We seek basic understandings of the cardiac thin filament, understandings that are relevant to the pathophysiology and treatment of highly prevalent cardiovascular disorders: ischemic heart disease, congestive heart failure, and hypertension. Thin filaments are large molecular assemblies that are challenging to understand in detail, but worth the requisite effort because of their high physiological and pathophysiological importance. The thin filament's sensitive response to variations in intracellular Ca2+ concentration is the most important rheostatic mechanism for adjusting force production in the heart. Furthermore, Ca2+-thin filament binding is the primary activation-inactivation switch for muscle contraction. The goal of this project is to elucidate the mechanism of regulation at the thin filament level, relating structure to cooperative function. To achieve this, structural insights must advance, and this is possible because of recent progress. Also, new functional data are needed. In this context, we propose four Aims: 1. Recently we proposed and provided evidence to support a thin filament structural model incorporating the high-resolution structure of troponin. However, other investigators have proposed a competing structural model of the thin filament. We will distinguish between these models, and provide tests of each, using single-molecule high resolution imaging with photobleaching; 2. Regardless of the outcome of Aim 1, it will remain important to obtain additional insight into thin filament structure. Electron microscopy and 3-D reconstruction will be performed with two independent types of experimental preparations: (a) Thin filaments reconstituted from actin, troponin, and tropomyosin. (b) True single particles, i.e., engineered mini-thin filament particles of single regulatory unit length; 3. Thin filament regulation depends on control of strong myosin binding to actin by troponin and tropomyosin, but the mechanism of this control is not understood. We will characterize the relative propensity of myosin to attach to each of the seven actins within the 7-actin long regulatory unit, using fluorescence resonance energy transfer between myosin and a panel of specifically engineered tropomyosins; 4. Recently we proposed an atomic model for the positioning of tropomyosin on F-actin.This proposal will be tested by functional and structural analyses of designed tropomyosin mutants and fragments.
