Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle

Abstract

Skeletal muscle disuse with space-flight and ground-based models (e.g., hindlimb unloading) results in dramatic skeletal muscle atrophy and weakness. Pathological conditions that cause muscle wasting (i.e., heart failure, muscular dystrophy, sepsis, COPD, cancer) are characterized by elevated “oxidative stress,” where antioxidant defenses are overwhelmed by oxidant production. However, the existence, cellular mechanisms, and ramifications of oxidative stress in skeletal muscle subjected to hindlimb unloading are poorly understood. Thus we examined the effects of hindlimb unloading on hindlimb muscle antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase), nonenzymatic antioxidant scavenging capacity (ASC), total hydroperoxides, and dichlorohydrofluorescein diacetate (DCFH-DA) oxidation, a direct indicator of oxidative stress. Twelve 6 month old Sprague Dawley rats were divided into two groups: 28 d of hindlimb unloading (n = 6) and controls (n = 6). Hindlimb unloading resulted in a small decrease in Mn-superoxide dismutase activity (10.1%) in the soleus muscle, while Cu,Zn-superoxide dismutase increased 71.2%. In contrast, catalase and glutathione peroxidase, antioxidant enzymes that remove hydroperoxides, were significantly reduced in the soleus with hindlimb unloading by 54.5 and 16.1%, respectively. Hindlimb unloading also significantly reduced ASC. Hindlimb unloading increased soleus lipid hydroperoxide levels by 21.6% and hindlimb muscle DCFH-DA oxidation by 162.1%. These results indicate that hindlimb unloading results in a disruption of antioxidant status, elevation of hydroperoxides, and an increase in oxidative stress.

Introduction

Loss of skeletal muscle mass occurs with disuse or “hypokinesia” [1] as well as muscle wasting pathologies including sepsis, muscular dystrophy, heart failure, COPD, cancer, and AIDS 2, 3, 4. Extreme disuse and muscle wasting occur with spaceflight, chronic bed rest, hindlimb unloading, and immobilization. Hindlimb unloading is the preferred animal model for extreme disuse and spaceflight [1]. There is agreement that muscle wasting with hindlimb unloading, and other disuse models, is a product of atrophy of skeletal muscle fibers and increased protein degradation 1, 5. However, the cellular and molecular triggers that lead to muscle wasting with hindlimb unloading are not well understood.

A potential mechanism that would trigger increased protein degradation and atrophy in skeletal muscle is “oxidative stress,” where antioxidant protein and scavenger protection are overwhelmed by oxidant production [6]. However, there are limited data in the literature concerning oxidative stress and skeletal muscle in disuse models. Girten et al. [7] first described a reduction in the antioxidant enzyme activities for catalase and total superoxide dismutase following 14 d of hindlimb unloading. However, the cytosolic Cu,Zn- and mitochondrial Mn-isoforms of superoxide dismutase were not distinguished, and glutathione peroxidase was not measured. In contrast, Kondo et al. 8, 9 reported a more complex response to 8 d of cast immobilization. Glutathione peroxidase and catalase did not change with immobilization, while the Cu,Zn isoform of superoxide dismutase increased by 41%. Immobilization also increased contributors to oxidative stress in skeletal muscle including lipid peroxidation, oxidized glutathione/reduced glutathione ratio, free iron, and xanthine oxidase 9, 10, 11, 12. In addition, Kondo et al. [13] and Appell et al. [14] reported that vitamin E supplementation blunted the amount of immobilization-induced muscle fiber atrophy ranging from 23 to 66%, suggesting that oxidative stress may play a role in muscle atrophy.

Thus the responses of the full spectrum of antioxidant enzymes to hindlimb unloading have not been fully delineated. In addition, there are disparate reported responses of the antioxidant enzyme system among limited disuse studies. It is very possible that differences between hypokinesia models are a factor. Moreover, there is lack of information regarding the effects of disuse on nonenzymatic antioxidant scavenging capacity. Finally, a direct marker of oxidant production and oxidative stress, such as dichlorohydrofluorescein diacetate (DCFH-DA), has not been used to confirm the existence of oxidative stress in hindlimb unloading. Thus our purpose was (i) to identify changes in direct and indirect indicators of oxidative stress in skeletal muscle with 28 d of hindlimb unloading, and (ii) to identify changes in muscle antioxidant enzyme and scavenger capacity with hindlimb unloading. We hypothesized that hindlimb unloading would increase direct and indirect indicators of oxidative stress, and oxidative stress would be linked to an imbalance in the antioxidant system and increased hydroperoxides.