In situ measurement of sarcomere operating range in passive and active muscle

描述(由申请人提供)：肌节长度是肌肉如何影响行为的主要决定因素之一。肌肉的主要功能是稳定运动还是发电，这很大程度上受肌肉活动的肌节长度的影响。此外，肌节属性的改变也会导致损伤后的肌肉损伤。尽管研究人员使用了各种方法来测量肌节长度，但能否在原位测量这些长度，以及当肢体移动或肌肉活跃时，这些长度是如何变化的，仍然是一个根本的挑战。这项提议中的研究将开发一种很有前途的新技术，即双光子显微镜，用于原位测量肌肉的肌节长度。这项技术将使我们能够表征肌肉、肢体结构和激活模式中肌节长度的变化。重要的是，这也将使我们能够开始描述这些措施在运动相关的肌肉损伤后如何变化。这项技术与目前表征肌肉肌节长度的技术相比有几个潜在的优势。首先，它可以在不干扰肌肉纤维的情况下测量肌节长度，因此可以在完整的肌肉中测量肌节长度。其次，肌节长度可以在功能正常的肌肉中测量，因此可以直接评估激活的效果。用目前测量肌节长度的技术，这样的测量是不可能的。第三，可以快速和重复地进行多个肌节长度测量，因此我们可以直接检查肌节的工作范围，而不需要求助于外推。这种重复性还使我们能够在与运动相关的现场活动中可视化肌肉损伤的发展；例如，跟踪同一肌肉中的肌节属性，因为损伤是通过偏心收缩逐渐诱导的。最后，我们将在我们最近开发的用于测量肌肉动作的体内准备中进行这些测量，以便我们能够在原位测量肌节长度和肌肉动作。在相同的准备中收集这些测量数据的能力将使我们对肌肉的功能及其对行为的贡献有前所未有的洞察力。我们在这项提案中有三个具体目标。首先，我们将测量大鼠后肢大量肌肉中肌节的工作范围，以确定肌节长度与假想的肌肉功能之间的关系。其次，我们将测量肌肉激活后肌节活动范围的变化，以确定神经控制改变肌节活动范围的程度。最后，我们将在偏心收缩引起的肌肉损伤的发展过程中对肌节进行成像。如果成功，这项研究有可能直接解决肌肉功能和疾病的这些基本问题，展示双光子显微镜在肌肉功能研究中的潜在力量。

DESCRIPTION (provided by applicant): Sarcomere lengths are one of the main determinants of how a muscle contributes to behavior. Whether a muscle functions primarily for movement stabilization or for power generation is strongly influenced by the sarcomere lengths over which that muscle operates. Moreover, alterations in sarcomere properties contribute to muscle damage following injury. Although investigators have used a variety of methods to measure sarcomere lengths, the ability to measure these lengths in situ and how they vary when the limb is moved or when the muscle is active, remains a fundamental challenge. The research in this proposal will develop a promising new technique, two photon microscopy, to measure sarcomere lengths in muscles in situ. This technique will allow us to characterize sarcomere length variations across muscles, limb configurations, and activation patterns. Importantly, it also will allow us to begin characterizing how these measures change following exercise related muscle damage. This technique has several potential advantages over current techniques of characterizing sarcomere lengths of muscles. First, it can measure sarcomere lengths without disturbing muscle fibers, so sarcomere lengths can be measured in an intact muscle. Second, sarcomere lengths can be measured in functioning muscles, so that effects of activation can be directly evaluated. Such measurements are impossible with current techniques of measuring sarcomere lengths. Third, multiple sarcomere length measurements can be made quickly and repeatedly, so that we can examine sarcomere operating ranges directly without resorting to extrapolation. This repeatability also allows us to visualize the development of muscle damage during exercise related activity in situ; e.g. tracking sarcomere properties in the same muscle as damage is progressively induced through eccentric contractions. Finally, we will perform these measurements in an in vivo preparation that we have recently developed for measuring muscle actions, so that we will be able to measure both sarcomere lengths and muscle actions in situ. The ability to collect these measurements together in the same preparation will give us unprecedented insight into the function of a muscle and how it contributes to behavior. We have three specific aims in this proposal. First, we will measure the operating range of sarcomeres across a large number of muscles in the rat hindlimb, to determine the relationship between sarcomere lengths and hypothesized muscle functions. Second, we will measure the change in sarcomere operating ranges after muscle activation, to determine the degree to which neural control alters sarcomere operating ranges. Finally, we will image sarcomeres during the development of muscle damage induced by eccentric contractions. If successful, this research has the potential to directly address these fundamental issues of muscle function and disease, demonstrating the potential power of two photon microscopy in studies of muscle function.