Plasticity in Airway Smooth Muscle

Indiana Univ-Purdue Univ At Indianapolis, Indianapolis IN

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Abstract

DESCRIPTION (Applicant's abstract): Smooth muscle contracts over a much wider length range than can be accommodated by the fixed filament arrays of striated muscle, suggesting that its filament lattice is plastic and leading to the following hypotheses: 1. Smooth muscle adapts to different lengths by varying the number of thick filaments in series. 2. Thick filament lengthening produces the well-known velocity slowing during the rise of activation. 3. A thin filament mechanism is required to selectively engage myosin in thick filaments. Hypothesis 1 will be tested by disrupting thick filament and assaying the relative amounts of contractile proteins in filaments vs. free I the cytoplasm using three complimentary methods: (1) optical birefringence to asses thick filament density; (2) electron microscopy to count thick- and thin-filaments in cell cross-sections; and (3) biochemical assays of myosin and actin diffusion from permeabilized muscle placed in rigor at different lengths. Maneuvers to alter thick filament length and density will include the normal contraction-relaxation cycle, sudden length changes, in adapted length, and alternations in myoplasmic calcium and cAMP. Hypothesis 2 will be tested by looking for increased shortening velocity under conditions shown to disrupt thick filaments in the experiments testing Hypothesis 1. In addition, biochemical alterations, such as variations in intracellular calcium and C-AMP will assessed to determine if they affect velocity. Hypothesis 3 will be assessed from tests of thin filament cooperativity, notably decreased activation following sudden length perturbations that detach crossbridges, particularly during relaxation, when activation is likely to be maintained at level higher than expected from the degree of myosin light chain phosphorylation. Activation will be assessed from the rate and extent of force recovery following the perturbations and from the maximum power capability determined from force-velocity curves. The results will provide insights into mechanisms and possible therapies of asthma and hypertension. These diseases result from increased activation of muscles encircling hollow conduits, but only if the muscles shorten to diminish circumference. Thus, the disease might be prevented if shortening could be avoided.