Reversible Modification of Thick‐ and Thin‐filament Proteins in Oxidant‐induced Changes to Skeletal Muscle Function

Exposure of skeletal muscle to reactive oxygen species (ROS) has been shown to modify the calcium sensitivity of force production. Calcium sensitivity changes were largely reversed by the reducing agent DTT, indicating modification of protein thiol groups. To determine which proteins are modified we used permeabilized rabbit psoas in which the intracellular and redox conditions can be tightly controlled. Oxidants hydrogen peroxide and dithiodypridine (DTDP) and reductants DTT and glutathione were used to modify thiols. We identified myofilament proteins with modified thiols using 3 gel electrophoresis techniques: 1d SDS page, 2d IEF/SDS page, and diagonal SDS page electrophoresis. To relate modifications of myofilament protein thiols to altered muscle mechanics, force‐pCa curves were measured. Exposure to oxidants caused increased actin and myosin oxidation. Treatment with glutathione produced gluathionylation of myofilament proteins including myosin. 2d diagonal gels showed that several myofilament proteins formed intermolecular disulfide bonds and that the regulatory myosin light chain could form an intramolecular disulfide bond. Force‐pCa curves confirmed that treatments that modified free thiol groups also modified calcium sensitivity. Results indicate that the reversible modification of both thick and thin filament proteins play a role in the oxidant‐induced changes to muscle function.