THE K104E MUTATION OF THE MYOSIN REGULATORY LIGHT CHAIN ALTERS KINETICS AND DISTRIBUTION OF ORIENTATIONS OF CROSS-BRIDGES IN TRANSGENIC CARDIAC MYOFIBRILS

My project aims at finding out the differences in a healthy and diseased heart. The disease I am focusing on is Familial Hypertrophic Cardiomyopathy(FHC), in particular, the K104E mutation, which is a mutation in the Regulatory Light Chain(RLC) of Myosin. The differences are established in terms of the rate of association and dissociation of myosin from actin, as well as the order of cross bridges. This study will help us to investigate the role of such mutations in causing FHC as well as offer us the opportunity to investigate other mutations causing this disease. Purpose (a): The purpose of my study is to examine the cross-bridge (XB) kinetics and the degree of order in contracting myofibrils from the ex-vivo left ventricles of transgenic (Tg) mice expressing Familial Hypertrophic Cardiomyopathy (FHC) Regulatory Light Chain (RLC) mutation K104E. Methods (b): 1. Myofibrils were prepared from the frozen hearts of Tg-WT mice and newly generated Tg-K104E mice. 2. Since the kinetics and degree of order are best studied when an individual cross bridge (XB) makes a significant contribution to the overall signal, the number of observed XBs was minimized to ~20 by sparsely labeling the Essential Light Chain(ELC) of myosin. Autofluorescence and photobleaching were minimized by labeling ELC with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule, SeTau 647. 3. Myofibrils were crosslinked with a cross linker prior to labeling. 4. Following labeling, fluorescence was measured by PicoQuant MT 200 inverse time-resolved fluorescence instrument coupled to Olympus IX 71 microscope. Results (c): We show that the K104E mutation, when compared with Wild Type (WT) ventricles, had significant effect on both the kinetics of the interaction between actin and myosin and on the degree of order of the myosin lever arm. In particular, the K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke, while decreasing the rate of XB dissociation from actin. Mutated XBs were significantly better ordered during steady-state contraction and during rigor but mutation had no effect on the degree of order in relaxed myofibrils. Conclusions (d): This implies that the mutated ventricle may be prone to decreased maximal tension and increased muscle relaxation time suggesting a potential for diastolic dysfunction in patients.