影响静息和颤抖产热。所有实验的一个关键特征是使用来自我们的群体的skMLCK“敲除”小鼠来提供一个强大的阴性控制条件，这在野生型小鼠中是根本不可行的。因此，我们将能够严格检查雌激素在肌球蛋白RLC磷酸化存在和/或不存在的情况下如何影响肌肉功能。我们还将能够评估环境干预（如饮食和寒冷）分别对骨骼肌中肌球蛋白RLC磷酸化和不磷酸化的小鼠静息和颤抖产热的影响。因此，这些研究将研究调节肌球蛋白RLC磷酸化的途径，以及反过来，这种分子机制如何调节肌肉代谢。

The long term goals of my research program are to further our understanding of how the posttranslational modification of myosin by phosphorylation modulates contractile function of skeletal muscle.  In mammalian striated muscle the transfer of a phosphate moiety from adenosine triphosphate (i.e. ATP) to a serine residue on the myosin regulatory light chain (RLC) is catalyzed by skeletal myosin light chain kinase (skMLCK). This reaction is regulated by a calcium and calmodulin signalling cascade that is activated during  contraction.  In turn, skMLCK catalyzed phosphorylation of the myosin RLC increases the calcium sensitivity of the contractile proteins, thus increasing submaximal force and producing what is known as "potentiation".   Strong associations between contraction-induced RLC phosphorylation and potentiation have been demonstrated in a variety of rodent fast twitch skeletal muscle models.  By contrast, although rodent slow twitch muscle has a limited enzymatic ability to phosphorylate the RLC they display no potentiation; accordingly, the presence of basal RLC phosphorylation in the slow twitch muscle phenotype may serve a different purpose that may or may not be shared with the fast muscle phenotype.  Because the same calcium signals that regulate muscle contraction also activate skMLCK activity it is possible that working skeletal muscle most often operates in the potentiated state produced by myosin RLC phosphorylation. Interesting in this regard is the possibility that skMLCK activity and thus RLC phosphorylation may be sensitive to hormonal influences, a signalling pathway linking system and muscle physiology. Accordingly, the goals of this application are to examine how hormones such as estrogen may influence myosin RLC phosphorylation (and thus potentiation) of mouse fast twitch skeletal muscle (as suggested by Lai et al. 2016) and if this influence involves skMLCK signalling. Another way we will study the system physiology-muscle physiology link is by examining how myosin RLC phosphorylation modulates muscle metabolic rate and heat production, i.e. thermogenesis.  The putative ability of myosin RLC phosphorylation to regulate muscle thermogenesis (McNamara et al. 2015) may influence both resting and shivering thermogenesis.  A key feature of all of the experiments is the use of skMLCK "knockout" mice from our colony to provide a robust negative control condition that is simply not feasible with wildtype mice. Thus, we will be able to critically examine how estrogen effects muscle function in the presence and/or absence of myosin RLC phosphorylation. We will also be able to assess the influence of environmental interventions such as diet and cold on resting and shivering thermogenesis, respectively, in mice with and without myosin RLC phosphorylation in their skeletal muscles. These studies will thus examine pathways regulating myosin RLC phosphorylation and how, in turn, this molecular mechanism may regulate muscle metabolism.