Due to reverse electron transfer, mitochondrial H2O2 release increases with age in human vastus lateralis muscle although oxidative capacity is preserved

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Abstract

Age-related changes in mitochondrial H2O2 release (MHR) could be responsible for an increase in oxidative stress in skeletal muscle and participate in the development of sarcopenia. We compared MHR in vastus lateralis biopsies obtained from young (23.5 ± 2.0 year, n = 6) and elderly (67.3 ± 1.5 year, n = 6) healthy sedentary men. Isolated mitochondria were incubated in the presence of glutamate/malate/succinate, with or without rotenone. Muscle fat oxidative capacity, citrate synthase, complex II, complex III, and cytochrome c oxidase activities were also measured. In parallel, we analyzed in gastrocnemius of young male Wistar rats (n = 6), the impact of lidocaine (local anesthetic used in humans) on mitochondrial respiration and MHR. In humans, muscle oxidative capacity was preserved with age but muscle MHR was markedly enhanced in elderly subjects compared to young adults (+175%, P < 0.05). Rotenone abolished this increase, demonstrating that it was due to a free radical release during reverse electron transfer from complex II towards complex I. Lidocaine can interfere with MHR measurements (intra-muscular injection in rats) but it can be avoided by minimizing contact with muscle (small multiple subcutaneous injections in humans). Physiologic consequences of the observed increase in muscle MHR with aging remain to be determined.

Introduction

Aging is characterized by several marked physiological changes, in particular by a loss of skeletal muscle mass and function termed sarcopenia (Morley et al., 2001). Many factors could be involved in this phenomenon. Among them, a loss of α-motor neuron input to muscle during aging (Roubenoff and Hughes, 2000), and a lower stimulation of muscle protein synthesis by feeding in elderly subjects (Mosoni et al., 1995, Dangin et al., 2003) were identified. Oxidative stress may also play a role in this process. It could lead to alterations of key metabolic enzymes (for instance those involved in the stimulation of muscle protein synthesis by feeding). It could also affect myofibrillar proteins and induce accelerated proteolysis, because oxidized proteins are prone to degradation (Levine et al., 1996). An increase in oxidative stress during aging may be related to an enhanced generation of reactive oxygen species (ROS). ROS are released by several pathways, but a large proportion is produced by the mitochondrial respiratory chain (Chance et al., 1979). It was demonstrated that respiratory chain complexes I and III generate superoxide anion (O2radical dot) (Barja, 1999), either from “normal” electron transfer after oxidation of complex I (glutamate, malate, pyruvate) or complex II (succinate)-linked substrates, but also from “reverse” electron transfer from complex II towards complex I (Liu et al., 2002). Spontaneously or by the action of superoxide dismutase (SOD), O2radical dot dismutates into H2O2, which is more stable and can diffuse through biological membranes.

Very few authors have analyzed age-related changes in skeletal muscle mitochondrial H2O2 release (MHR). Most often, complex I-linked substrates were used, and no significant age-related changes were found in glycolytic muscles in human (Tonkonogi et al., 2003) and in rats (Drew et al., 2003, Capel et al., 2004), although an increase has been described in one occasion in rats (Bejma and Ji, 1999). We recently showed in rats that MHR increased with age in an oxidative muscle (using complex I-linked substrates) (Capel et al., 2004). However, the effect of aging on skeletal muscle MHR in a situation where reverse electron transfer from complex II can occur has never been studied while the recent literature has suggested that this phenomenon may play a predominant role in MHR (Liu et al., 2002, St-Pierre et al., 2002, Lambert and Brand, 2004).

Therefore, the aim of the present study was to analyze the age-related changes in human muscle MHR using a mixture of complex I (glutamate, malate) and complex II (succinate) substrates, which is closer to the in vivo situation than when using only one type of substrate. In this context, all electron transfers within the respiratory chain are possible. We also performed measurements in presence of rotenone which blocks electron transfer to and from complex I. In addition, because the muscle biopsies were obtained using the local anesthetic lidocaine which can have uncoupling effects on mitochondria (Dabadie et al., 1987) and may therefore reduce MHR, we analyzed in young rats how lidocaine interferes with mitochondrial respiration and MHR measurements.

Section snippets

Subjects

Young (23.5 ± 2.0 year, n = 6) and elderly (67.3 ± 1.5 year, n = 6) healthy men participated in the study. All subjects had normal physical and medical examination. They were nonsmokers, not suffering from any diagnosed disease, and under no medication. They were sedentary, i.e., they did not participate in any regular physical exercise program. In order to rigorously select sedentary subjects, a maximal oxygen consumption (VO2max) test was performed before final inclusion. According to Coggan et al.

State III oxygen consumption

There was no significant effect of intra-muscular injection of lidocaine on state III respiration (Fig. 1A). There was only a tendency (P = 0.06) for a reduced respiration in the presence of glutamate + malate as substrates. Rotenone significantly stimulated succinate-supported state III oxygen consumption in control and lidocaine injected groups (Fig. 1A). When the three substrates were used together, rotenone significantly reduced oxygen consumption in both groups (Fig. 1A).

State IV oxygen consumption

The only significant

Discussion

The present study showed that mitochondrial H2O2 release (MHR) is increased with age in human vastus lateralis muscle using a combination of substrates close to the in vivo situation, and allowing normal and reverse electron transfer. The evolution of mitochondrial H2O2 release (MHR) in muscle during aging is still under debate and was never studied with such a substrate combination. Previously, in glycolytic muscles and using complex I-linked substrates, no changes with age were observed for

Acknowledgements

The authors are grateful to X. Leverve and R. Favier for their valuable scientific advice. We also thank Dr. I. Delcourt, M. Brandolini, C. Giraudet, G. Manlhiot, N. Mathieu for their skillful medical and technical support, and H. Lafarge for literature management. This study was funded by a grant “ATC Nutrition INSERM – INRA 2002”, AIP n°275.

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