Smooth muscle molecular mechanics

平滑肌存在于身体的所有中空器官（例如肠、膀胱、血管）中。平滑肌在其以低能量成本长时间产生和维持力的能力方面是独特的。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，称为闭锁状态，是平滑肌的一个重要特征，但仍然知之甚少。因此，这项研究计划的首要目标是了解闩锁状态背后的分子机制。 肌肉收缩是通过肌球蛋白分子马达与肌动蛋白丝的相互作用来完成的。据信，当肌球蛋白分子在附着于肌动蛋白时酶促失活时，锁存状态发生。这一理论来自于在整个肌肉水平上进行的测量，但从未在分子水平上得到验证。其他几种蛋白质（钙调蛋白、钙调蛋白、非肌肉肌球蛋白等）也被假设在闩锁状态中起作用，但同样，在分子水平上没有证据证明它们的贡献。因此，我们的目标是确定力是否可以保持在失活肌球蛋白直接在分子水平上进行力的测量。在我之前的NSERC资助下，我们开发了一种微室（微流体设备），允许添加化学品而不会产生任何大流量。因此，我们现在建议使用该室在分子力测量过程中注入失活酶，并确定是否发生力维持（锁存状态）。我们还将确定其他蛋白质，如telokin，caldesmon等是否需要维持力，或者肌球蛋白和肌动蛋白是否足够。 活化和非活化肌球蛋白具有非常不同的构象。因此，失活的肌球蛋白也可能具有其自身特定的临时构象，有利于力的维持。我们建议使用原子力显微镜来解决这个问题。沿着上面所列的分子力学研究，这种成像将帮助我们获得闩锁状态的完整图像。 最后，一个更近的闩锁状态理论从分子测量中发展出来，表明部分失活的肌球蛋白（两个中的一个失活的头部）与肌动蛋白的相互作用时间比双重或未激活的肌球蛋白更长。这样的肌球蛋白将保持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验证。因此，我们建议进行整个肌肉的机械和生物化学测定，以验证是否存在这样的单一激活的头部在闩锁状态。 总之，这项研究计划提出了一种多层次（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量的独特能力。

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.