Effects of Actin-Binding Compounds on the ATPase Activity of Myosin from Skeletal and Cardiac Muscle

We have used an ATPase activity assay to assess the effects of actin-binding compounds on striated muscle protein structure and function. The human muscular system has been shown be affected by drugs used to treat disease. Our high-throughput and time-resolved fluorescence resonance energy transfer (TR-FRET) assay, used in a small-molecule screen, detected several compounds that affect actin-myosin structure and function in both cardiac and skeletal systems. The objective of the current study is to determine the specificity of the identified compounds in more physiological environments, particularly in cardiac and skeletal myofibrils. A malachite green assay for actin-activated myosin ATPase activity was used to test the enzymatic function of myosin. Tests were done on both skeletal (rabbit psoas) and cardiac (bovine ventricle) myofibrils with 0-100 μM concentration of test compounds. We found that the concentration-dependent responses of several compounds were different for skeletal and cardiac myofibrils, suggesting that the mode of action is different for the two muscle types. This study is designed to elucidate mechanisms of action for these compounds, in preparation of future drug design studies. This work was supported by NIH grants to DDT (R01AR032961, R01HL129814, R37AG026160, and T32GM008244) and LAP (F30AG057108)