Reduced mitochondrial ROS, enhanced antioxidant defense, and distinct age-related changes in oxidative damage in muscles of long-lived Peromyscus leucopus

Abstract

Comparing biological processes in closely related species with divergent life spans is a powerful approach to study mechanisms of aging. The oxidative stress hypothesis of aging predicts that longer-lived species would have lower reactive oxygen species (ROS) generation and/or an increased antioxidant capacity, resulting in reduced oxidative damage with age than in shorter-lived species. In this study, we measured ROS generation in the young adult animals of the long-lived white-footed mouse, Peromyscus leucopus (maximal life span potential, MLSP = 8 yr) and the common laboratory mouse, Mus musculus (C57BL/6J strain; MLSP = 3.5 yr). Consistent with the hypothesis, our results show that skeletal muscle mitochondria from adult P. leucopus produce less ROS (superoxide and hydrogen peroxide) compared with M. musculus. Additionally, P. leucopus has an increase in the activity of antioxidant enzymes superoxide dismutase 1, catalase, and glutathione peroxidase 1 at young age. P. leucopus compared with M. musculus display low levels of lipid peroxidation (isoprostanes) throughout life; however, P. leucopus although having elevated protein carbonyls at a young age, the accrual of protein oxidation with age is minimal in contrast to the linear increase in M. musculus. Altogether, the results from young animals are in agreement with the predictions of the oxidative stress hypothesis of aging with the exception of protein carbonyls. Nonetheless, the age-dependent increase in protein carbonyls is more pronounced in short-lived M. musculus, which supports enhanced protein homeostasis in long-lived P. leucopus.

Fig. 1.

Hind limb muscle fiber types. Representative images of hind limb muscle cross sections of succinate dehydrogenase (SDH) activity staining (A, B), cytochrome-c oxidase (COX) staining (C, D) and immunofluorescence with antibodies against myosin heavy chain I (MHC I; blue), MHC IIa (green), and MHC IIb (red) (E). An illustration of different muscles in the cross-section view is shown on the right. A, C, E: top four panels are images of M. musculus. B, D, E: bottom four panels are images of P. leucopus. Scale bar = 100 μm. E, bottom: quantitative result of the percentage of different fiber types in the gastrocnemius muscle from M. musculus (black bars) and P. leucopus (gray bars). Quantification was performed from n = 4 animals per species and by counting at least 1,000 fibers per animal. Data are expressed as means ± SE. Representative transmission electron micrograph (TEM) images of gastrocnemius muscles from P. leucopus (F) and M. musculus (G). Scale bar equals 2 μm. All images were captured with same exposure settings and scaled equivalently postacquisition without gamma adjustment.

Fig. 2.

Activity of glycolytic and oxidative enzymes and electron transport chain complexes. A: skeletal muscle homogenates were used to test glycolytic enzyme activities: hexokinase (HK), pyruvate kinase (PK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and oxidative enzymes, cytochrome-c oxidase (COX), and citrate synthase (CS) (n = 6–12). B: activities of respiratory complex chains, including NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinone:cytochrome-c oxidoreductase (complex III), cytochrome-c oxidase (complex IV), and ATPase. Mitochondrial proteins were solubilized with dodecyl maltoside (1%), and membrane-enriched soluble proteins were then used to measure electron transport chain (ETC) activity either using a spectrophotometric assay or an in-gel assay, see details in materials and methods (n = 6–10). C: representative blue-native gel image of electron transport chain complexes. D: quantification of protein contents of ETC (n = 8). E: production of ATP was measured in isolated skeletal muscle mitochondria using a luciferase/luciferin assay kit as described in materials and methods (n = 6). F: mitochondrial membrane potential (ΔΨ_m) was measured using fluorescent safarine O dye. The quenching of safarine O fluorescence is proportional to the electrical potential across the mitochondrial inner membrane (n = 6). Black bar represents M. musculus, and gray bar represents P. leucopus. Data are expressed as means ± SE. *P < 0.05 compared with M. musculus.

Fig. 3.

Mitochondrial reactive oxygen species (ROS) production. Mitochondrial H₂O₂ production was measured using Amplex-Red probe at nonphosphorylating state (A) and in the presence of inhibitors (B) (n = 6). Mitochondrial superoxide release was measured by electron paramagnetic resonance spectroscopy (EPR) with spin trap DIPPMPO with substrates (C) and with antimycin A (D) (n = 8); matrix aconitase activity was measured in the presence of succinate, succinate+rotenone and succinate+rotenone+antimycin A (E) (n = 8). The inactivation reflects the net production of superoxide in the mitochondrial matrix compartment. F: uncoupling protein 3 (UCP3) was blotted in mitochondrial fractions from muscles of M. musculus and P. leucopus. Top: representative images of Western blot analysis. Bottom: quantification results (n = 3). Black bar represents M. musculus, and gray bar represents P. leucopus. Data are expressed as means ± SE. *P < 0.05 compared with M. musculus.

Fig. 4.

Antioxidant enzyme activities. Activities of CuZnSOD (A), MnSOD (B), catalase (C) and glutathione peroxidase (D) were measured in skeletal muscle homogenates from M. musculus and P. leucopus, as described in detail in materials and methods. n = 4 for (A) and (B); n = 6 for (C) and (D). Black bar represents M. musculus, and gray bar represents P. leucopus. Data are expressed as means ± SE. *P < 0.05 compared with M. musculus.

Fig. 5.

Age-associated oxidative damage to lipids and proteins. The scatterplots of skeletal muscle isoprostanes in M. musculus and P. leucopus over chronological age (A) and physiological age (B). The association between protein carbonyl levels in muscle with chronological age (C) and physiological age (D) in M. musculus and P. leucopus. Each point in the graphs represents one individual animal. Black triangles (▲) represent P. leucopus and open diamonds (◇) represents M. musculus. Linear regression line is shown on top of each data set. The correlation was tested using Pearson two-tailed test with P < 0.05 considered to be significant.