Actin-binding compounds, discovered by FRET-based high-throughput screening, differentially affect skeletal and cardiac muscle

Abstract

We have used spectroscopic and functional assays to evaluate the effects of a group of actin-binding compounds on striated muscle protein structure and function. Actin is present in every human cell, and its interaction with multiple myosin isoforms and multiple actin-binding proteins is essential for cellular viability. A previous highthroughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay from our group identified a class of compounds that bind to actin and affect actomyosin structure and function. In the current study, we tested their effects on the two isoforms of striated muscle α-actins, skeletal and cardiac. We found that a majority of these compounds affected the transition of monomeric G-actin to filamentous F-actin, and that these effects were different for the two actin isoforms, suggesting a different mode of action. To determine the effects of these compounds on sarcomeric function, we further tested their activity on skeletal and cardiac myofibrils. We found that several compounds affected ATPase activity of skeletal and cardiac myofibrils differently, suggesting different mechanisms of action of these compounds for the two muscle types. We conclude that these structural and biochemical assays can be used to identify actin-binding compounds that differentially affect skeletal and cardiac muscles. The results of this study set the stage for screening of large chemical libraries for discovery of novel compounds that act therapeutically and specifically on cardiac or skeletal muscle.

Actin-binding compounds that affect the kinetics of the interaction of cardiac myosin with actin

“…This was confirmed by the observation that actin-activated ATPase activity was decreased by several of the compounds (14). In a subsequent study, the same authors showed that several of these compounds showed significantly different effects on the Ca 2+dependence of steady-state ATPase activities of cardiac and skeletal myofibrils (16). This motivated the present study, in which we have used PFA and transient kinetics to define more clearly the mechanisms of action of these compounds, focusing on cardiac actin-myosin interaction.…”

“…A subsequent study by the same authors showed that some of these actin-binding compounds have distinct effects on the polymerization properties of skeletal and cardiac actin isoforms, and on the steady-state ATPase activities of skeletal and cardiac myofibrillar ATPase activity (16). Given the very small sequence differences in structure between cardiac and skeletal muscle actin, this is a surprising finding.…”