Dietary restriction attenuates age-associated muscle atrophy by lowering oxidative stress in mice even in complete absence of CuZnSOD

Abstract

Age-related loss of muscle mass and function, sarcopenia, has a major impact on the quality of life in the elderly. Among the proposed causes of sarcopenia are mitochondrial dysfunction and accumulated oxidative damage during aging. Dietary restriction (DR), a robust dietary intervention that extends lifespan and modulates age-related pathology in a variety of species, has been shown to protect from sarcopenia in rodents. Although the mechanism(s) by which DR modulates aging are still not defined, one potential mechanism is through modulation of oxidative stress and mitochondrial dysfunction. To directly test the protective effect of DR against oxidative stress-induced muscle atrophy in vivo, we subjected mice lacking a key antioxidant enzyme, CuZnSOD (Sod1) to DR (60% of ad libitum fed diet). We have previously shown that the Sod1(-/-) mice exhibit an acceleration of sarcopenia associated with high oxidative stress, mitochondrial dysfunction, and severe neuromuscular innervation defects. Despite the dramatic atrophy phenotype in the Sod1(-/-) mice, DR led to a reversal or attenuation of reduced muscle function, loss of innervation, and muscle atrophy in these mice. DR improves mitochondrial function as evidenced by enhanced Ca2+ regulation and reduction of mitochondrial reactive oxygen species (ROS). Furthermore, we show upregulation of SIRT3 and MnSOD in DR animals, consistent with reduced mitochondrial oxidative stress and reduced oxidative damage in muscle tissue measured as F2-isoprostanes. Collectively, our results demonstrate that DR is a powerful mediator of mitochondrial function, mitochondrial ROS production, and oxidative damage, providing a solid protection against oxidative stress-induced neuromuscular defects and muscle atrophy in vivo even under conditions of high oxidative stress.

Figure 1. Calorie restriction (CR) attenuates age-related muscle atrophy

(A) A comparison of gastrocnemius (left) and vastus lateralis (right) muscle wet-weight normalized to body weight (n=6–7). (B) Transmission electron micrographs (TEM) of interfibrillar mitochondria (C) and neuromuscular junctions (D) from gastrocnemius muscle of young, old, and old DR. Arrows denote abnormal mitochondria (n=3). Scale bar=2μm (E) A representative image of neuromuscular junction from gastrocnemius muscle. Scale bar=20μm. (F) A quantification of postsynaptic endplate area. Young mice are 7 months and old mice are 33 months old (n=6). (G) Oxidative stress is reduced by DR as measured by F₂-isoprostanes as a marker of lipid peroxidation (n=6). Young vs. Old **p<0.01 and Old vs. Old DR ^#p<0.05. All values are represented as mean ± SEM.

Figure 2. Dietary restriction (DR) attenuates age-related muscle atrophy and degeneration in Sod1^−/− mice

(A) Bodyweight measured throughout the lifespan (n=6–8). (B) A comparison of gastrocnemius wet weight measured throughout the lifespan (n=6–8). (C–G) A comparison of morphological features of WT, Sod1^−/−, and Sod1^−/− CR at 11–14 months. (C) A representative images of hematoxylin and eosin staining of gastrocnemius muscle (n=4). (D) A representative staining of Gomori trichrome staining of gastrocnemius muscle (n=4). Black arrows denote increased fibrosis. % Fibrosis is defined as the area of blue relative to red fiber area in a given field of view. 20–30 random fields of view were chosen for quantification. (E) A representative immunofluorescence staining of gastrocnemius muscle. Sarcolemma membrane is stained with anti-laminin pseudo colored in red, myonuclei are colored in blue using DAPI, and oxidative fibers are stained with anti-cytochrome c oxidase in green. White arrows represent fiber grouping and yellow arrowhead denote centralized nuclei (n=4). (F) A quantification of cross sectional area shown in (C). (G) A quantification of fibrotic area shown in (D). (H) A quantification of centralized nuclei shown in (E). WT vs. Sod1^−/− ***p<0.001, Sod1^−/− vs. Sod1^−/− DR ^#p<0.05, and WT vs. Sod1^−/− DR ^*p<0.05. All values are represented as mean ± SEM.

Figure 3. Dietary restriction (DR) attenuates age-related defects in neuromuscular junction (NMJ) in Sod1^−/− mice

(A) A representative images of neuromuscular junction from gastrocnemius from WT, Sod1^−/−, and Sod1^−/− DR at 11–14 months. Motoneuron and presynaptic nerve terminals were stained using anti-neurofilament and anti-synaptophysin (pseudo-colored in red) and postsynaptic acetylcholine receptors (AChR) were stained using fluorophore conjugated α-Bungarotoxin (pseudo-colored in green). Arrows denote sprouting of motoneuron and arrowheads denote thinning of motor neurons. (B) TEM of sciatic nerve cross section. Red arrows denote small demyelinated motor neurons. Scale bar=2μm (n=4) (C) Representative images of individual neuromuscular junctions. Arrows denote fragmentations of pre- and post-synaptic endplates. Scale bar = 20μm (D) A quantification of endplate area. At least 60 endplates and 10 random images per animal were scored blindly (n=4). Endplate size was measured by calculating the surface area that are colocalized by AChR (Bungarotoxin) and presynaptic motoneuron (neurofilament/synaptophysin) using Nikon Element software. (E) A comparison of rotarod performance at 16–18 months as measurement of overall neurological function (n=6). WT vs. Sod1^−/− **p<0.01, Sod1^−/− vs. Sod1^−/− DR ^#p<0.05, and WT vs. Sod1^−/− DR ^*p<0.05. (F) A representative electron micrograph images of gastrocnemius neuromuscular junctions at 11 months. Arrows denote abnormal subsynaptic mitochondria (n=3). All values are represented as mean ± SEM.

Figure 4. Dietary restriction (DR) ameliorates age-related defects in mitochondrial Ca²⁺ homeostasis in Sod1^−/− mice

(A) Live cell imaging from single fibers from flexor digitorum brevis (FDB) (WT in top panels, Sod1^−/− middle panels, and Sod1^−/− CR bottom panels). Cytosolic Ca²⁺ were stained with Fluo-4 (green), mitochondrial membrane potential were assessed with tetramethylrhodamine methyl esters (TMRM) stained in red, and NMJ stained in blue with α-Bungarotoxin. Merged images of three stains shown in right column. At least 20 fibers were analyzed from each animal (n=5). (B) A representative traces of calcium retention assay in mitochondria isolated from hindlimb muscle at 14 months (n=5). Arrows denote number of times Ca²⁺ (2μM) was added to mitochondria. (C) A quantification of time to reach mitochondrial permeability transition (MPT) (n=6). (D) A representative electron micrograph of alterations in interfibrillar mitochondria from gastrocnemius muscle at 13–14 months (n=3). WT vs. Sod1^−/− *p<0.05, **p<0.01, ***p<0.001, and Sod1^−/− vs. Sod1^−/− DR ^#p<0.05.

Figure 5. Dietary restriction (DR) attenuates age-related defects in mitochondrial ROS in Sod1^−/− mice

(A) State 1 and (B) State 2 complex I-linked (glutamate/malate) mitochondrial H₂O₂ production at different age points (n=4–6). (C) Mitochondrial free radical leak (H₂O₂ emission/State 2 O₂ consumption) assessed from hind-limb muscle at 18 months (n=6). (D) Oxidative stress is measured by F₂-isoprostanes as a marker of lipid peroxidation (n=6). (E) Western blot analyses of different antioxidant enzymes. WT vs. Sod1^−/− *p<0.05, **p<0.01, ***p<0.001, and Sod1^−/− vs. Sod1^−/− DR ^#p<0.05.

Figure 6. Dietary restriction (DR) upregulates MnSOD activity and decreases mitochondrial protein acetylation

(A) Top, A representative image of gel-based superoxide dismutase activity. Top band represents MnSOD (tetramer) activity and lower band represents CuZnSOD (dimer) activity. Bottom, Western blot analysis of MnSOD, aconitase, and lon protease (n=6). Actin was used as loading control. (B) A quantification of MnSOD activity shown in (A). (C) A quantification of aconitase content shown in (A). (D) Top, A representative immunoblot of total acetylated lysine residues in mitochondrial fractions. Bottom, A representative immunoblot of SIRT3 content (n=4). mtHSP70 was used as loading control. (E) A quantification of total acetylated lysine shown in (D) (n=5). (F) A quantification of SIRT3 content shown in (D) (n=5). (G) A quantitative real-time PCR analysis of PGC-1α. (H) Endurance exercise capacity measured at 14–16 months (n=4–5). WT vs. Sod1^−/− *p<0.05, **p<0.01, Sod1^−/− vs. Sod1^−/− DR ^#p<0.05, and WT vs. Sod1^−/− DR **p<0.01.