Original ContributionAge-related alterations in oxidatively damaged proteins of mouse skeletal muscle mitochondrial electron transport chain complexes
Abstract
Age-associated mitochondrial dysfunction is a major source of reactive oxygen species (ROS) and oxidative modification to proteins. Mitochondrial electron transport chain (ETC) complexes I and III are the sites of ROS production and we hypothesize that proteins of the ETC complexes are primary targets of ROS-mediated modification which impairs their structure and function. The pectoralis, primarily an aerobic red muscle, and quadriceps, primarily an anaerobic white muscle, have different rates of respiration and oxygen-carrying capacity, and hence, different rates of ROS production. This raises the question of whether these muscles exhibit different levels of oxidative protein modification. Our studies reveal that the pectoralis shows a dramatic age-related decline in almost all complex activities that correlates with increased oxidative modification. Similar complex proteins were modified in the quadriceps, at a significantly lower level with less change in enzyme and ETC coupling function. We postulate that mitochondrial ROS causes damage to specific ETC subunits which increases with age and leads to further mitochondrial dysfunction. We conclude that physiological characteristics of the pectoralis vs quadriceps may play a role in age-associated rate of mitochondrial dysfunction and in the decline in tissue function.
Introduction
Sarcopenia, or muscle weakness, is a major physiological sign of aging in humans [1], [2], [3], [4]. The mitochondrial theory of aging proposes that increasing oxidative stress resulting from progressive mitochondrial dysfunction is a mechanism of mammalian aging and may be an important physiological characteristic of muscle aging [5], [6]. It is well established that although ROS are generated by multiple compartments, e.g., the plasma membrane NADPH oxidases [7], peroxisomal lipid metabolism, and cytosolic enzymes such as cyclogenases, the majority (∼90%) of ROS is generated by mitochondrial dysfunction [8]. The generation of mitochondrial ROS, in vivo, is linked to the processes of oxidative phosphorylation carried out by the mitochondrial electron transport chain complexes. Thus, leakage of electrons, i.e., mitochondrial ROS, is produced in vivo, from ETC complexes during normal respiration, particularly from Complex I (CI) and Complex III (CIII) [9], [10], [11]. Increased ROS production and oxidative stress during the aging process are, therefore, attributed to mitochondrial dysfunction, and the age-associated decline in tissue function is proposed to be a consequence of dysfunctional mitochondria [12], [13], [14].
Mitochondrially generated ROS are difficult to measure directly both in vitro and in vivo. Although the production of ROS in CI and CIII has been estimated by electron paramagnetic resonance [15], [16], [17], the short half-life of these radicals makes their accurate measurement difficult. Since the products of ROS reaction with proteins are stable and easily measured we have proposed that the amount of modified protein may be indicative of the level of oxidative modification in tissues [13]. Furthermore, since oxidatively modified proteins have been shown to impair the functions of these proteins [18], [19], [20], [21] we have initiated these studies to characterize the role of these modifications on the development of mitochondrial dysfunction in aged pectoralis (aerobic) and quadriceps (anaerobic) skeletal muscle.
The covalent oxidative protein modifications caused by reactions with free radicals are more stable and easily detectable, and thus have been used as molecular markers of oxidative stress [17], [22], [23], [24]. The relative abundance of modified proteins has been used in our laboratory to indicate the level of oxidatively modified proteins that accumulate in aged tissues [22], [23], [24]. Protein modifications caused by ROS include the formation of lipid peroxidation adducts (4-hydroxynonenal or HNE and malondialdehyde or MDA) on lysine, histidine, and cysteine, nitration of tyrosine and cysteine, and carbonylation oflysine, arginine, proline, and threonine [18], [19], [25], [26]. Oxidatively modified proteins have been detected by immunoblotting using antibodies specific for these modifications and subsequently identified by mass spectrometry [17], [22], [23], [24]. In addition, such oxidative modifications to proteins can result in reduction of function associated with aging and age-associated diseases [18], [19], [21], [22], [23], [24], [25], [26], [27]. These oxidative modifications are, therefore, molecular markers that provide insight into the cumulative effects of oxidative stress on the molecular mechanisms of aging and development of age-associated diseases.
In this study we analyzed mitochondria isolated from young, middle-aged, and old mouse pectoralis and quadriceps skeletal muscles because of their aerobic vs anaerobic physiological characteristics, to determine whether aging affects the activities of their ETC complexes CI–CV. We chose, specifically, the pectoralis which is an adductor, internal rotator, and flexor of the shoulder; its fibers, which are slow twitch (type I), consist of high myoglobin levels which improves the delivery of oxygen, and high mitochondrial content, are primarily red muscle and highly aerobic. Secondly, we chose the quadriceps which consists of fast-twitch type I fibers whose physiological characteristics include fewer mitochondria and less myoglobin. The large store of glycogen and high levels of glycolytic enzymes enable these fibers to respire anaerobically. We tested, therefore, whether specific ETC CI–CV proteins of the highly aerobic pectoralis and primarily anaerobic quadriceps muscles are differentially susceptible to oxidative modification due to their location within the mitochondria, whether the levels of these modifications increase, and whether their functions are altered with aging. To achieve this we identified the oxidatively modified subunits of ETC CI–CV and correlated the levels of protein modification with changes in enzyme activities. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was used to resolve intact ETC complexes [28] followed by second-dimension denaturing SDS-PAGE to resolve individual complex subunits. Protein abundance of each complex was measured using antibodies to specific complex subunits to quantify age-related changes [22], [23]. Using immunobloting with antibodies recognizing specific types of oxidative damage, we detected proteins that were carbonylated, and modified by HNE, MDA, and tyrosine nitration. Proteins shown to be differentially modified were identified by MALDI-TOF-TOF mass spectrometry. These studies identify whether there is a differential susceptibility of highly aerobic skeletal muscle ETC complexes compared to the anaerobic skeletal muscle. Finally, we determined whether there is a direct correlation between increased protein modification on enzyme function associated with the ETC complexes that might suggest a progressive increase in endogenous oxidative stress during aging. Our study systematically identifies the effects of oxidative protein modification on ETC activity, on possible specificity of targeted oxidative modification and whether these modifications play a role in causing symptoms of the aging, of the pectoralis and quadriceps.
Section snippets
Animals
Young (3–5 months), middle–aged (12–14 months), and old (20–22 months) male C57BL/6 mice, purchased from the National Institute on Aging (Bethesda, MD), were maintained in a pathogen-free facility at 27°C, 45–55% humidity, on a 14/10 light/dark cycle and fed ad libitum on an NIH low-fat diet (Purina) 4% fat by weight.
Mitochondrial isolation
Mice were sacrificed by cervical dislocation and their skeletal muscles were harvested immediately, rinsed in ice-cold PBS, and prepared for subcellular fractionations.
Inhibitor-sensitive enzyme activities from pectoralis and quadriceps
To evaluate the effects of aging on mitochondrial ETC complexes derived from the physiologically unique pectoralis (red-aerobic) and quadriceps (white-anaerobic) we compared the enzyme activities of all five complexes as well as the coupled activities of CI–III and CII–III for all three ages (Figs. 1 and 2).
Pectoralis enzyme activities
The data, in all cases, indicate that the enzyme activity in pectoralis decreased from young to middle age and in some cases (CI, CIII) further decline was noted from middle-age to aged
Discussion
Our studies show that the levels of oxidatively modified mitochondrial ETC proteins in the mouse pectoralis, whose physiological characteristics include high levels of myoglobin and mitochondria, hence the high oxygen-carrying capacity and aerobic respiration, are significantly lower than those of the quadriceps, a relatively anaerobic muscle, which contains considerably less amounts of myoglobin and mitochondria. We propose that the differences in the levels of their oxidatively modified
Acknowledgments
This publication was supported by U.S.P.H.S. grant 1P01 AG021830-04 awarded by the National Institute on Aging, and the National Institute on Aging 1 P30 AG024832-03 Claude D. Pepper Older Americans Independence Center grant and by the Sealy Center on Aging. J.E.N. thanks the Kempner Foundation and the National Institutes of Environmental Health Sciences Training Grant (T32-07254) for additional fellowship support.
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