Skeletal muscle atrophy has been observed in muscle disuse during unloading, immobilization, denervation, fasting, aging, and several disease conditions. Unloading-related muscle loss caused by prolonged bed rest or spaceflight specifically occurs in antigravity muscles including in slow muscle fibers [1
]. It has been known that the number of mitochondria in slow muscle fibers is higher than that in fast muscle fibers [3
]. Mitochondria are the main energy source of skeletal muscles that produce adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). In this process, 0.2–2.0% of diatomic oxygen passes through the electron transport chain complexes I and III and incompletely reduces leaked electrons to superoxide anions [4
]. Mitochondria play a vital role in disused skeletal muscle atrophy [5
] and mitochondrial signaling can contribute to disuse muscle atrophy in three major ways [6
]: (1) increased mitochondrial reactive oxygen species (ROS) production, (2) energy stress: decrease in ATP production and the activation of adenosine 5′-monophosphate–activated protein kinase (AMPK), (3) mitochondria release of pro-apoptotic factors: release of cytochrome c into the cytosol and active caspase 3. In fact, the increase of mitochondrial ROS production could participate in both the energy stress and apoptosis [9
]. Therefore, mitochondrial ROS has major impact on disuse-related muscle atrophy, and the development of antioxidants to prevent the increase in mitochondrial ROS in muscle due to inactivity has been extensively researched.
Astaxanthin (AX; 3,3′-dihydroxy-β, β′-carotene-4,4′-dione), a ketone carotenoid with 13 conjugated double bonds, naturally accumulates in microalgae, yeast, crustaceans, fish epidermis, and other biologicals. Natural AX is mainly obtained from Haematococcus pluvialis
, and its biological effects as an antioxidant have been widely studied [10
]. The unique molecular structure of AX allows for its insertion through the lipid bimolecular layer of cell membranes, leading to stronger protection and free-radical-scavenging effects at the cell membrane than other antioxidants such as β-carotene, α-tocopherol, and vitamin C [11
]. AX reportedly maintains mitochondrial integrity by reducing oxidative stress, prevents the loss of mitochondrial membrane potential, and increases mitochondrial oxygen consumption, which inhibits mitochondrial dysfunction [13
]. Additionally, AX suppresses bleomycin-induced ROS generation and apoptosis mediated by the disturbed mitochondrial signaling pathway in type II alveolar epithelial cells [16
]. These reports raise the possibility that AX acts in both the mitochondrial and cell membranes.
Although antioxidants have been shown to prevent muscle atrophy [17
], their mechanisms of action vary owing to their different characteristics. Some studies have shown that AX might have an effect on muscle atrophy [19
]; however, its mechanism of action in mitochondria remains unclear. This study investigated the effect of AX on muscle atrophy induced by mitochondrial oxidative stress and dysfunction. Sol8 cells (slow-type muscle cells extracted from the soleus muscle) were used to explore the underlying mechanisms.
The novel findings of this research revealed that dietary AX supplementation attenuated the decrease in muscle mass and myofibers in the SO muscle, preventing mitochondrial dysfunction caused by oxidative stress. AX particularly inhibited the reduction of mitochondrial complexes I and III protein content and regulated mitochondrial oxidative phosphorylation and biogenesis in the SO muscle of tail-suspension mice. In addition, AX treatment mitigated the generation of mitochondrial ROS, cytochrome c release into the cytosol, and caspase 3 activation in Sol8 myotubes.
Although the body weight of mice decreased after tail suspension, EDL muscle weight did not influence this reduction. This meant that muscle atrophy induced by tail suspension was not due to decreased body weight stemming from starvation. Indeed, numerous studies demonstrate that the weight loss in EDL is hardly detectable in suspended animals [31
]. Consistent with these findings, tail suspension induced a loss of skeletal muscle, including the GA, TA, and SO, but not the EDL muscle (Figure 2
). Although GA and TA are mostly composed of fast-twitch fibers, they also contain intermediate muscle fibers such as IIa (GA: 20.9% ± 1.6%, TA: 18.2% ± 2.4%). In contrast, EDL is occupied by fast-twitch fibers such as IIb. Indeed, we found that the size of IIa in muscle fibers in the S-ND group showed significantly decreased, compared with the C-ND group in the SO muscle (Figure 3
). Thus, muscle atrophy caused by tail suspension preferentially affected slow-type rather than fast-type muscles. Moreover, AX prevented atrophy in the muscle containing type I and IIa myofibers. These findings suggest that AX acts in slow-twitch and intermediate muscle fibers.
The CSA of type I and IIa myofibers in S-AX group significantly increased, compared with S-CN or other groups (Figure 3
). SO muscle contains type IIx (11.8% ± 1.7%) and type IIb (3.1% ± 1.1%) as well as type I (30.6% ± 1.2%), type IIa (49.1% ± 1.2%) [21
]. Given the influence of AX in type I and IIa muscle fibers, AX is likely to contribute to the transformation of type IIx to type I and IIa in SO muscle of tail-suspension mice, indicating increased CSA in S-AX. Likewise, TA muscle contains type IIx (44.7% ± 11.9%, CSA: 2186.0 ± 35.2 μm2
), type I (0.6% ± 1.6%, CSA: 1501.4 ± 7.1 μm2
) and type IIa (18.2% ± 2.4%, CSA: 1369.6 ± 22.4 μm2
). Additionally, GA muscle contains type IIx (19.6% ± 2.1%, CSA: 2186.0 ± 35.2 μm2
), type I (7.9% ± 0.5%, CSA: 1743.4 ± 28.2 μm2
) and type IIa (41.6% ± 1.3%, CSA: 1346.2 ± 22.8 μm2
]. We found that the wet weight of TA and GA in the C-AX group was significantly decreased, compared with C-ND group. AX is presumed to cause the transformation of type IIx to type I and IIa muscle fibers, resulting in the shift to small-sized fibers. However, further studies are necessary to define this mechanism.
We found that the ratio of body weight/food intake decreased significantly at 5 and 6 weeks in the tail-suspension group (S-ND and S-AX). Previous studies showed that there was an increased reliance on carbohydrate metabolism for energy associated with muscle unloading [33
]. In addition, shift of fiber phenotype was related to the downregulation of mitochondrial proteins and upregulation of glycolytic protein, suggesting a shift from oxidative to glycolytic metabolism [26
]. Consistent with these results, we found the decreased ratio of body weight/food intake and the shift of type fibers caused by tail suspension. Additionally, we found that the mRNA expression of oxidative metabolism–related transcription factors was decreased in the tail-suspension group. Thus, these results showed that the decreased ratio of body weight/food intake was associated with the energy shift from oxidative to glycolytic metabolism.
Numerous studies have demonstrated that mitochondria play an important role in muscle atrophy [5
]. In oxidative phosphorylation, mitochondrial respiratory chain complexes I and III are believed to be the major sites of ROS leakage, although other components of oxidative phosphorylation also contribute to the production of ROS in the mitochondria [36
]. In this study, we found that the amount of H2
production was significantly increased by tail suspension in normal diet mice (S-ND group). However, the AX-supplemented diet inhibited the increase of H2
production in the S-AX group. In addition, there was a significant reduction in mitochondrial respiratory chain complexes I and III in the SO muscle of the S-ND group, compared to that of the C-ND group (Figure 4
). Kanazashi et al. reported that the SO muscle displays a decreased succinate dehydrogenase activity, an integral component of the mitochondrial respiratory chain, and increased oxidative stress during hindlimb suspension in rats [19
]. Our previous study also demonstrates that mitochondrial dislocation during unloading conditions has deleterious effects on muscle fibers leading to atrophy and ROS leakage from the mitochondria [5
]. The weakening of mitochondrial oxidative phosphorylation is usually accompanied with changes in mitochondrial biosynthesis. We found that the expression of energy and oxidative metabolism genes significantly decreased in the SO muscle of tail-suspension mice, while AX showed the regulating effect on the mitochondria biogenesis (Figure 6
). Therefore, muscle atrophy stimulated by unloading stress in tail suspension is associated with the disturbance of mitochondrial ability including mitochondrial oxidative phosphorylation and mitochondrial biogenesis.
Numerous reports have proven that AX exerts its effects on mitochondria in fatty-liver disease, caused due to a high-fat diet, nonalcoholic steatohepatitis [38
], gastric inflammation by oxidative stress [40
], and cardiovascular diseases [41
]. These unique characteristics of AX on mitochondria may relate to itself membrane structure. AX consists of conjugated double bonds and hydroxyl and keto groups that can embed in the cell membrane, from the inside to the outside. This feature confers strong antioxidant activity, which enables AX to react with the free radicals [43
]. Our results indicated that AX was preferentially detected in the mitochondrial fraction and is consistent with previous reports of its accumulation in the mitochondria of normal human mesangial cells and blastocysts [15
]. These findings strengthened the possibility that AX reacted to ROS produced from mitochondrial respiratory chain complexes, leading to the prevention of oxidative stress–related diseases, including muscle atrophy.
AnA is an inhibitor of the mitochondrial respiratory chain complex III, a major site of mitochondrial ROS generation, and strongly activates the production and release of superoxide anions into the inner mitochondria membrane space [45
]. We examined the effect of AX on AnA-induced mitochondrial O2-
production using succinate as a complex II substrate. AX significantly suppressed mitochondrial complex III–driven ROS production in Sol8 myotubes (Figure 7
a), whereas its effect was not observed in C2C12 myotubes (data not shown), which is likely to be involved in muscle fiber type. Sol8 and C2C12 cells were derived from SO and adult dystrophic mouse muscles, respectively. Indo et al. showed that the SO muscle, which is enriched with slow-twitch fibers, exhibits a higher production of ROS than fast-twitch fibers [3
]. Some studies have also reported that dietary antioxidants reduce ROS production and ameliorate atrophy in the SO muscle more than other fast-twitch fibers [46
]. These results indicate that AX could target the mitochondria to eliminate O2-
production and inhibit muscle atrophy induced due to mitochondrial oxidative stress in slow-twitch fibers.
Loss of MMP and excess ROS production in mitochondria leads to cytochrome c release from the mitochondria into the cytosol, resulting in the induction of apoptosis [49
]. It has been revealed that overproduction of mitochondrial ROS, mitochondrial dysfunction, and mitochondria-mediated apoptosis play vital roles in skeletal muscle atrophy [54
]. Caspase 3 is downstream of cytochrome c; the release of cytochrome c activates caspase 3, which induces apoptosis [56
]. It has been reported that a deficiency in caspase 3 prevents denervation-induced muscle atrophy [58
]. In addition, disturbed TUNEL-positive nuclei, increased caspase 3 protein level, and decreased Bcl-2, anti-apoptotic members that inhibit the release of cytochrome c by unloading were improved by AX [48
]. In our present study, AX showed improvement of disturbed MMP as well as increased mitochondrial ROS by AnA treatment, thereby inactivating caspase 3 through an inhibition of cytochrome c release into cytosol in Sol8 myotubes. In agreement with these findings, AX has been shown to protect against decreased MMP by virtue of improving mitochondrial function in cancer and neural cells [14
]. These results suggested that AX targeted and protected mitochondria by scavenging free oxygen radicals, regulating MMP, and inhibiting apoptosis in muscle cells.