Testing Predictions of a Simple Two-State Model of Thin Filament Regulation: Inhibitors that can Activate Thin Filament Motility

Abstract

Striated muscle is activated by calcium binding and further activated when myosin heads strongly bind the regulated thin filament (RTF). Several intermediate states of the myosin-RTF binding pathway continue to be debated, however for an accurate mesoscopic kinetic model it is imperative to define only stable chemical states without inclusion of transient intermediate structures. Our novel simplified model of thin filament regulation (SMoR) is a streamlined, two-state system with calcium- and myosin-dependent binding rates. This minimalist model is consistent with both the Hill and the McKilloop and Geeves models. It is easily incorporated into larger scale models of skeletal and cardiac mechanics and provides a simple analytical expression with powerful predictive value for accurate hypothesis testing. SMoR predicts amrinone, an inhibitor of ADP release, will increase calcium sensitivity; sucrose, an inhibitor of myosin attachment, will decrease calcium sensitivity as a result of modulations of attachment and detachment kinetics, respectively. Our experimental data shows pCa50 shifts from a standard value of 5.65 to 6.44 with 4mM amrinone; and to 5.14 with 180mM sucrose. Our model predicts that altering both attachment and detachment kinetics will have compensatory effects, allowing calcium sensitivity to be “rescued”. Complex modulations of myosin kinetics were tested experimentally with 4mM amrinone which was rescued with low myosin density and 120mM sucrose to restore the pCa50 to 5.64 and 5.66 respectively. SMoR predicts that decreasing the ADP release rate at low calcium can increase actin-sliding velocities. Consistent with the prediction, we show amrinone increase actin-sliding velocities at sub-activating calcium concentrations. The ability to make complex predictions from fundamental principles has yet to be shown by any other model. SMoR is an accessible and accurate alternative to more complex models of regulation.