We plan to utilize ADR to determine how this drug affects the activity and assembly of the major ultrastructural proteins composing the myofibrils in cardiac cells. We will attempt to determine the manner by which ADR binds to the plasma membrane of both cardiac tissue and cardiac cells in culture, and the mechanism by which ADR is internalized so readily in the cytoplasm. We will establish the site of binding and storage of this drug by isolating distinct vesicular organelles and the regulatory and contractile structures of the myofibrils. By using ADR as a probe, we will dissect the mechanism by which cardiac cells assemble their fibrillar structures and how ADR interferes with this assembly. For this study, we will use our established laboratory experience and techniques that include biochemical, biophysical and immunological determinations. In vitro our studies will focus on alterations by ADR on the basic properties of Alpha-actinin, myosin, actin, tropomyosin, and the troponin complex. They will include ADR binding and uptake by subcellular organelles such as plasmalemma or sarcoplasmic reticulum vesicles. Ultrastructurally, we plan to dissect the location of ADR-target proteins in the sarcomeres and to determine with labeled antibodies alterations in their arrangement or structural integrity. OUr ultimate objective is to understand the basic events that lead to human ADR cardiac muscle disease.
