Genetically enhancing mitochondrial antioxidant activity improves muscle function in aging

Abstract

Age-related skeletal muscle dysfunction is a leading cause of morbidity that affects up to half the population aged 80 or greater. Here we tested the effects of increased mitochondrial antioxidant activity on age-dependent skeletal muscle dysfunction using transgenic mice with targeted overexpression of the human catalase gene to mitochondria (MCat mice). Aged MCat mice exhibited improved voluntary exercise, increased skeletal muscle specific force and tetanic Ca(2+) transients, decreased intracellular Ca(2+) leak and increased sarcoplasmic reticulum (SR) Ca(2+) load compared with age-matched wild type (WT) littermates. Furthermore, ryanodine receptor 1 (the sarcoplasmic reticulum Ca(2+) release channel required for skeletal muscle contraction; RyR1) from aged MCat mice was less oxidized, depleted of the channel stabilizing subunit, calstabin1, and displayed increased single channel open probability (Po). Overall, these data indicate a direct role for mitochondrial free radicals in promoting the pathological intracellular Ca(2+) leak that underlies age-dependent loss of skeletal muscle function. This study harbors implications for the development of novel therapeutic strategies, including mitochondria-targeted antioxidants for treatment of mitochondrial myopathies and other healthspan-limiting disorders.

Keywords: aging; exercise capacity; muscle weakness; oxidation; skeletal muscle.

Fig. 1.

Improved exercise capacity in aged MCat mice. Mice were housed in individual cages equipped with running wheels for three weeks. Exercise distance (A) and running wheel time (B) were recorded. Data are mean ± SEM (*P < 0.01 vs. young WT; ^#P < 0.05 vs. aged WT; n: young WT = 7, young MCat = 8, aged WT = 8, aged MCat = 8, ANOVA).

Fig. 2.

Preserved skeletal muscle function in aged MCat mice. (A and B) Tetanic contractions (70 Hz) in isolated EDL muscles from MCat and WT littermates (force normalized to cross-sectional area). (C and D) Average specific force in EDL muscles from the same mice as in A and B. Data are mean ± SEM (n: young WT = 4, young MCat = 4, aged WT = 8; aged MCat = 7; t test was performed for each individual point: *P < 0.05 vs. aged WT).

Fig. 3.

Improved tetanic Ca²⁺ in skeletal muscle from aged MCat mice. (A–D) Representative traces of normalized Fluo-4 fluorescence in FDB muscle fibers during a 70 Hz tetanic stimulation in young WT (A), young MCat (B), aged WT (C), and aged MCat (D). (E) Peak Ca²⁺ responses in FDB fibers stimulated at 70 Hz (fibers taken from the same animals as in A–D, n = 15–21 cells from at least three mice in each group). (F) Resting cytosolic Ca²⁺ (measured ratiometrically). Data are mean ± SEM (*P < 0.05 vs. young WT; ^#P < 0.05 vs. aged WT, ANOVA).

Fig. 4.

Reduced SR Ca²⁺ leak and increased SR Ca²⁺ load in muscle from aged MCat mice. (A) Representative images of line scans of Fluo-4 fluorescence from permeabilized FDB muscle fibers showing Ca²⁺ spark activity. The heat diagram indicates the normalized change in fluorescence intensity (ΔF/F₀). (B) Bar graph showing average Ca²⁺ spark frequency (n = 15–25 cells from at least three mice in each group). (C) Representative time course of Ca²⁺ leak from SR microsomes following Ca²⁺ uptake. (D) Ca²⁺ leak as calculated by the percentage of uptake. (E) SR Ca²⁺ load (measured by applying 1 mM 4-CmC). Data are mean ± SEM (*P < 0.05, **P < 0.01 vs. young WT; ^#P < 0.05 vs. aged WT, ANOVA).

Fig. 5.

Skeletal muscle RyR1 isolated from aged MCat mice is remodeled and exhibits reduced single-channel open probability (P_o). (A) Representative immunoblots from triplicate experiments of immunoprecipitated RyR1 from aged murine EDL. (B) Bar graphs showing quantification of the immunoblots in A; DNP: 2,4-dinitrophenylhydrazone. (C) Representative RyR1 single-channel current traces. Channel openings are shown as upward deflections and the closed (c-) state of the channel is indicated by horizontal bars in the beginning of each trace. Tracings from over 2 min of recording for each condition showing channel activity at two time scales (5 s in upper trace and 500 ms in lower trace) as indicated by dimension bars, and the respective P_o (open probability), T_o (average open time), and T_c (average closed time) are shown above each trace. The activity of the channel indicated by the thick black bar is shown on the expanded time scale (the 500 ms trace below). (D) Bar graph summarizing P_o at 150 nM cytosolic [Ca²⁺] in young WT (n = 6), aged WT (n = 5), young MCat (n = 7), and aged MCat (n = 5) channels. Data are mean ± SEM (*P < 0.05, **P < 0.01 vs. young WT, ^#P < 0.05, ^#P < 0.01 vs. aged WT, ANOVA).

Fig. 6.

Antioxidant application to aged WT skeletal muscle reduces age-associated SR Ca²⁺ leak. (A) Representative immunoblot of immunoprecipitated RyR1 from aged murine skeletal muscle. For DTT treatment, SR vesicles were preincubated with 1 mM DTT. (B) Bar graphs showing quantification of the immunoblots in A. (C) Bar graph representing Ca²⁺ leak in SR microsomes of skeletal muscles from aged WT mice. For N₂ treatment, solutions was prebubbled with 100% N₂ for 1 h. (D) Bar graph representing average Ca²⁺ spark frequency in permeabilized FDB muscle fibers from aged WT mice. Data are mean ± SEM (n = 19–22 cells from three mice per group; *P < 0.05 vs. aged WT; **P < 0.01 vs. aged WT, ANOVA).