The Effect of Glutathione on Skeletal Muscle Calcium Sensitivity and Myofilament Sulfhydryl Groups

Abstract

Glutathione, a critical reducing agent present in relatively high levels(∼5mM) in skeletal muscle, can also attach to protein thiols via a disulfide bond. This process is referred to as glutathionylation, and is thought to be a protective mechanism to prevent irreversible protein oxidation. Prior studies have shown that when skinned fibers are exposed to reduced glutathione there is an increase in calcium sensitivity with no significant change in maximal force. These calcium sensitivity changes were largely reversible by the reducing agent DTT, indicating modification of protein thiols. We measured the force-pCa relationship of permeabilized rabbit psoas fibers treated with DTDP a thiol-specific oxidizing agent and glutathione (5mM). 2D gel electrophoresis using either IEF 4-6.5 or NEPHGE 3-10 in the first dimension was used to identify myofilament proteins whose sulfhydryl groups were modified with the oxidant-glutathione treatment. Additionally, phosphorylation of the regulatory myosin light chain was analyzed using 2d gels (IEF 4-6.5) and the phosphorylation specific stain Diamond Pro-Q. The pCa 50 of skinned psoas fibers was decreased upon exposure to DTT. Following DTT, the addition of DTDP and GSH sequentially, increased the calcium sensitivity. 2D gel electrophoresis, indicated myosin as a critical target for glutahionylation under these conditions. The RMLC was phosphorylated in the psoas fibers and the level remained constant during oxidant - glutathione treatment. In summary, our data suggest that glutathionylation of myofilament proteins can modulate calcium sensitivity, and may play an important role in maintaining muscle function during oxidative stress.