oa Three-Dimensional Structure Of The Human Cardiac Myosin Thick Filament - Clinical Implications

All muscles involve the interaction between two sets of filamentous proteins, actin and myosin, that leads to muscle contraction and force production mediated by the hydrolysis of ATP (Adenosine triphosphate). Actin filament structure is understood to high resolution, but myosin filament structure is much less well defined. The myosin filaments are formed from complicated arrangements of myosin molecules and accessory proteins. Myosin molecules are hexameric polypeptide chains, each consisting of two myosin heavy chains (MHC) and four myosin light chains (MLC). Accessory proteins are the myosin binding protein C (MyBP-C) and Titin. We have previously resolved the three-dimensional (3D) structure of myosin filaments in normal human heart muscles (AL-Khayat et al., Proc. Natl. Acad. Sci., USA, 110, 318-323). Mutations in cardiac muscle myosin (MHC or MLC) and its associated proteins (MyBP-C or Titin) are known to be associated with a number of myopathies (e.g. familial hypertrophic cardiomyopathy and dilated cardiomyopathy) which change the proteins involved in producing and regulating heart muscle contraction. In order to understand the effect of myosin-associated heart disease, it is important to understand the 3D structure of myosin filaments in both the normal as well as in the diseased human heart muscles. The aim of this project is to study and compare the arrangement of the myosin molecules and the accessory proteins in the human heart muscle myosin filaments and how these arrangements change in diseased human heart muscles, suffering from either hypertrophic or dilated cardiomyopathies. A laboratory method to isolate myosin filaments from normal undiseased human cardiac muscle that preserves the highly ordered pseudo-helical structure of the myosin filaments has already been developed. This led, for the first time, to the detailed analysis of the 3D structure of myosin filaments from normal human heart muscles by utilizing the experimental technique of transmission electron microscopy (EM) as well as the computational single particle image analysis, 3D reconstruction and structural interpretation. Knowledge of this 3D structure serves as the starting point from which myosin filaments isolated from human cardiomyopathic samples, with known mutations in either myosin or its associated proteins, will be studied later in detail. The 3D structure of mutated myosin filaments will be resolved by a state of the art Electron Microscopy Facility at QCRC which is now underway. This will have both the highest spatial and time resolution for collecting EM images. A laboratory method will also be developed to isolate myosin filaments from human cardiomyopathic samples, which will be examined by EM and single particle image analysis so that to determine the overall 3D structure of myosin filaments in diseased heart muscles. By direct comparison to the known 3D structure of myosin filaments from normal undiseased human cardiac muscle, this would eventually permit the structural effects of known myosin filaments-associated mutations to be investigated in detail as well as to relate structure-to-function-to the overall disease process. Detailed understanding of the disease process would then allow us to design possible.