Modulation of Ki67 and myogenic regulatory factor expression by tocotrienol-rich fraction ameliorates myogenic program of senescent human myoblasts

Abstract

Introduction: Replicative senescence results in dysregulation of cell proliferation and differentiation, which plays a role in the regenerative defects observed during age-related muscle atrophy. Vitamin E is a well-known antioxidant, which potentially ameliorates a wide range of age-related manifestations. The aim of this study was to determine the effects of tocotrienol-rich fraction (TRF) in modulating the expression of proliferation- and differentiation-associated proteins in senescent human myoblasts during the differentiation phase.

Material and methods: Human skeletal muscle myoblasts were cultured until senescence. Young and senescent cells were treated with TRF for 24 h before and after differentiation induction, followed by evaluation of cellular morphology and efficiency of differentiation. Expression of cell proliferation marker Ki67 protein and myogenic regulatory factors MyoD and myogenin were determined.

Results: Our findings showed that treatment with TRF significantly improved the morphology of senescent myoblasts. Promotion of differentiation was observed in young and senescent myoblasts with TRF treatment as shown by the increased fusion index and larger size of myotubes. Increased Ki67 and myogenin expression with TRF treatment was also observed in senescent myoblasts, suggesting amelioration of the myogenic program by TRF during replicative senescence.

Conclusions: TRF modulates the expression of regulatory factors related to proliferation and differentiation in senescent human myoblasts and could be beneficial for ameliorating the regenerative defects during aging.

Keywords: differentiation; myoblasts; myotubes; replicative senescence; tocotrienols.

Figure 1

Effects of replicative senescence and TRF treatment on the cellular morphology of myoblasts. The micrographs of myoblasts were captured from A – young control, B – TRF-treated young, C – senescent control, and D – TRF-treated senescent (magnification: 200×, scale bar: 200 µm). Myoblasts were displayed were stained for desmin (green) and Hoechst (blue). Regardless TRF treatment, young myoblasts were displayed in spindle shape. Meanwhile, untreated senescent control cells manifested broader and flatter cytoplasm with prominent striations. On contrary, TRF-treated senescent myoblasts exhibited more spindle shaped cytoplasm which resembled the morphology of young myoblasts

Figure 2

Effects of replicative senescence and TRF treatment on the expression of proliferation marker Ki67. A – Myoblasts were labelled with anti-Ki67 antibody (green) and Hoechst (blue) to detect the presence of Ki67 protein, as indicated by white arrow (magnification 200×, scale bar: 200 µm). B – Quantitative analysis of Ki67 expression in myoblasts at day 0, day 1, day 3 and day 5 of differentiation ^ap < 0.05, significantly different compared to young control at corresponding day of differentiation; ^bp < 0.05, significantly different compared to TRF-treated young myoblasts at corresponding day of differentiation; ^cp < 0.05, significantly different compared to senescent control at corresponding day of differentiation; ^#p < 0.05, significantly different compared to the corresponding group at day 0 of differentiation; and ^*p < 0.05, significantly different compared to the corresponding group at the day 1 of differentiation. Data are presented as the mean ± SD, n = 3.

Figure 3

Effects of replicative senescence and TRF treatment on the expression of MyoD. A – Myoblasts were labelled with anti-MyoD antibody (green) and Hoechst (blue) to detect the presence of MyoD protein, as indicated by white arrow (magnification 200×, scale bar: 200 µm). B – Quantitative analysis of MyoD expression in myoblasts at day 1, day 3 and day 5 of differentiation ^aDenotes p < 0.05, significantly different compared to young control at corresponding day of differentiation; ^bp < 0.05, significantly different compared to TRF-treated young myoblasts at corresponding day of differentiation; ^cp < 0.05, significantly different compared to senescent control at corresponding day of differentiation; ^#p < 0.05, significantly different compared to the corresponding group at day 1 of differentiation; and ^*p < 0.05, significantly different compared to the corresponding group at the day 3 of differentiation. Data are presented as the mean ± SD, n = 3.

Figure 4

Effects of replicative senescence and TRF treatment on the expression of myogenin. A – Myoblasts were labelled with anti-Myogenin antibody (green) and Hoechst (blue) to detect the presence of myogenin protein, as indicated by white arrow (magnification 200×, scale bar: 200 µm). B – Quantitative analysis of myogenin expression in myoblasts at day 1, day 3 and day 5 of differentiation ^aDenotes p < 0.05, significantly different compared to young control at corresponding day of differentiation; ^bp < 0.05, significantly different compared to TRF-treated young myoblasts at corresponding day of differentiation; ^#p < 0.05, significantly different compared to the corresponding group at day 1 of differentiation; and ^*p < 0.05, significantly different compared to the corresponding group at the day 3 of differentiation. Data are presented as the mean ± SD, n = 3.

Figure 5

Effects of replicative senescence and TRF treatment on the efficiency of myogenic differentiation. A – Micrographs showed the formation of myotubes in each group (magnification 200×, scale bar: 200 µm). Multinucleated myotubes were observed in young groups, conversely myogenic differentiation was impaired during replicative senescence. B – The fusion index and C – the size of myotubes were determined, showing that TRF was able to increase the differentiation potential of senescent myoblasts ^ap < 0.05, significantly different compared to young control at corresponding day of differentiation; ^bp < 0.05, significantly different compared to TRF-treated young myoblasts at corresponding day of differentiation; ^cp < 0.05, significantly different compared to senescent control at corresponding day of differentiation; ^#p < 0.05, significantly different compared to the corresponding group at day 1 of differentiation; and ^*p < 0.05, significantly different compared to the corresponding group at the day 3 of differentiation. Data are presented as the mean ± SD, n = 3.