Department of Cell Biology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center , Fort Worth, TX , USA.
Department of Mathematics, Computer Science, and Physics, Texas Wesleyan University , Fort Worth, TX , USA.
Front Cardiovasc Med. 2015 Nov 18;2:35. doi: 10.3389/fcvm.2015.00035. eCollection 2015.
Contraction of muscles results from the ATP-coupled cyclic interactions of the myosin cross-bridges with actin filaments. Macroscopic parameters of contraction, such as maximum tension, speed of shortening, or ATPase activity, are unlikely to reveal differences between the wild-type and mutated (MUT) proteins when the level of transgenic protein expression is low. This is because macroscopic measurements are made on whole organs containing trillions of actin and myosin molecules. An average of the information collected from such a large assembly is bound to conceal any differences imposed by a small fraction of MUT molecules. To circumvent the averaging problem, the measurements were done on isolated ventricular myofibril (MF) in which thin filaments were sparsely labeled with a fluorescent dye. We isolated a single MF from a ventricle, oriented it vertically (to be able measure the orientation), and labeled 1 in 100,000 actin monomers with a fluorescent dye. We observed the fluorescence from a small confocal volume containing approximately three actin molecules. During the contraction of a ventricle actin constantly changes orientation (i.e., the transition moment of rigidly attached fluorophore fluctuates in time) because it is repetitively being "kicked" by myosin cross-bridges. An autocorrelation functions (ACFs) of these fluctuations are remarkably sensitive to the mutation of myosin. We examined the effects of Alanine to Threonine (A13T) mutation in the myosin regulatory light chain shown by population studies to cause hypertrophic cardiomyopathy. This is an appropriate example, because mutation is expressed at only 10% in the ventricles of transgenic mice. ACFs were either "Standard" (Std) (decaying monotonically in time) or "Non-standard" (NStd) (decaying irregularly). The sparse labeling of actin also allowed the measurement of the spatial distribution of actin molecules. Such distribution reflects the interaction of actin with myosin cross-bridges and is also remarkably sensitive to myosin mutation. The result showed that the A13T mutation caused 9% ACFs and 9% of spatial distributions of actin to be NStd, while the remaining 91% were Std, suggesting that the NStd performances were executed by the MUT myosin heads and that the Std performances were executed by non-MUT myosin heads. We conclude that the method explored in this study is a sensitive and valid test of the properties of low prevalence mutations in sarcomeric proteins.
肌肉收缩是由肌球蛋白横桥与肌动蛋白丝的 ATP 偶联循环相互作用引起的。当转基因蛋白表达水平较低时，收缩的宏观参数，如最大张力、缩短速度或 ATP 酶活性，不太可能揭示野生型和突变型（MUT）蛋白之间的差异。这是因为宏观测量是在包含数万亿个肌动蛋白和肌球蛋白分子的整个器官上进行的。从这样一个大型组件收集的信息的平均值必然会掩盖由一小部分 MUT 分子施加的任何差异。为了避免平均问题，我们在分离的心室肌原纤维（MF）上进行了测量，其中薄丝用荧光染料稀疏标记。我们从心室中分离出一个 MF，将其垂直定向（以便能够测量取向），并用荧光染料标记 100,000 个肌动蛋白单体中的 1 个。我们观察了包含大约三个肌动蛋白分子的小共焦体积的荧光。在心室收缩过程中，肌动蛋白不断改变取向（即刚性附着荧光团的跃迁时刻随时间波动），因为它不断被肌球蛋白横桥“踢动”。这些波动的自相关函数（ACFs）对肌球蛋白的突变非常敏感。我们检查了肌球蛋白调节轻链中的丙氨酸到苏氨酸（A13T）突变的影响，该突变在人群研究中显示会导致肥厚型心肌病。这是一个合适的例子，因为突变在转基因小鼠的心室中仅表达 10%。ACFs 要么是“标准”（Std）（随时间单调衰减），要么是“非标准”（NStd）（不规则衰减）。肌动蛋白的稀疏标记还允许测量肌动蛋白分子的空间分布。这种分布反映了肌动蛋白与肌球蛋白横桥的相互作用，并且对肌球蛋白突变也非常敏感。结果表明，A13T 突变导致 9%的 ACFs 和 9%的肌动蛋白空间分布为 NStd，而其余 91%为 Std，这表明 NStd 表现由 MUT 肌球蛋白头执行，而 Std 表现由非 MUT 肌球蛋白头执行。我们得出结论，本研究中探索的方法是对肌节蛋白中低流行突变特性的敏感且有效的测试。
