PHOSPHORYLATION OF THE REGULATORY LIGHT CHAIN PLAYS A DECISIVE ROLE IN CHOOSING BETWEEN ORDER AND CHAOS OF CROSS-BRIDGES IN MICE TIBIALIS MUSCLE

Purpose: Myosin consists of two heavy chains and four light chains. Two of the light chains, so called Regulatory Light Chains (RLC), get phosphorylated during muscle contraction. The role of this phosphorylation is currently unknown. In this work we describe our attempts to clarify this matter. Methods: A transgenic mouse was produced in which myosin RLC was devoid of serines that are normally necessary for phosphorylation. Glycerinated muscle bundles were dissected from the tibialis muscle of the mouse, and homogenized to produce myofibrils. Phosphorylation status in W.T muscle was preserved by adding phosphatase inhibitors. Essential light chain (the other two light chains of myosin, ELC1) was specifically labeled with SeTau fluorescent dye and exchanged with native ELC1. Cross-bridges of contracting myofibrils were analyzed for an error of the mean orientation. Results: Number of occurrences (histograms) of a given orientation were plotted vs. the mean orientation. The Full Width at Half Maximum (FWHM) of the mean was calculated. FWHM is an index of cross-bridge order/chaos. Small FWHM means order, large FWHM means chaos. During active contraction FWHM values in de-phosphorylated and phosphorylated muscle were 0.363 ± 0.049 and 0.456 ± 0.083, respectively. Two-tailed t test revealed statistically extremely significant difference (P<<0.001, t=4.83 with 63 degrees of freedom). Confidence intervals were from 0.0545 to 0.131. Conclusions: Phosphorylation of RLC causes considerable increase in the cone of angles within which myosin head interacts with thin filaments to produce force.