REGULATION OF ACTOMYOSIN INTERACTIONS IN CARDIAC MUSCLES

脊椎动物的心脏和横纹肌收缩已被假想 通过改变肌球蛋白结合位点的数量来调节 肌动蛋白或力产生反应的动力学。此外, 这一规定也被假设为不仅是由 钙离子与肌钙蛋白C(TNC)结合，也是肌钙蛋白C的主要效应因子 通过交叉桥连接到细丝上。我们最近的实验 观察结果导致了一个新的、更简单的模型来解释钙离子 肌动球蛋白结合和动力学的调节(概述于 导言)：认为分子动力学对细丝的调节 单位直接影响肌动球蛋白功能的表观动力学 次极值激活。在项目II中，我们将使用渗透化 准备研究动态的分子调控机制 肌肉收缩过程中的三个机械性过程 动力学参数：等长张力恢复速率(k/tr)； 无负荷缩短率(V/US)；肌球蛋白“力量卒中”， 通过对小幅度响应的张力瞬变的第二阶段进行评估 长度步长。选择了三个化学力学参数进行测试 除了稳态等长力外，调节机制还可以 检查横桥循环的多个方面，因为每个方面 这些参数随细丝的变化而明显变化。 激活水平，只要他们已经被检查过。第一个具体 需要检验的假设是k/tr处于亚最大钙激活状态 反映各个法规单位的动态属性。直接 与我们的初步数据支持的这一假设相关的是， 监管单位中跨国公司的类型是决定 该单位在次最大激活期间的动态。第二 假说是肌球蛋白一元性“力量卒中”的动力学， 由一步后的毫秒时间刻度张力瞬变反映 长度的变化，都是由钙离子调节的。至少，这些 实验将允许我们区分不同阶段的假设 2动力学受以下两种因素之一的调节：(A) 最初附着、低力产生肌动球蛋白中间体，与(B) 交叉桥之间的协作交互作用使得回速率 产生力的转变的常数随力的分数减小。 附连的交叉桥增加。第三个假设是，卸货 缩短速度主要由不同的机制调节。 心脏和骨骼肌的对比。这些实验使用的是带皮的纤维 准备工作将辅之以细丝滑动的测量。 在项目IV中使用纯化的体外运动分析中的速度 蛋白质。总而言之，我们将测试以下各项的有效性和普遍性 一种新的假说--个体调控的动态特性 单位在决定人口数量方面发挥着迄今未被认识到的重要作用。 肌肉收缩的宏观动力学。

Vertebrate cardiac and striated muscle contraction has been hypothesized to be regulated by altering either the number of myosin-binding sites on actin or the kinetics of the force generating reaction. In addition, this regulation has also been hypothesized to be modulated not just by the primary effector, Ca2+ binding to troponin C (TnC), but also in turn by crossbridge binding to the thin filament. Our recent experimental observations have led to a new and simpler model to explain the Ca2+ regulation of actomyosin binding and kinetics (outlined in the Introduction): that the molecular dynamics of thin filament regulatory units directly influence the apparent kinetics of actomyosin function at submaximal activation. In Project II, we will use permeabilized preparations to examine the molecular regulatory mechanism of dynamic processes during muscle contraction as indicated by three mechanical kinetic parameters: the rate of isometric tension redevelopment (k/TR); unloaded shortening velocity (V/US); and the myosin 'power stroke,' assessed by phase 2 of tension transients in response to small amplitude length steps. Three chemomechanical parameters have been chosen to test the regulatory mechanism in addition to steady state isometric force to examine on multiple facets of the crossbridge cycle and because each of these parameters exhibits an apparent variation with thin filament activation level, insofar as they have been examined. The first specific hypothesis to be tested is that k/TR at submaximal Ca2+-activation reflects dynamic properties of individual regulatory units. Directly related to this hypothesis, as supported by our Preliminary data, is that the type of TnC in a regulatory unit is a major determinant of the dynamics of that unit during submaximal activations. The second hypothesis is that the kinetics of the myosin unitary 'power stroke,' as reflected by millisecond time scale tension transients following a step change in length, are regulated by Ca2+. At the very least, these experiments will allow us to distinguish between hypotheses that phase 2 kinetics are modulated by either (a) a transient population of an initial attached, low force producing actomyosin intermediate, vs. (b) cooperative interactions between crossbridges such that the back rate constant of the force-producing transition decreases as the fraction of attached crossbridges increases. The third hypothesis is that unloaded shortening velocity is primarily regulated by different mechanisms in cardiac vs. skeletal muscle. These experiments using skinned fiber preparations will be complemented with measurements of filament sliding velocity in Project IV using in vitro motility assays on purified proteins. In total, we will be testing the validity and universality of a new hypothesis--that the dynamic properties of individual regulatory units play a major and heretofore unrecognized role in determining the macroscopic kinetics of muscle contraction.