The investigator will learn the technique of high resolution NMR spectroscopy of macromolecules. This technique, coupled with the investigators current expertise of the techniques proposed in this grant application brings biophysical, biochemical, and physiological techniques to bear on an important clinical issue - heart failure. Heart failure is characterized by decreased cardiac output and elevated left ventri-cular filling pressure. Patients with heart failure in NYHA class IV have less than 50 percent survival rate at one year. The proposed experiments have never been performed on human myocardium. The experiments will provide novel and important insights into the mechanisms of heart failure and may assist in future drug development for the treatment of heart disease. The research environment is challenging as well as supportive for academic career develop-ment. Alterations in the structure and function of myofibrillar proteins have been recently shown to be important in several cardiac myopathies. This has led to a greater understanding the contribution of myofibrilla proteins to failing myocardium and to the identification of potential sites for targeting therapy. We propose to study the structure and function of two important thin filament regulatory proteins, troponin (Tn) and tropomyosin (Tm), in normal and failing human myocardium. Using recombinant DNA technology, we will purify and characterize tropomyosin and troponin subunits from normal and failing human hearts. We will the investigate functional differences and similarities between regulatory proteins obtained from normal human myocardium and those from diseased human myocardium biochemically and physiologically (e.g., myo-fibrillar ATPase activity, pCa-force relationship in skinned fiber, pCa-fluo-rescence relationship). Ca2+ sensitizes will be selected to probe subunit interaction and to study changes in molecular structure. The proteins which demonstrate significant differences in myofibrillar function and response to Ca2+ sensitizes will then be studied structurally using spectroscopic methods, namely fluorescence and NMR spectroscopy.
The specific aims for this proposal are:
Specific Aim 1 : To isolate and determine the differences in regulatory proteins between thin filament from normal and disease human myocardium.
Specific Aim 2 : To investigate functional differences between normal and diseased human myocardium in terms of the interaction among regulatory proteins.
Specific Aim 3 : To identify and characterize Ca2+ sensitizers that exert a positive inotropic effect on diseased myocardium.
Specific Aim 4 : To investigate effect of Ca2+ sensitizers on the solution structure of troponin C and its complexes with other regulatory proteins obtained from normal and diseased human myocardium using fluorescence and NMR spectroscopy.
