Smooth muscle molecular mechanics

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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.******