Craniofacial Muscle Fiber Specializations: A Comparative Approach

头面部肌肉共同服务于一套极其不同的功能。这组肌肉包括眼外肌，负责在视觉跟踪移动对象的过程中高度精确的眼球运动，喉部肌肉，用于发声和呼吸道保护，以及下颌肌，驱动咀嚼。这些肌肉在结构、生化特性和生理特性方面都非常复杂，因此与用于运动的四肢肌肉明显不同。这个项目的总体目标是更全面地了解头面部肌肉的分子和细胞特性，使这些肌肉能够提供如此广泛的功能，并以非凡的精度做到这一点。肌球蛋白是一种大型蛋白质，由两个重链和四个轻链亚基组成，是肌肉收缩特性的关键调节因子。表达“超快”和“眼外/喉”肌球蛋白亚型的整个头面部肌肉被认为具有明显的收缩特性。然而，与描述肢体肌肉肌球蛋白在生理特性调节中的作用的大量文献相反，超快肌球蛋白和眼外/喉部肌球蛋白的具体功能意义尚不清楚。该项目旨在测试哺乳动物骨骼肌纤维是否具有明显的收缩特性，其中哺乳动物骨骼肌纤维被鉴定为表达在食肉动物闭口肌中发现的“超快”肌球蛋白，或在眼外肌和喉内肌中表达的“眼外/喉外”肌球蛋白。单个肌肉纤维的收缩特性包括最大缩短速度、快速松弛和再拉伸后的张力再发展速度、张力产生能力、僵硬和肌原纤维ATPase活性。随后将使用超灵敏的凝胶电泳法测定每个纤维的肌球蛋白重链和轻链亚型组成。这一结果将被用来计算根据肌球蛋白亚型组成确定的纤维中的交叉桥附着和分离的速率常数。主要目的是测试超快肌球蛋白、眼外肌/喉肌球蛋白和四肢肌球蛋白之间的交叉桥动力学是否有显著差异。这些结果有望通过确定基本的交叉桥速率常数，为表达不同肌球蛋白异构体的纤维中交叉桥特性的差异提供机制上的理解。该项目将在一定程度上基于比较方法。食肉动物(狗、猫)闭嘴肌肉中的单个肌肉纤维将与已知不表达超快肌球蛋白的杂食动物(猪)的肌肉纤维进行比较。这两种肌肉纤维收缩特性的差异将说明超快肌球蛋白为适应食肉动物的进食行为所赋予的独特特性。这个项目将导致对高度专业化的肌肉群的更全面的了解，到目前为止，这些肌肉群得到的关注相对较少。特别是，这些结果有望为肌肉的功能提供有价值的细胞和分子见解，这些肌肉服务于肉食动物攻击性的进食方式，并奠定与眼睛运动、发声和呼吸道保护相关的极其精确的运动功能的基础。了解这些高度专门化肌肉收缩的细胞和分子基础将极大地扩展我们对脊椎动物肌肉纤维复杂性的理解，并扩大已知的肌肉收缩特性。

Craniofacial muscles collectively serve an extremely diverse set of functions. This set of muscles includes extraocular muscles which are responsible for highly precise eye movements during visual tracking of a moving object, laryngeal muscles that serve vocalizations and airway protection, and mandibular muscles that drive mastication. These muscles are highly complex with respect to structure, biochemical properties and physiological properties and, as such, are markedly different from limb muscles that serve locomotion. The overall goal of this project is to acquire a more comprehensive understanding of the molecular and cellular properties of craniofacial muscles that allow these muscles to serve such a vast array of functions and to do so with an extraordinary level of precision. Myosin is a large protein, being composed of two heavy chain and four light chain subunits, and is a key regulator of contractile properties of muscles. Whole craniofacial muscles which express "superfast" and "extraocular/laryngeal" myosin isoforms are well recognized as having distinct contractile properties. However, the specific functional significance of superfast myosin and of extraocular/laryngeal myosin remains unknown, in contrast to a large body of literature describing the roles of limb muscle myosins in the regulation of physiologic properties. The project aims are designed to test whether mammalian skeletal muscle fibers that are identified as expressing either "superfast" myosin, found in carnivore jaw-closing muscles, or "extraocular/laryngeal" myosin, expressed in extraocular and intrinsic laryngeal muscles, have distinct contractile properties. The contractile properties to be measured in single muscle fibers include the maximal velocity of shortening, the rate of tension redevelopment following a rapid slackening and re-stretch, tension-generating ability, stiffness and myofibrillar ATPase activity. The myosin heavy chain and light chain isoform composition of each fiber will subsequently be determined using an ultrasensitive gel electrophoresis protocol. The results will be utilized to calculate the rate constants for crossbridge attachment and detachment in fibers that are identified on the basis of their myosin isoform composition. The primary objective is to test whether crossbridge kinetics differ significantly between superfast, extraocular/laryngeal and limb muscle myosins. The results are expected to provide a mechanistic understanding of differences in crossbridge properties in fibers that express different myosin isoforms through the determination of fundamental crossbridge rate constants. This project will be based, in part, on a comparative approach. Single muscle fibers from jaw-closing muscles of carninvores (dogs, cats) will be compared to those from an omnivore (pig) that is known to not express superfast myosin. Differences in contractile properties between these two sources of muscle fibers will illustrate the unique properties imparted by superfast myosin to accommodate the feeding behavior of carnivores. This project will result in a more comprehensive understanding of a highly specialized group of muscles that has thus far received relatively little attention. In particular, the results are expected to provide valuable cellular and molecular insights into the function of muscles that serve the aggressive feeding style characteristic of carnivores and underlie the extremely precise motor functions associated with eye movements, vocalization and airway protection. Understanding the cellular and molecular bases of contraction in these highly specialized muscles will greatly expand our understanding of the complexity of muscle fibers among vertebrates and broaden the known repertoire of their contractile properties.