Greater Susceptibility of the Sarcoplasmic Reticulum to H₂O₂ Injuries in Diaphragm Muscle from mdx Mice

Abstract

The aim of the present study was to investigate the direct effects of a reactive oxygen species, H 2 O 2 , on the contractile function and sarcoplasmic reticulum properties of dystrophindeficient diaphragm using chemically skinned fibers and sarcoplasmic reticulum vesicle preparations. The results obtained using Triton X-100-skinned fibers demonstrate that exposure to 1 mM H 2 O 2 had similar effects on the maximal Ca 2ϩ -activated tension and on the Ca 2ϩ sensitivity of the contractile apparatus of diaphragm fibers in Bl10 and mdx mice. The effects of H 2 O 2 were also assessed on sarcoplasmic reticulum function using saponin-skinned fibers and sarcoplasmic reticulum vesicle preparations. We found that H 2 O 2 induced changes in sarcoplasmic reticulum properties, particularly in the Ca 2ϩ pump function. The most important finding was that diaphragm muscle from mdx mice displayed increased sensitivity to the oxidant. Furthermore, in isolated superfused diaphragm muscle from mdx mice, the data demonstrate that the amount of superoxide anion produced under fatiguing conditions was increased. Our study shows that the sarcoplasmic reticulum, and the Ca 2ϩ pump in particular, in dystrophin-deficient muscles display increased susceptibility to H 2 O 2 injuries. This suggests that free radicals might, therefore, be involved in the pathophysiological pathway and dysregulation of Ca 2ϩ homeostasis of muscular dystrophy.

Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers

“…H 2 O 2 is naturally produced in many tissues, including skeletal muscle. It is involved in multiple physiological processes necessary for normal functioning of cells (29,69,76), but it can also have deleterious effects and has been used to study the mechanism of cardiac and smooth muscle dysfunction (37,57,59,98).…”

“…H 2 O 2 is naturally produced in many tissues, including skeletal muscle. It is involved in multiple physiological processes necessary for normal functioning of cells (29, 69, 76), but it can also have deleterious effects and has been used to study the mechanism of cardiac and smooth muscle dysfunction (37,57,59,98).Previously reported physiological studies on the effects of H 2 O 2 on skeletal muscle contractility have been performed with a variety of muscles, such as permeabilized extensor digitorum longus (EDL) and soleus muscle from rat, intact fibers from mouse foot, and permeabilized rat diaphragm fibers (2, 14, 38, 61) We have selected as our model muscle permeabilized rabbit psoas muscle fibers because these fibers have been routinely used to determine molecular changes in myosin during muscle contraction (20, 55), and because permeabilization diminishes the number of possible targets of peroxide by removing soluble proteins and the membrane-based excitationcontraction system. The majority of proteins remaining in the permeabilized psoas are actin (22%) and myosin (43%) (99).…”