Protective Effect of Delta-Like 1 Homolog Against Muscular Atrophy in a Mouse Model

Figures

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  Fig. 1.. Effects of delta-like 1 homolog (DLK1) on body composition and histopathological morphology of tibialis anterior muscle in the dexamethasone-induced muscle atrophy mouse model. (A, B, C, D) Effects of DLK1 on body composition in dexamethasone-induced tibialis anterior (TA) skeletal muscle atrophy mice using dual energy X-ray absorptiometry (n=4 per each group). (E) Size comparison of TA muscles from a mouse hindlimb. (F) TA muscle weights (n=6 per each group). (G) Representative histopathological images of TA muscle (200× magnification; scale bar 200 μm) using Masson’s trichrome stain. (H) Mean cross-sectional area of the TA muscle (μm2). Results are presented as mean±standard error of the mean. Treatment groups were as follows: control=oral distilled water and intraperitoneal (IP) phosphate buffered saline (PBS); dexamethasone (Dex)=oral Dex (1 mg/kg) and IP PBS; Dex+DLK1=oral Dex (1 mg/kg) and IP DLK1 (0.8 mg/kg/day). CSA, cross-sectional area; NS, non-significant. aP<0.05 was considered statistically significant.

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  Fig. 2.. Effects of delta-like 1 homolog (DLK1) on muscle atrophic and myogenic factors in the dexamethasone-induced muscle atrophy mouse model. A graph showing relative mRNA expression of muscle atrophic (myostatin, atrogin1, and muscle-specific RING finger protein 1 [MuRF1]) and myogenic (myogenic differentiation [MyoD] and myogenin) factors in the mouse tibialis anterior (TA) muscle. Results are presented as mean±standard error of the mean. Treatment groups were as follows: control=oral distilled water and intraperitoneal (IP) phosphate buffered saline (PBS); dexamethasone (Dex)=oral Dex (1 mg/kg) and IP PBS; Dex+DLK1=oral Dex (1 mg/kg) and IP DLK1 (0.8 mg/kg/day). NS, non-significant. aP<0.05 was considered statistically significant.

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  Fig. 3.. Effects of delta-like 1 homolog (DLK1) on tibialis anterior muscle morphology and mitochondrial biogenesis markers in the dexamethasone-induced muscle atrophy mouse model. (A) Representative electron microscopic images of myofibrils within the tibialis anterior (TA) muscle (5,000×, 10,000×, and 20,000× magnification; M=myofilament, yellow arrow=Z line, red arrow=mitochondria, dashed arrow=a broken structure, hollow red arrow=a swollen structure). Graphs showing lengths of (B) sarcomeres and (C) mitochondria in TA muscle cells (n=30 per each group). (D) Relative mRNA expression of mitochondrial fusion (Opa1) and fission (Drp1) markers (n=10 per each group). Results are presented as mean±standard error of the mean. Treatment groups were as follows: control=oral distilled water and intraperitoneal (IP) phosphate buffered saline (PBS); dexamethasone (Dex)=oral Dex (1 mg/kg) and IP PBS; Dex+DLK1=oral Dex (1 mg/kg) and IP DLK1 (0.8 mg/kg/day). NS, non-significant. aP<0.05 was considered statistically significant.

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  Fig. 4.. Effect of delta-like 1 homolog (DLK1) on dexamethasone-induced myotube atrophy. (A) Representative histopathological images of H&E staining showing myoblast (Day 0) and differentiated multi-nucleated myotubes (Day 5) according to the type of differentiation medium. (B) Thickness of myotubes (µm). (C) Relative mRNA expression of muscle atrophic (myostatin, atrogin1, and muscle-specific RING finger protein 1 [MuRF1]) and myogenic (myogenic differentiation [MyoD] and myogenin) factors in myotubes. Results are presented as mean±standard error of the mean. Myotube cultures were divided into three groups on differentiation Day 4 and cultured in different media for 24 hours: control=Dulbecco’s Modified Eagle Medium (DMEM)+ethanol (0.02% v/v); dexamethasone (Dex)=DMEM+Dex (10 μM dissolved in ethanol); and Dex+DLK1=DMEM+Dex (10 μM dissolved in ethanol)+DLK1 (5.0 μg/mL). NS, non-significant. aP<0.05 was considered statistically significant.

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  Fig. 5.. Effects of delta-like 1 homolog (DLK1) on mitochondrial biogenesis markers in dexamethasone-induced myotube atrophy. (A) Representative electron microscopic images of myotube cells (5,000×, 15,000×, and 40,000× magnification). (B) Relative mRNA expression of mitochondrial fusion (Opa1) and fission (Drp1) markers (n=10 per each group). Results are presented as mean±standard error of the mean. Myotube cultures were divided into three groups on differentiation Day 4 and cultured in different media for 24 hours: control=Dulbecco’s Modified Eagle Medium (DMEM)+ethanol (0.02% v/v); dexamethasone (Dex)=DMEM+Dex (10 μM dissolved in ethanol); and Dex+DLK1=DMEM+Dex (10 μM dissolved in ethanol)+DLK1 (5.0 μg/mL). NS, non-significant. aP<0.05 was considered statistically significant.

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  Fig. 6.. Effect of delta-like 1 homolog (DLK1) on muscle-related markers in atrophied myotubes with or without knockdown of myostatin expression. (A) Western blot images with (B) quantification of myostatin and myogenin protein after transfection with small interfering ribonucleic acids (siRNAs) of negative control or myostatin (n=3 to 6 per group). Results are presented as mean±standard error of the mean. Transfected myotube cultures were divided into three groups on differentiation Day 4 and cultured in different media for 24 hours: control=Dulbecco’s Modified Eagle Medium (DMEM)+ethanol (0.02% v/v); dexamethasone (Dex)=DMEM+Dex (10 μM dissolved in ethanol); and Dex+DLK1=DMEM+Dex (10 μM dissolved in ethanol)+DLK1 (5.0 μg/mL). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NS, non-significant. aP<0.05 was considered statistically significant.

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