The Impact of SRT2104 on Skeletal Muscle Mitochondrial Function, Redox Biology, and Loss of Muscle Mass in Hindlimb Unloaded Rats

Abstract

Mechanical unloading during microgravity causes skeletal muscle atrophy and impairs mitochondrial energetics. The elevated production of reactive oxygen species (ROS) by mitochondria and Nox2, coupled with impairment of stress protection (e.g., SIRT1, antioxidant enzymes), contribute to atrophy. We tested the hypothesis that the SIRT1 activator, SRT2104 would rescue unloading-induced mitochondrial dysfunction. Mitochondrial function in rat gastrocnemius and soleus muscles were evaluated under three conditions (10 days): ambulatory control (CON), hindlimb unloaded (HU), and hindlimb-unloaded-treated with SRT2104 (SIRT). Oxidative phosphorylation, electron transfer capacities, H₂O₂ production, and oxidative and antioxidant enzymes were quantified using high-resolution respirometry and colorimetry. In the gastrocnemius, (1) integrative (per mg tissue) proton LEAK was lesser in SIRT than in HU or CON; (2) intrinsic (relative to citrate synthase) maximal noncoupled electron transfer capacity (E_CI+II) was lesser, while complex I-supported oxidative phosphorylation to E_CI+II was greater in HU than CON; (3) the contribution of LEAK to E_CI+II was greatest, but cytochrome c oxidase activity was lowest in HU. In both muscles, H₂O₂ production and concentration was greatest in SIRT, as was gastrocnemius superoxide dismutase activity. In the soleus, H₂O₂ concentration was greater in HU compared to CON. These results indicate that SRT2104 preserves mitochondrial function in unloaded skeletal muscle, suggesting its potential to support healthy muscle cells in microgravity by promoting necessary energy production in mitochondria.

Keywords: SRT2104; antioxidants; electron transfer system; hindlimb unloading; mitochondria; oxidative stress; respiration; skeletal muscle; spaceflight.

Figure 1

Effect of the Sirtuin-1 Agonist SRT2104 on mitochondrial enzyme activities in hindlimb unloaded rats. (A,B) Citrate synthase and (C,D) integrative (relative to mg protein) and (E,F) intrinsic (relative to CS activity) cytochrome c oxidase activities in soleus and gastrocnemius muscles of ambulatory control rats (CON; n = 6), rats hindlimb unloaded for 10 days (HU; n = 6), and rats hindlimb unloaded for 10 days while treated with SRT2104 (SIRT; n = 4). Values are means ± SEM. ns indicates no significant difference (p > 0.05).

Figure 2

Effect of the Sirtuin-1 Agonist SRT2104 on integrative (relative to tissue wet weight) mitochondrial capacities as measured by high-resolution respirometry in the gastrocnemius muscle of hindlimb unloaded rats. (A) Integrative proton LEAK, (B,C) oxidative phosphorylation supported by complex I (P_CI and P_CIG), (D) maximal coupled oxidative phosphorylation (P_CI+II), (E) maximal noncoupled electron transfer (E_CI+II), and (F) electron transfer supported by complex II only (E_CII) capacities in the gastrocnemius muscle of ambulatory control rats (CON; n = 6), rats hindlimb unloaded for 10 days (HU; n = 6), and rats hindlimb unloaded for 10 days while treated with SRT2104 (SIRT; n = 4). Values are means ± SEM. ns indicates no significant difference (p > 0.05).

Figure 3

Effect of the Sirtuin-1 Agonist SRT2104 on intrinsic (relative to CS activity) mitochondrial capacities as measured by high-resolution respirometry in the gastrocnemius muscle of hindlimb unloaded rats. (A) Intrinsic proton LEAK, (B,C) oxidative phosphorylation supported by complex I (P_CI and P_CIG), (D) maximal coupled oxidative phosphorylation (P_CI+II), (E) maximal noncoupled electron transfer (E_CI+II), and (F) electron transfer supported by complex II only (E_CII) capacities in the gastrocnemius muscle of ambulatory control rats (CON; n = 6), rats hindlimb unloaded for 10 days (HU; n = 6), and hindlimb unloaded for 10 days while treated with SRT2104 (SIRT; n = 4). Values are means ± SEM. ns indicates no significant difference (p > 0.05).

Figure 4

Effect of the Sirtuin-1 Agonist SRT2104 on mitochondrial flux control ratios in the gastrocnemius muscle of hindlimb unloaded rats. (A) The ratio of LEAK to maximal electron transfer (flux control ratio; FCR_LEAK), (B) FCR for oxidative phosphorylation supported by complex I (FCR_PCI), (C) FCR when glutamate was added as an additional complex I substrate (FCR_PCIG), (D) FCR for maximal coupled oxidative phosphorylation (FCR_PCI+II) and (E) FCR for noncoupled electron transfer supported by complex II only (FCR_ECII) of ambulatory control rats (CON; n = 6), rats hindlimb unloaded for 10 days (HU; n = 6), and rats hindlimb unloaded for 10 days while treated with SRT2104 (SIRT; n = 4). Values are means ± SEM. ns indicates no significant difference (p > 0.05).

Figure 5

Effect of the Sirtuin-1 Agonist SRT2104 on reactive oxygen species in hindlimb unloaded rats. H₂O₂ production and concentration in isolated mitochondria from soleus (A,C) and gastrocnemius (B,D) muscles of ambulatory control rats (CON; n = 6), rats hindlimbs unloaded for 10 days (HU; n = 3), and rats hindlimbs unloaded for 10 days while treated with SRT2104 (SIRT; n = 4). Values are means ± SEM. ns indicates no significant difference (p > 0.05).

Figure 6

Effect of the Sirtuin-1 Agonist SRT2104 on antioxidant enzyme activities in hindlimb unloaded rats. (A) Total superoxide dismutase and (B) glutathione peroxidase activities in the gastrocnemius muscle of ambulatory control rats (CON; n = 6), rats hindlimbs unloaded for 10 days (HU; n = 6), and rats hindlimbs unloaded for 10 days while treated with SRT2104 (SIRT; n = 4). Values are means ± SEM. ns indicates no significant difference (p > 0.05).

Figure 7

Effect of the Sirtuin-1 Agonist SRT2104 on hindlimb unloaded rats’ muscle weights. Muscle weights of the right soleus (A), the left soleus (B), and the left gastrocnemius (C) of ambulatory control rats (CON; n = 6), rats hindlimbs unloaded for 10 days (HU; n = 6), and rats hindlimbs unloaded for 10 days while treated with SRT2104 (SIRT; n = 4). Values are means ± SEM. ns indicates no significant difference (p > 0.05).