Myosin light chain phosphorylation facilitates in vivo myosin filament reassembly after mechanical perturbation

It is generally believed that the myosin thick filaments in smooth muscle are structurally less stable than those in striated muscle. In vitro studies have shown that phosphorylation of the 20-kD myosin light chain (MLC) facilitates formation of the thick filaments from monomeric myosins in solution. It appears that the structural integrity of the thick filaments can be enhanced by phosphorylation of the MLC. It is however not known whether the transition from monomeric to filamentous myosin occurs in intact smooth muscle when the light chains are phosphorylated during muscle activation. The physiological significance of the thick filament lability and the role of MLC phosphorylation in modulating the filament integrity in intact smooth muscle are still being debated. It has been shown in our laboratory that mechanical strain is able to induce partial dissolution of unphosphorylated myosin thick filaments in intact smooth muscle; repolymerization of the thick filaments occurs when the muscle is subjected to cycles of contraction and relaxation. The objective of my thesis research is to determine whether MLC phosphorylation is required for repolymerization of the thick filaments after they have been partially disassembled by mechanical agitation. We used the conventional electron microscopy to quantify the cross-sectional density of myosin thick filaments in airway smooth muscle; partial dissolution of the thick filaments was induced by applying length oscillations to the muscle preparation; recovery of the thick filament density after oscillation was examined in the presence and absence of wortmannin, a potent inhibitor of myosin light chain kinase (MLCK). The results showed that isometric force production in airway smooth muscle was totally dependent on MLC phosphorylation. The inhibition of MLC phosphorylation alone did not cause disassembly of myosin filaments. The unphosphorylated thick filaments however partially dissolved when the muscle was subjected to oscillatory strains, as evidenced by a 25% decrease in the filament density. The post-oscillation filament density did not recover when wortmannin was present; it recovered to the pre-oscillation level when wortmannin was removed. Based on the above findings, we conclude that thick filament formation in vivo is MLC phosphorylation dependent.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat