3.1. Construction of Skeletal Muscle Tissues on Devices
Skeletal muscle tissue was prepared on the microdevices for measuring muscle contractile forces. The tissue shifted to the upper parts of the posts on the first day of growth culture and formed a ribbon-shaped tissue by self-organization (Figure 2
A). The tissue contained several myotubes with sarcomere structure, which were aligned along the long axis direction of the tissue (Figure 2
After differentiation was induced (day 0), the contractile force of the muscle tissues with respect to the number of days elapsed was measured (Figure 2
C). A contractile force of 6.6 ± 2.2 μN was observed from the third day of differentiation, which increased to 77.5 ± 11.1 μN by the eighth day of differentiation (Figure 2
C). As a sufficiently stable contractile force was obtained on the sixth day (57.5 ± 12.8 μN) as reported previously [13
], it was decided that muscle tissue prepared by differentiation culture for six days is to be used in subsequent experiments.
3.3. Evaluation of Muscle Tissue with Reduced Contractile Force Due to Addition of Dex
The addition of Dex to skeletal muscle cells cultured in 2D is known to increase the expression of atrogenes including ubiquitin ligases Atrogin-1
and muscle-specific RING-Finger protein, MuRF-1
]. The expression profile of these genes in the skeletal muscle tissues with reduced contractile force (treated with 100 μM Dex for 24–48 h) was examined. The obtained values were normalized using β-actin
. Compared with the control, expression levels increased significantly by 2.6 times for Atrogin-1
and 2.2 times for MuRF-1
because of Dex addition (Figure 4
As the increased expression of Atrogin-1 and MuRF-1 by Dex addition was established, it was presumed that muscle protein degradation was promoted in these muscle tissues.
Next, it was examined if the number of myotubes retaining the sarcomere structure is altered by the addition of Dex. As a result, it was determined that compared to the control state (without the addition of Dex), the proportion of myotubes retaining the sarcomere structure was significantly lower in the muscle tissue 24 h after the addition of Dex (Figure 4
B). It has been reported that periodic electrical pulse stimulation of myotubes significantly increases the proportion of myotubes retaining the sarcomere structure [27
]. Furthermore, it is reported that interruption of electrical stimulation causes the sarcomere structure to disappear in a short time and the contractile force of the myotubes to decreases sharply [27
]. Although it is not evident how the addition of Dex decreases the proportion of sarcomeric myotubes in a three-dimensional muscle tissue (Figure 4
B), this phenomenon is believed to be one of the factors affecting the decrease in muscle contractile force upon the addition of Dex.
Thus, these results suggested that proteolytic degradation of the ubiquitin-proteasome system and alterations in sarcomere structure is likely to be involved in the reduction of contractile force due to Dex addition and that Dex effectively induces atrophy in 3D skeletal muscle tissue.
3.4. Verification of Contractile Force Recovery Effect by Addition of IGF-I to Muscle Atrophy Model
Finally, in order to investigate if the atrophic muscle tissues induced by Dex can be applied to drug screening, it was verified if the contractility was recovered by adding the model compound IGF-I. IGF-I is a peptide growth hormone and plays a major role in promoting skeletal muscle growth and differentiation [31
]. It has been reported elsewhere that IGF-I increases contractile force of engineered normal skeletal muscle tissues [13
]. It has also been reported that IGF-I prevents a Dex-induced increase in proteolysis and blocks Dex induction of atrogenes, including Atrogin-1
in C2C12 myotubes [34
]. Thus, although it is not evident that IGF-I prevents Dex-induced decrease of contractile force of atrophic tissues, it was used as a model compound in this study.
100 μM Dex was added to the muscle tissue on the device, prepared by a differentiation culture for 24 h, to induce atrophy. Then, 8 ng/mL IGF-I was added. The mixture was incubated for 24 h, and muscle contractile force was measured (Figure 5
A). The relative value was determined by assigning the value 1 to the contractile force before addition of Dex. As a result, the group to which only Dex was added yielded a relative value of 0.23 ± 0.07, whereas the group to which both Dex and IGF-I were added yielded 0.42 ± 0.10. Thus, any decrease in contractile force was not completely eliminated but significantly reduced by the addition of IGF-I to the Dex-treated tissue (p
< 0.01) (Figure 5
A). It should be elucidated in the future whether the treatment with IGF-I for longer than 24 h fully recovers the contractile force of the Dex-treated tissue.
In addition, variations in the expression levels of atrogenes in Dex and Dex + IGF-I were examined (Figure 5
B). The expression level of each gene in Dex + IGF-I was significantly lower compared to that in Dex (Atrogin-1
: 3.83 ± 0.39 for Dex and 2.89 ± 0.20 for Dex + IGF-I; MuRF-1
: 2.79 ± 0.68 for Dex and 1.61 ± 0.33 for Dex + IGF-I). These results indicate that IGF-I suppresses muscle weakness by reducing the proteolytic effect of ubiquitin proteasome, induced by Dex addition through the phosphatidylinositol 3-kinase (PI3K)-Akt pathway [35
]. Thus, the atrophic muscle tissues induced by Dex are suitable for the application of screening the drugs.
As illustrated in Figure 5
A, when IGF-I was added to the normal skeletal muscle tissues, the contractile force displayed a tendency to increase, albeit not significantly (0.61 ± 0.10 for control and 0.81 ± 0.20 for IGF-I). On the contrary, in previous studies, it was reported that IGF-I increased the contractile force of engineered normal skeletal muscle tissue; Vandenburgh et al. reported that the contractile force of the fibrin gel-based skeletal muscle tissue was significantly increased two days after the addition of IGF-I to the medium at a concentration of 100 ng/mL [13
]; Huang et al. directly embedded IGF-I in the fibrin gel (25 ng/mL) and reported that it results in a significant increase in contractile force—50% over untreated tissues—after 14 days [33
]; and Sato et al. constructed skeletal muscle tissue using IGF-I gene-engineered myoblast cells, which secreted IGF-I at approximately 6.5 ng/(mL·d) to the medium and reported that the tissue on day 7 generated significantly higher (1.5 times) contractile force than the control [32
]. In the present study, IGF-I was added to the tissue at 8 ng/mL for 24 h, and no significant increase in the contractile force was observed (Figure 5
A). Comparing these results, it can be concluded that addition of IGF-I is likely to be marginally effective in increasing the contractile force of normal skeletal muscle tissues under shorter culture durations. Therefore, although further investigation is necessary to examine the effects of longer culture durations on the contractile force, by using skeletal muscle tissues, in which atrophy was induced by adding Dex, high sensitivity screening is likely to be possible in a shorter period than by using normal skeletal muscle tissue.
In the present study, we suggest the usability of the Dex-induced atrophic tissues for drug screening by using mice C2C12 cells (Figure 5
). In the future, it is expected that human skeletal muscle cells, including primary cells and the cells derived from induced pluripotent stem cells (iPSCs) [36
], will be used. It has been reported that Dex induced the expression of Atrogin-1
in primary human myotubes [39
]. Thus, the system used in this study would be applicable to the human cells although the optimization of the process for constructing tissues and inducing atrophy should be examined. Furthermore, miniaturization of the system, especially the devices and electrodes, will be needed to improve the throughput. Nonetheless, since the device fabrication, tissue construction and atrophy induction were simple, and the equipment for the electrical stimulation was commercially available, we believe that the system used in this study shows high versatility.