| Notes: | Advisor: Julian Solway.
Includes supplementary digital materials.
Thesis (Ph.D.)--The University of Chicago, Division of the Biological Sciences, and The Pritzker School of Medicine, Department of Pathology, 2012.
Dissertation Abstracts International, Volume: 74-02(E), Section: B.
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| Summary: | Asthma is chronic lung disorder characterized by excessive airway narrowing and inflammation, both of which make breathing difficult during an asthma exacerbation. Airway narrowing is largely a result of contraction of the airway smooth muscle, which is found helically wrapped around the airways. Breathing is known to functionally antagonize bronchoconstriction caused by airway muscle contraction. In vitro, experimental application of force fluctuations to contracted airway smooth muscle strips causes them to relengthen. Such force fluctuation-induced relengthening (FFIR) likely represents the mechanism by which breathing antagonizes bronchoconstriction. Here I propose that the connectivity between actin filaments, connected through intervening myosin filaments in contracting airway myocytes, is a key determinant of FFIR. First, I first demonstrate that MEK, an enzyme previously implicated in the regulation of smooth muscle contraction, augments FFIR in canine tracheal smooth muscle strips. Furthermore, MEK inhibition blunts phosphorylation of h-caldesmon, a downstream target of MEK thought to inhibit actin and myosin interaction in its non phosphorylated state. Next, I describe a novel method to study the contraction of intraparenchymal human airways subjected to dynamic conditions that simulate breathing. The results demonstrate that breathing-induced reversal of bronchoconstriction depends upon the degree to which each breath actually stretches the airway tidally, which in turn depends upon both the depth of breathing and the severity of bronchoconstriction. Finally, I describe two projects that, when complete, will allow for direct evaluation of the above hypothesis in human airway smooth muscle. In the first, I show that treating human airways with latrunculin B, a compound that is thought to shorten actin, enhances breathing induced reversal of bronchoconstriction. In the second, I have developed a system to allow for direct evaluation of actin filament lengths, myosin filament lengths, and of their interconnectivity, using 3D reconstructions of smooth muscle contractile myofilament ultrastructure built from transmission electron micrographic tomograms of human tracheal smooth muscle strips that have been high pressure frozen at physiological length. Results show that acetylcholine stimulation results in an increase in both actin filament and myosin filament total content and total numbers. These studies shed new light on the role of contractile filament connectivity in regulating airway smooth muscle relengthening in response to breathing.
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