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磷酸化翻译后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）上的丝氨酸残基由骨骼肌球蛋白轻链激酶（skMLCK）催化。该反应由在收缩期间激活的钙和钙调蛋白信号级联调节。 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，从而增加了次最大力并产生了所谓的“增强作用”。 收缩诱导的RLC磷酸化和增强之间的强关联已在各种啮齿动物快收缩骨骼肌模型中得到证实。 相比之下，尽管啮齿动物慢缩肌具有有限的酶促能力来磷酸化RLC，但它们不显示增强作用;因此，慢缩肌表型中基础RLC磷酸化的存在可能服务于不同的目的，其可能与快肌表型共享或不共享。 因为调节肌肉收缩的相同钙信号也激活skMLCK活性，所以工作骨骼肌可能最常在由肌球蛋白RLC磷酸化产生的增强状态下工作。在这方面有趣的是，skMLCK活性，从而RLC磷酸化可能是敏感的激素的影响，信号通路连接系统和肌肉生理学的可能性。因此，本申请的目的是研究激素（如雌激素）如何影响小鼠快缩骨骼肌的肌球蛋白RLC磷酸化（从而增强）（如Lai et al. 2016所述），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式是通过检查肌球蛋白RLC磷酸化如何调节肌肉代谢速率和产热，即产热。 肌球蛋白RLC磷酸化调节肌肉产热的推定能力（McNamara et al. 2015）可能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群体的skMLCK“敲除”小鼠来提供用野生型小鼠根本不可行的稳健阴性对照条件。因此，我们将能够严格审查雌激素如何影响肌肉功能的存在和/或不存在的肌球蛋白RLC磷酸化。我们还将能够评估环境干预的影响，如饮食和寒冷的休息和颤抖产热，分别在小鼠和无肌球蛋白RLC磷酸化在他们的骨骼肌。因此，这些研究将检查调节肌球蛋白RLC磷酸化的途径，以及这种分子机制如何反过来调节肌肉代谢。

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.