Habitual Exercise Attenuates the Aging Associated Muscle Atrophy and Muscle Strength Decline in SAMP8

Loss of muscle mass and strength are progressing with aging. Exercise is a beneficial method to prevent physical disfunction and habitual exercise improve the muscle quality. Therefore, we evaluated the effects of a long-term habitual exercise on the senescence-accelerated mice prone8 (SAMP8). 27wk SAMP8 were used in this study. Mice were classified into 28 (28w) and 44 weeks old. The 44-week group was divided into the sedentary group (44w) and a group exercising for 16 weeks (44w+Ex). The 44w+Ex performed habitual exercise from 28 to 44 weeks. Additionally, grip strength tests were performed with mice aged 28 and 44 weeks. Mice were dissected and collected muscle samples and measured muscle weight at 44w. Gastrocnemius was decreased in 44w but were unchanged in 44w+Ex. Grip strength in 44w was lower trend, but there was no change in 44w+Ex. The phosphorylation levels of Akt and p70S6K as a protein synthesis marker were decreased in 44w. Cytochrome c oxidase subunit IV(COXIV) mRNA and protein levels decreased in 44w. These results suggested that long-term habitual exercise attenuated muscle mass and strength decline through improving muscle protein synthesis and mitochondrial function. In conclusion, long-term habitual exercise attenuated muscle mass and strength decline.


Introduction
Aging is an inevitable process in animals, including humans, and has various effects on the body. For example, aging affects the functioning of the brain, nerves, and intestines. Skeletal muscle aging includes skeletal muscle loss and muscular weakness, known as sarcopenia [1], Sarcopenia was initially described by Rosenberg [2]. The European Working Group on Sarcopenia in Older People (EWGSOP) provides a definition of age-related sarcopenia and a statement on diagnostic criteria. The EWGSOP recommend using both the loss of skeletal muscle mass and the loss of muscle function (muscle and physical ability) for diagnosis of sarcopenia. This definition is more complex because muscle strength is not only dependent on muscle mass, but also function [3,4]. In recent years, studies have reported that various forms of exercise have positive effects in preventing sarcopenia. For example, resistance training can stimulate muscle protein synthesis [5,6]. Kryger reported that resistance training by women at an advanced age could increase skeletal muscle mass and muscle strength [7]. More recently, the effects of habitual exercise have also been studied [8,9,10].
Aging affects insulin sensitivity and reduces muscle protein synthesis [11]. Akt is one of the muscle protein synthesis related protein, and is upstream protein of muscle protein synthesis. On the other hands, downstream of Akt is 70kDa S6 kinase (p70S6K), which activates muscle protein synthesis. Phosphorylation levels of Akt is decreasing in the old mice has been reported. As a result, decrease of Akt phosphorylation suppress the activation in downstream of Akt [12]. However, habitual exercise improves the phosphorylation levels of Akt and activate the muscle protein synthesis [11]. Furthermore, mitochondria contents and function decline with aging [13,14]. Reductions in mitochondrial function causes muscle ATP synthesis reduction and muscle weakness [15,16]. However, habitual exercise regulates the mitochondrial contents and function during aging [17]. Short et al. reported that long-term habitual exercise can increase cytochrome c oxidase subunit IV (CoxIV) mRNA levels regardless of age [18]. Moreover, habitual exercise increases the Pgc-1a mRNA levels, promoting mitochondria production [19,20]. Positive correlations between muscle ATP synthesis and muscle function have been reported [16], therefore, if mitochondrial functions improve, muscle function may also increase.
In the study of sarcopenia, aging model animals are required, however, natural aging models require a lot of time to evaluate. We used senescence-accelerated mice (SAM). SAM represent a multifactor approach and are an accepted model for accelerated senescence and study of age-related pathologies [21]. In particular, SAMP8 (prone 8 of SAM) were reported as useful in the sarcopenia model [22,23]. To our knowledge, there are no studies on the effects of long-term habitual exercise programs on the sarcopenia model. Hence, the aim of this study was to evaluate the effects of longterm habitual exercise on the sarcopenia model.

Animals and Experimental Grouping
The animal experiments in this study were approved by the Animal Care Committee, University of Tsukuba. An overview of the experiments is shown in Figure 1. Male SAMP8 aged 28 weeks (Japan SLC, Hamamatsu, Japan) were kept under conventional in individually at 20-26℃ with a humidity level of 40-60% and a 12-h light/dark cycle. Female mice were excluded from this study due to the effects of menstruation and menopause. Mice were freely fed a standard feed (MF, Oriental Yeast, Tokyo, Japan). Mice were divided into the 28 weeks old group (28w) and the 44 weeks old group, split into the sedentary group (44w) and group undergoing exercise for 16 weeks (44w+Ex). At the age of 28 weeks, the number of mice were 28 w (n = 10), 44 w (n = 10) and 44 w + Ex (n = 10), but there are individuals which died after the initial grip strength measurement and during study. Therefore, the data used in this study were 28w (n = 8), 44w (n = 7), and 44w + Ex (n = 9).

Exercise Protocol
The mice ran on a treadmill for small animals (FVRO, 4E9S-6, Fuji Medical Science, Chiba, Japan). The mice learned to run for 5 days followed by training performed at 15 m/min for 30 min a day in light cycle, 5 days per week from 28 to 44 weeks of age. Training protocol were modified from Murase et al [18]. The back of each treadmill lane contained an electrified grid, which delivered a shock stimulus to mice if mice are resting on grid. The mice were monitored during running training to prevent escape. If mice are caught in a treadmill, immediately rescued and excluded from the experiment.

Grip Strength Test
The grip strength test reflected a muscle strength index of all four limbs and was conducted using a small animal grip measurement device (GMP-100B, MELQUEST, Toyama, Japan). Mice were held in the limb-grip, the tail was pulled backward, and the maximum value of the force applied to release the grip was taken as the measurement value. The measurement was performed five times, and the average value, excluding the maximum value and the minimum value, was taken as the index for each mouse. The same researcher conducted the measurements each time. Measurements were conducted so that the researcher was blind to the individual mouse and measured value to remove bias.

Sampling
At the time of physiological investigation (28 and 44 weeks old; Figure 1), mice were anaesthetized by an intraperitoneal injection of pentobarbitone sodium (Kyoritsu Seiyaku Corporation, Tokyo, Japan). Soleus muscles, plantaris muscles, and gastrocnemius muscles were harvested, following Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 31 July 2020 doi:10.20944/preprints202007.0741.v1 which mice were euthanized by cervical dislocation. The muscle wet weights were measured at the time of euthanasia and then immediately frozen in liquid nitrogen and stored at -80℃ for later biochemical assays.

Western Blot Analysis
Total proteins were extracted from gastrocnemius muscle with radioimmunoprecipitation assay (

Statistical Analysis
Data are shown as the mean ± SEM. Data were subjected to a one-way analysis of variance was used to evaluate significance. Post hoc test was used Tukey's method. Statistical analyses were performed using SPSS statistics ver. 26 for Mac (SPSS Japan, Tokyo, Japan).

Body Weight
There were no changes in body weights when the groups were compared. The body weight graphs are displayed in Figure 2.

Skeletal Muscle Mass and Muscle Strength Reduction
To evaluate the effect of aging and habitual exercise on muscle, skeletal muscle mass (Figure3-A), relative skeletal muscle mass and muscle strength were measured (Figure 3-B). In the 44w group, gastrocnemius muscle mass declined with aging and grip strength tended to decrease. In the 44w+Ex group, there were no significant changes in skeletal muscle and muscle strength. Thus, habitual exercise prevented reductions in skeletal muscle mass and muscle strength associated with aging.

Protein Synthesis Related Protein Expression
To evaluate the effect of aging and habitual exercise on the phosphorylation level of Akt and p70S6K were measured (Figure 4). In Akt and p70S6K, 44w was significant lower compared to both 28w and 44w+Ex group.

Mitochondrial Functioning Gene and Protein Expression
Habitual exercise increased the expression level of genes related to mitochondrial functioning. To evaluate the effect of aging and habitual exercise on the expression levels of genes related to mitochondrial function, the expression levels of Pgc-1a, CoxIV and Atp5a1 were measured ( Figure 5-A). In the 44w group, CoxIV mRNA levels declined with aging and there were no changes in Pgc-1a and Atp5a1. In the 44w+Ex group, there were no changes in CoxIV and Atp5a1. CoxIV genes expression was changed 44w so we examined the protein expression in COXIV. In COXIV, 44w was significant lower compared to both 28w and 44w+Ex group.

Discussion
Muscle aging, known as sarcopenia, is significant problem in terms of quality of life, health, and decreased life expectancy. Muscle aging studies and muscle aging animal models are required since human life is of considerable length, making examinations very difficult. In this study, we used the SAMP8 for a sarcopenia model animal. First, we examined muscle mass and muscle strength changes following a habitual exercise program. Guo et al. reported that SAMP8 muscle mass peaked at 7 months old and muscle strength decline began at 8 months old; therefore, we used 28 weeks old (7 months old) SAMP8 [22]. Relative gastrocnemius muscle mass decreased with aging (Figure 3-B). Additionally, muscle strength decreased in the 44w group. These results were consistent with previous studies [22], and both aspects are important since EWGSOP refers to sarcopenia as not only a decrease in muscle mass, but also a decline in muscle strength. Although the absolute weights tended to be significant, there was no significant difference. This point was different from previous studies. Liu et al. reported that fat accumulates in SAMP8's muscles with aging [24]. Hence, ectopic fat may have accumulated in this study. Additionally, differences in breeding environment affected the amount of physical activity, etc., and there may have been no clear difference in changes in muscle weight. Nevertheless, it is useful to be able to evaluate the effects of aging on muscles in a short period of time, so SAMP8 is a useful model animal for sarcopenia.
To evaluate why sarcopenia was prevented by long-term, habitual exercise, muscle protein synthesis related proteins and genes related to mitochondrial function were investigated.
Muscle atrophy is caused by a decline in muscle protein synthesis [14], therefore, phosphorylation levels of Akt and p70S6K, which are related to muscle protein synthesis, were measured. In this study, the phosphorylation level of proteins showed decling with aging and exercise suppressed its decline. Therefore, muscle atrophy at this age of SAMP8 may be primarily due to declined muscle protein synthesis, and contrary to the aging, exercise maintained the muscle protein synthesis. Previous study referred that 18~19 m/min is lactate threshold (LT) in mice [19]. Exercise below LT was low to moderate intensity, and it is considered that this study was low to moderate intensity of 15 m / min. Hence, this study revealed that habitual exercise of low intensity exercise can maintain muscle mass by maintaining muscle synthesis.
Long-term, habitual exercise can improve mitochondrial function and increase mitochondria content, therefore, we examined the genes and proteins related to mitochondrial function. Mitochondria is one of the important organellar to produce ATP, and mitochondrial function and muscle function are related [16]. During aging, mitochondrial function decline and this causes muscle strength to also decline. In this study, the mRNA and protein expression level of CoxIV, which is a mitochondrial marker, were decreased in 44w ( Figure 5). In this sarcopenia model, aging may have caused a decrease in mitochondrial function and / or a decrease in mitochondrial content, but exercise had inhibitory effect. Generally, it is difficult for elderly people to exercise with high intensity. However, our results suggested that even though low intensity exercise that is safety and executable for elderly could attenuate the aging associated muscle mass and strength decline through the maintenance of mitochondrial function and maintaining muscle protein synthesis.
Despite these findings, the mitochondrial genome level and mitochondria content, were not measured in this study. In addition, muscle fiber type and fiber cross sectional area could not be examined using muscle sections, creating a limitation which must be addressed in future study.

Conclusions
The effects of long-term, habitual exercise in SAMP8 mice as a sarcopenia model were evaluated in this study. Long-term, habitual exercise preserved muscle mass and muscle strength. The mechanisms underlying this phenomenon could be preservation of the muscle protein synthesis and mitochondrial function. Funding: This research was funded by Zenyaku kogyo Co., Ltd.