Targeting myomiRs by tocotrienol-rich fraction to promote myoblast differentiation

Abstract Background Several muscle-specific microRNAs (myomiRs) are differentially expressed during cellular senescence. However, the role of dietary compounds on myomiRs remains elusive. This study aimed to elucidate the modulatory role of tocotrienol-rich fraction (TRF) on myomiRs and myogenic genes during differentiation of human myoblasts. Young and senescent human skeletal muscle myoblasts (HSMM) were treated with 50 μg/mL TRF for 24 h before and after inducing differentiation. Results The fusion index and myotube surface area were higher (p < 0.05) on days 3 and 5 than that on day 1 of differentiation. Ageing reduced the differentiation rate, as observed by a decrease in both fusion index and myotube surface area in senescent cells (p < 0.05). Treatment with TRF significantly increased differentiation at days 1, 3 and 5 of young and senescent myoblasts. In senescent myoblasts, TRF increased the expression of miR-206 and miR-486 and decreased PTEN and PAX7 expression. However, the expression of IGF1R was upregulated during early differentiation and decreased at late differentiation when treated with TRF. In young myoblasts, TRF promoted differentiation by modulating the expression of miR-206, which resulted in the reduction of PAX7 expression and upregulation of IGF1R. Conclusion TRF can potentially promote myoblast differentiation by modulating the expression of myomiRs, which regulate the expression of myogenic genes

The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts.

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts

Effects of replicative senescence and vitamin E treatment at the early phase of myogenic differentiation.

<p>The <i>MYF5</i> (a) and <i>MYOD1</i> (b) mRNA expression levels on day 0 and day 1 of differentiation were determined, while the <i>MYOG</i> (c) mRNA expression level was determined on day 0 and day 2 of differentiation. The percentage of nuclei that stained for myogenin (green) on day 3 of differentiation is shown in (d). Photomicrographs were taken from all groups using a fluorescence microscope (magnification: 200×) (e). TRF significantly increased the number of myogenin-labeled nuclei on day 3 of differentiation, as indicated by arrows. *p<0.05 significantly different compared to young control at corresponding day of differentiation, ^#p<0.05 significantly different compared to senescent control at corresponding day of differentiation, ^£p<0.05 significantly different compared to TRF-treated young myoblasts at the corresponding day of differentiation, ^§p<0.05 significantly different compared to TRF-treated senescent myoblasts at corresponding day of differentiation, **p<0.05 significantly different compared to the corresponding treatment at day 0 of differentiation. The data are presented as the means ± SD.</p

Effects of serial passaging on population doubling, cell proliferation and expression of SA-β-gal in myoblasts.

<p>During extensive expansion, myoblasts significantly lost their proliferation capacity as represented by a hyperbolic proliferative lifespan curve (a) and decreasing percentage of BrdU incorporation (b), while the percentage of senescent cells increased, as represented by positive SA-β-gal staining (c). For (b) and (c), the data are presented as the means ± SD, n = 3. *p<0.05 compared to myoblasts at PD14 (young), ^#p<0.05 compared to myoblasts at PD18 (pre-senescent) with a <i>post-hoc</i> Dunnett T3.</p

Effects of replicative senescence and TRF treatment on intracellular ROS generation.

<p>ROS was normally generated in myoblasts, however the elevated ROS level may disturb proliferation and survival of cells. The amount of intracellular ROS was significantly increased in senescent myoblasts. The fluorescence intensity of positive stained cells in senescent myoblasts was significantly reduced in TRF-treated cells, revealing free radical scavenging properties exerted in TRF. *p<0.05 significantly different compared to young control, ^#p<0.05 significantly different compared to senescent control, with <i>post-hoc</i> LSD test. Data are presented as mean ± SD, n = 3.</p

Effects of replicative senescence and vitamin E treatment on the differentiation capacity of myoblasts.

<p>The photomicrographs of myotubes were taken from young control (a), TRF-treated young (b), ATF-treated young (c), senescent control (d), TRF-treated senescent (e) and ATF-treated senescent (f) cells (magnification: 200×). Desmin was stained green, and the nuclei were stained blue (Hoechst). The myotubes that formed from young myoblasts were significantly bigger than the myotubes from senescent myoblasts. The fusion index (g) and the size of myotubes (h) were determined to evaluate the efficiency of muscle differentiation. TRF significantly increased the fusion index (n = 3), which was not shown with ATF treatment. No changes was observed in the size of the myotubes that formed (n = 12) with the TRF and ATF treatments. *p<0.05 significantly different compared to young control, ^#p<0.05 significantly different compared to senescent control, ^§p<0.05 significantly different compared to TRF-treated senescent myoblasts, with <i>post-hoc</i> Tukey HSD. The data are presented as the means ± SD.</p

Effects of replicative senescence and vitamin E treatment on myoblasts phenotype.

<p>The photomicrographs of myoblasts were taken from a young control (a), TRF-treated young (b), ATF-treated young (c), senescent control (d), TRF-treated senescent (e) and ATF-treated senescent (f) cells (magnification: 400×). Myoblasts were stained for desmin (green) and Hoechst (blue). Both TRF- and ATF-treated senescent myoblasts resembled the morphology of young cells. The width and length of cells were measured (g). The width of senescent myoblasts significantly increased in the untreated control and decreased with TRF and ATF treatment. The spindle-shaped cells can be maintained in culture for two days after withdrawal of treatments and retained in the following passage (h). *p<0.05 significantly different compared to untreated young myoblasts, ^#p<0.05 significantly different compared to untreated senescent myoblasts, with <i>post-hoc</i> Dunnett T3. The data are presented as the means ± SD, n = 30.</p

Effects of the TRF and ATF treatments on cell viability and proliferation.

<p>Dose-response curve of TRF (a) and ATF (b) treatments (24 h) in young and senescent myoblasts (n = 9). The prolonged treatments of TRF and ATF (48 h) at optimal dose were unlikely to further improve cell viability in senescent myoblasts (c). Therefore, 24 h treatment was used in subsequent experiment. Comparison of cell viability between myoblasts from donor 1, a 17 year-old, female Caucasian and donor 2, a 16 year-old male Caucasian (d). There were no significant different observed between the two cell lines in response to both TRF and ATF treatments based on the viability assessment. <i>Post-hoc</i> Dunnett T3 test for (a) and (b). *p<0.05 significantly different compared to untreated young myoblasts, ^#p<0.05 significantly different compared to untreated senescent myoblasts, ^§p<0.05 significantly different compared to TRF-treated senescent myoblasts. The data are presented as the means ± SD.</p

Effects of TRF on the MRFs mRNA expression levels during 5 days of differentiation induction.

<p>The <i>MYF5</i> (a), <i>MYOD1</i> (b) and <i>MYOG</i> (c) mRNA expression levels in senescent control were significantly lower than young control (p<0.05). TRF significantly increased the expression of both <i>MYOD1</i> and <i>MYOG</i> mRNA at day 3 (D3) and day 5 (D5) of differentiation, resembled the expression in young control, while the expression in senescent myoblasts remained low, even after 5 days of differentiation. ^ap<0.05 significantly different compared to young control at corresponding day of differentiation, ^bp<0.05 significantly different compared to senescent control at corresponding day of differentiation, The data are presented as the means ± SD.</p