The Influence of Myosin Regulatory Light Chain Phosphorylation on Skeletal Muscle Energetics, Mechanics and Function

这个申请是为了资助做的工作，检查活力，机械和功能方面的活动依赖增强（或简称为“增强”）的小鼠骨骼肌。增强被定义为在快收缩肌肉的收缩活动期间或之后观察到的肌肉力量的增加。增强是快速收缩表型的特征性质，并且这些肌肉类型在体内通常可以在这种状态下操作。在快收缩肌中容易诱导增强，并且存在不同形式的增强。例如，阶梯增强是在延长的低频刺激期间观察到的力的逐渐增加，而强直后增强（PTP）是在短暂但高频刺激之后观察到的力的增加。在这两种情况下，增强的主要细胞内机制是磷酸化的调节轻链（RLC）亚基所包含的快速肌球蛋白类型。RLC的磷酸盐含量受到调节肌肉力量的相同钙信号的高度调节，所述钙信号还激活催化磷酸盐转移到RLC的骨骼肌球蛋白轻链激酶。磷酸盐的去除是不受调节的，并以稳定的速率发生;因此RLC的磷酸盐含量直接随收缩活动而变化。RLC的磷酸化增加了收缩器官的钙敏感性，从而解释了这种分子机制如何以及为什么增强力。事实上，对骨骼肌球蛋白轻链激酶敲除小鼠进行的研究表明，在不存在RLC磷酸化的情况下，PTP完全不存在，楼梯增强大大降低（~ 50%）。这一有趣的结果表明，即使RLC磷酸化可能是PTP的主要机制，次级机制可能涉及阶梯增强。这种次要机制可能是刺激诱导的静息钙水平增加，尽管发生这种情况的机制尚不清楚。有趣的是，除了增加力，增强也可能增加收缩的能量成本，尽管RLC磷酸化相对于改变静息钙稳态对这一重要作用的相对贡献是未知的。这项拨款申请的主要目标之一是研究增强对肌肉收缩能量学的影响。这些研究的新颖之处在于，我们将使用缺乏骨骼肌球蛋白轻链激酶（“敲除”）的肌肉来研究这个问题，因此这些肌肉不显示刺激诱导的RLC磷酸化增加。因此，使用这些独特的肌肉将提供明确的答案，如何或如果增强改变小鼠肌肉的能量需求，以及这种变化是否可以归因于RLC磷酸化或改变钙稳态。在其他实验中，我们将比较正常和骨骼肌肌球蛋白轻链激酶敲除肌肉的力反应，以确定这些各自的机制在各种条件下增强的相对贡献。这项研究计划的一个相关目标是分离出RLC磷酸化与静息钙改变的机械/功能后果。在其他实验中，我们将试图确定在蚓状肌中观察到的增强作用，我们可以在收缩过程中量化细胞内钙的肌肉，是否可以从静息钙的变化中解偶联，并确定这种变化如何在没有RLC磷酸化的情况下增强力。总之，这项工作利用正常和敲除肌肉（分别有和没有RLC磷酸化）将回答许多问题有关的增强。

This application is for funding to do work examining the energetic, mechanistic and functional aspects of activity dependent potentiation (or simply "potentiation") of mouse skeletal muscle. Potentiation is defined as the increase in muscle force observed during or following contractile activity of fast twitch muscles. Potentiation is a characteristic property of the fast twitch phenotype and these muscle types may often operate in this state in vivo. Potentiation is readily induced in fast twitch muscle and different forms of potentiation exist. For example, staircase potentiation is the progressive increase in force observed during prolonged, low frequency stimulation while posttetanic potentiation (PTP) is the increase in force observed following brief but high frequency stimulation. In both cases, the primary intracellular mechanism for potentiation is phosphorylation of the regulatory light chain (RLC) subunits contained by fast myosin types. The phosphate content of the RLC is highly regulated with the same calcium signals that regulate muscle force also activate a skeletal myosin light chain kinase enzyme that catalyzes the transfer of a phosphate to the RLC. The removal of the phosphate is unregulated and occurs at a steady rate; thus the phosphate content of the RLC varies directly with contractile activity. Phosphorylation of the RLC increases the calcium sensitivity of the contractile apparatus, thus accounting for how and why this molecular mechanism potentiates force. Indeed, studies performed on skeletal myosin light chain kinase knockout mice show that, in the absence of RLC phosphorylation, PTP is completely absent and staircase potentiation is greatly reduced (~ 50%). This interesting outcome indicates that even though RLC phosphorylation may be the primary mechanism for PTP, secondary mechanisms may be involved in staircase potentiation. This secondary mechanism may be stimulation - induced increase in resting calcium levels although the mechanism by which this happens is unknown. Interestingly, in addition to increasing force, potentiation may also increase the energetic cost of contraction although the relative contribution of RLC phosphorylation versus altered resting calcium homeostasis to this important effect is unknown. One of the main objectives of this grant application are to examine the influence of potentiation on the energetics of muscle contraction. The novel aspect of these studies is that we will examine this issue using muscles devoid of skeletal myosin light chain kinase ("knockouts") and thus which do not display stimulation induced increases in RLC phosphorylation. Thus, use of these unique muscles will provide definitive answers as to how or if potentiation alters the energetic demands of mouse muscle and whether this change can be attributed to RLC phosphorylation or to altered calcium homeostasis. In other experiments, we will compare the force responses of normal and skeletal myosin light chain kinase knockout muscles to determine the relative contribution of these respective mechanisms to potentiation under a variety of conditions. A related objective of this research program is to separate out the mechanical / functional consequences of RLC phosphorylation versus altered resting calcium. In other experiments we will attempt to determine whether the potentiation observed in the lumbrical, a muscle with which we can quantify intracellular calcium during contraction, can be uncoupled from changes in resting calcium and also to determine how this change potentiates force in the absence of RLC phosphorylation. In summary, this work utilizing normal and knockout muscles (with and without RLC phosphorylation, respectively) will answer many questions related to potentiation.