DITHIOTHREITOL EFFECTS ON TEMPERATURE-INDUCED CHANGES IN THE CONTRACTILE ACTIVATION CHARACTERISTICS OF MAMMALIAN SKELETAL MUSCLE AS RESULT OF SUPEROXIDE PRODUCTION

Chris van der Poel, D. George Stephenson, School of Zoology, Faculty of Science and Technology, La Trobe University, Bundoora, Vic, 3086.

Brief exposure of fast-twitch rat skeletal muscle to supraphysiological temperatures results in marked reversible changes in contractile activation characteristics¹. The purpose of this study was to investigate whether these changes could be linked to the production of superoxide (O₂^-) and reversed by the reducing agent dithiothreitol (DTT), which is a sulphydryl (SH) specific reducing agent.

Long-Evans hooded rats were killed by an overdose of halothane. Extensor digitorum longus muscles were dissected and underwent temperature treatment at 46°C for 5 min. The production of O₂^- was measured using a cytochrome C assay². For measurements of contractile activation characteristics (maximum Ca²⁺-activated force, Ca²⁺-sensitivity and steepness of force-pCa (-log₁₀[Ca²⁺]) curves, single muscle fibres were dissected, mechanically skinned and activated in a series of Ca²⁺-buffered solutions with and without 10 mM DTT.

During the temperature treatment O₂^- was produced with an apparent maximum rate of 1.8 ± 0.2 nmol/min/gww and the presence of O₂^- was directly linked to the observed changes in contractile activation characteristics because the use of an O₂^- scavenger (20 mM Tiron) completely prevented any temperature-induced changes of the contractile activation characteristics.

Results show that :(i) the temperature (O₂^-)-induced effects on the ability of the contractile apparatus to develop force are not due to the reducing action of O₂^- on myofibrillar proteins but rather to O₂^- oxidising SH groups because DTT can reverse this action of O₂^-, (ii) the temperature (O₂^-)-induced force depression is dependent on the activation of the contractile apparatus because once the contractile apparatus was activated, DTT could not reverse this effect and (iii) the temperature (O₂^-)-induced effects on the steepness of the force-pCa curves could not be reversed by DTT indicating that O₂^- acts also on groups other than those responsible for the depression in force.

(1) van der Poel C, Stephenson DG. Proceedings of the Australian Physiological and Pharmacological Society. 2000;31(2):104P.

(2) Zuo L, Christofi FL, Wright VP, Liu CY, Merola AJ, Berliner LJ, Clanton TL. American Journal of Cell Physiology. 2000;279:C1058-C1066.