Mechanical loading of tissue engineered skeletal muscle prevents dexamethasone induced myotube atrophy

Abstract

Skeletal muscle atrophy as a consequence of acute and chronic illness, immobilisation, muscular dystrophies and aging, leads to severe muscle weakness, inactivity and increased mortality. Mechanical loading is thought to be the primary driver for skeletal muscle hypertrophy, however the extent to which mechanical loading can offset muscle catabolism has not been thoroughly explored. In vitro 3D-models of skeletal muscle provide a controllable, high throughput environment and mitigating many of the ethical and methodological constraints present during in vivo experimentation. This work aimed to determine if mechanical loading would offset dexamethasone (DEX) induced skeletal muscle atrophy, in muscle engineered using the C2C12 murine cell line. Mechanical loading successfully offset myotube atrophy and functional degeneration associated with DEX regardless of whether the loading occurred before or after 24 h of DEX treatment. Furthermore, mechanical load prevented increases in MuRF-1 and MAFbx mRNA expression, critical regulators of muscle atrophy. Overall, we demonstrate the application of tissue engineered muscle to study skeletal muscle health and disease, offering great potential for future use to better understand treatment modalities for skeletal muscle atrophy.

Keywords: C2C12; Dexamethasone; Hypertrophy; Myotubes; Skeletal muscle; Ubiquitin–proteasome.

Fig. 1

Loss of contractile force and myotube cross sectional area (CSA) (µm²) of 50 µl engineered skeletal muscle cultures 24 h post DEX treatment (0, 10, 20, 40, 80 and 100 µM). All cultures were compared to no DEX controls (CON) at day 14 within individual experimental repeats to calculate relative force. Significant values are identified using * where a significance of P ≤ 0.05 was achieved. All data presented as mean ± SD, from n = 5 engineered muscles, obtained from three independent experiments

Fig. 2

MAFbx and MuRF-1 ΔΔCT expression level of CON(control day 14, n = 6), 40 µM DEX administration over 24 h (DEX, n = 11), 40 µM DEX administration over 24 h replaced with differentiation media for 45 h (DEX + DM, n = 10) and 40 µM DEX administration over 24 h replaced with differentiation media and mechanically loaded for 3 h and sampled after 45 h (DEX + STRETCH, n = 11). Significant values are identified using * where a significance of P ≤ 0.05 was achieved. All data presented as mean ± SD, obtained from three independent experiments

Fig. 3

Immunohistochemical fluorescent staining of the nucleic DNA (blue) and muscle specific protein filament MyHC (green) in cross sections of engineered muscles (× 10 magnification) a representative CON (no DEX administration at day 14, n = 7), b 40 µM DEX administration over 24 h (DEX, n = 8), c 40 µM DEX administration over 24 h replaced with differentiation media for 48 h (DEX + DM, n = 7), d 40 µM DEX administration over 24 h replaced with differentiation media and mechanically loaded for 3 h and sampled after 48 h (DEX + STRETCH, n = 6), a average myotube width (µm), f average cross sectional area (CSA) of the myotubes (μm²) of CON (Control at day 14), DEX, DEX + DM and DEX + STRETCH. Scale bar represents 100 μm. Significantly different values are identified using*. All data presented as mean ± SD, obtained from three independent experiments. (Color figure online)

Fig. 4

MAFbx and MuRF-1 ΔΔCT expression level of CON (Control day 14, n = 5), 40 µM DEX administration over 24 h (DEX, n = 12), and mechanically loaded for 3 h then administered 40 µM DEX over 24 h (STRETCH + DEX, n = 9). Significant values are identified using * where a significance of P ≤ 0.05 was achieved. All data presented as mean ± SD, obtained from three independent experiments

Fig. 5

Immunohistochemical fluorescent staining of the nucleic DNA (blue) and muscle specific protein filament MyHC (green) in cross sections of engineered muscles (× 10 magnification) a representative CON (no DEX administration at day 14), b 40 µM DEX administration over 24 h (DEX), c Mechanical load for 3 h following by 40 µM DEX administration over 24 h (STRETCH + DEX), d average myotube width (µm), e average cross sectional area (CSA) of the myotubes (μm²) of CON (Control at day 14), DEX and STRETCH + DEX. Scale bar represents 100 μm. Significantly different values are identified using*. All data presented as mean ± SD, from n = 6 engineered muscles, obtained from three independent experiments. (Color figure online)

Fig. 6

Maximal contractile tetanic force of engineered skeletal muscle at CON (control day 14, n = 6), 40 µM DEX administration over 24 h (DEX, n = 10), mechanically loaded for 3 h then administered 40 µM DEX over 24 h (STRETCH + DEX, n = 9) and 40 µM DEX administration over 24 h replaced with differentiation media and mechanically loaded for 3 h and sampled after 48 h (DEX + STRETCH, n = 6). All cultures were compared to CON at day 14 within individual experimental repeats to calculate relative tetanic force. All data presented as mean ± SD, obtained from three independent experiments