Smooth muscle molecular mechanics

平滑肌存在于身体的所有中空器官中（如肠、膀胱、血管）。平滑肌的独特之处在于它能以低能量消耗长时间产生和保持力量。一旦刺激收缩，平滑肌可以在单次收缩的时间过程中调节其能量消耗速率。这种特性被称为锁存状态，是平滑肌的一个重要特征，但人们对其了解甚少。因此，这个研究项目的首要目标是了解锁存状态背后的分子机制。*******肌肉收缩是由肌凝蛋白分子马达与肌动蛋白丝的相互作用完成的。据信，当肌凝蛋白分子在与肌动蛋白结合时酶失活时，就会发生锁存状态。这一理论来自于对整个肌肉水平的测量，但从未在分子水平上得到证实。其他几种蛋白质（钙钙蛋白、钙desmon、非肌肉肌球蛋白等）也被假设在闩锁状态中起作用，但同样没有证据表明它们在分子水平上起作用。因此，我们的目的是通过直接在分子水平上进行力测量来确定在肌凝蛋白失活期间是否可以保持力。在我之前的NSERC资助下，我们开发了一种微室（微流体装置），可以在不产生任何散装流动的情况下添加化学品。因此，我们现在建议在分子力测量期间使用该室注射灭活酶，并确定是否发生力维持（闭锁状态）。我们还将确定是否需要其他蛋白质，如端粒蛋白，caldesmon等来维持力量，或者是否需要肌凝蛋白和肌动蛋白。*******活化的和非活化的肌凝蛋白有非常不同的构象。因此，失活的肌凝蛋白也可能有自己的特定的临时构象，有利于维持力。我们建议用原子力显微镜来解决这个问题。与上面列出的分子力学研究一起，这种成像将帮助我们获得闩锁状态的完整图像。*******最后，最近的锁存状态理论从分子测量中进化而来，表明部分失活的肌凝蛋白（两个失活的头中的一个）与肌动蛋白的相互作用时间比双重或未激活的肌凝蛋白更长。这样的肌凝蛋白会附着在肌动蛋白上并保持力量。然而，这一理论尚未在整个肌肉水平上得到证实。因此，我们建议进行全肌肉机械和生化分析，以验证在闩锁状态下是否存在这种单一激活的头部。*******总之，这个研究项目提出了一个多层次（从分子到整个肌肉）和多学科的方法来了解平滑肌有效维持力的独特能力******

Smooth muscle is found in all hollow organs of the body (e.g. intestine, bladder, blood vessels). Smooth muscle is unique in its ability to generate and maintain force for long periods of time, at low energy cost. Once stimulated to contract, smooth muscle can modulate its rate of energy consumption within the time course of a single contraction. This property, called the latch-state, is an important characteristic of smooth muscle but remains poorly understood. Thus, the overarching goal of this program of research is to understand the molecular mechanism behind the latch-state.*******Muscle contraction is accomplished by the interaction of the myosin molecular motors with actin filaments. It is believed that the latch-state occurs when myosin molecules get enzymatically deactivated while attached to actin. This theory came from measurements performed at the whole muscle level but was never verified at the molecular level. Several other proteins (calponin, caldesmon, non-muscle myosin, etc.) have also been hypothesized to play a role in the latch state but again, no proof of their contribution has been made at the molecular level. Thus, we aim to determine whether force can be maintained during deactivation of myosin by making force measurements directly at the molecular level. With my previous NSERC grant, we developed a micro-chamber (microfluidic device) that allows the addition of chemicals without creating any bulk flow. Thus, we now propose to use this chamber to inject deactivating enzymes during molecular force measurements and determine whether or not force maintenance (latch-state) occurs. We will also determine whether other proteins such as telokin, caldesmon etc. are needed for force maintenance or if myosin and actin are sufficient.*******Activated and non-activated myosins have very different conformations. Thus, it is possible that deactivated myosin also has its own specific temporary conformation that favours force maintenance. We propose to address this question using atomic force microscopy. Along with the molecular mechanics studies listed above, this imaging will help us get a complete picture of the latch state.*******Finally, a more recent theory of the latch-state evolved from molecular measurements that showed that partially deactivated myosin (one deactivated head out of two) interacts longer with actin than doubly- or unactivated myosin. Such myosin would remain attached to actin and maintain force. This theory has however not been verified at the whole muscle level. Thus, we propose to perform whole muscle mechanical and biochemical assays to verify whether or not such single activated heads are present during the latch state.*******In summary, this program of research proposes a multi-level (from molecule to whole muscle) and multi-disciplinary approach to understand the unique ability of smooth muscle to efficiently maintain force.******