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Epicatechin elicits MyoD-dependent myoblast differentiation and myogenic conversion of fibroblasts

View Article: PubMed Central - PubMed

ABSTRACT

Prevention of age-associated reduction in muscle mass and function is required to manage a healthy life. Supplemental (-)-Epicatechin (EC) appears to act as a potential regulator for muscle growth and strength. However, its cellular and molecular mechanisms as a potential muscle growth agent have not been studied well. In the current study, we investigated a role of EC in differentiation of muscle progenitors to gain the molecular insight into how EC regulates muscle growth. EC enhanced myogenic differentiation in a dose-dependent manner through stimulation of promyogenic signaling pathways, p38MAPK and Akt. EC treatment elevated MyoD activity by enhancing its heterodimerization with E protein. Consistently, EC also positively regulated myogenic conversion and differentiation of fibroblasts. In conclusion, EC has a potential as a therapeutic or nutraceutical remedy to treat degenerative muscle diseases or age-related muscle weakness.

No MeSH data available.


EC induces activation of p38MAPK and Akt in a dose-dependent manner.(A) C2C12 myoblasts were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to p-p38MAPK (pp38), p38MAPK (p38), p-Akt (pAkt) and Akt. The experiment was repeated three times with similar results. (B) Quantification of blots from three experiments similarly performed as shown in panel A. The relative signal intensity of pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01. (C) Primary mouse MEFs were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to pp38, p38, pAkt and Akt. (D) Quantification of blots from three experiments similarly performed as shown in panel C. The relative signal intensity for pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01 and **P < 0.05. (E) C2C12 myoblasts were treated with 2.5 μM SB203580 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pp38, p38, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (F) Quantification of three blots, similarly performed as shown in panel E. The signal intensity of pp38, myogenic proteins such as MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (G) C2C12 myoblasts were treated with 1 μM LY294002 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pAkt, Akt, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (H) Quantification of three blots from experiments similarly performed as shown in panel G. The signal intensity of pp38, MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (I) C2C12 cells from replica experiments as shown in panel E and G were immunostained for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. (J) Quantification of myotube formation from data shown in panel I. Data from three independent experiments were presented as the means ± 1 SD. Asterisks indicate significant difference from the control. *P < 0.01, **P < 0.05.
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pone.0175271.g002: EC induces activation of p38MAPK and Akt in a dose-dependent manner.(A) C2C12 myoblasts were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to p-p38MAPK (pp38), p38MAPK (p38), p-Akt (pAkt) and Akt. The experiment was repeated three times with similar results. (B) Quantification of blots from three experiments similarly performed as shown in panel A. The relative signal intensity of pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01. (C) Primary mouse MEFs were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to pp38, p38, pAkt and Akt. (D) Quantification of blots from three experiments similarly performed as shown in panel C. The relative signal intensity for pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01 and **P < 0.05. (E) C2C12 myoblasts were treated with 2.5 μM SB203580 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pp38, p38, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (F) Quantification of three blots, similarly performed as shown in panel E. The signal intensity of pp38, myogenic proteins such as MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (G) C2C12 myoblasts were treated with 1 μM LY294002 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pAkt, Akt, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (H) Quantification of three blots from experiments similarly performed as shown in panel G. The signal intensity of pp38, MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (I) C2C12 cells from replica experiments as shown in panel E and G were immunostained for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. (J) Quantification of myotube formation from data shown in panel I. Data from three independent experiments were presented as the means ± 1 SD. Asterisks indicate significant difference from the control. *P < 0.01, **P < 0.05.

Mentions: It has been well documented that two promyogenic kinases, p38MAPK and Akt play essential roles in myoblast differentiation [17, 18]. To assess the molecular regulatory pathways of EC-mediated myoblast differentiation, C2C12 myoblasts were treated with the indicated concentration of EC and assessed for the activation status of p38MAPK and Akt by using antibodies recognizing the active phosphorylated form of p38MAPK (pp38) or Akt (pAkt) by immunoblotting. The treatment of EC increased the levels of pp38 and pAkt in a dose-dependent manner, whereas the level of total proteins stayed relatively constant (Fig 2A and 2B). In agreement with EC-treated myoblasts, primary MEFs treated with EC displayed enhanced levels of pp38 and pAkt (Fig 2C and 2D). Especially, the effective concentration of EC to activate p38MAPK in primary MEFs appears to range from 1 to 5 μM.


Epicatechin elicits MyoD-dependent myoblast differentiation and myogenic conversion of fibroblasts
EC induces activation of p38MAPK and Akt in a dose-dependent manner.(A) C2C12 myoblasts were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to p-p38MAPK (pp38), p38MAPK (p38), p-Akt (pAkt) and Akt. The experiment was repeated three times with similar results. (B) Quantification of blots from three experiments similarly performed as shown in panel A. The relative signal intensity of pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01. (C) Primary mouse MEFs were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to pp38, p38, pAkt and Akt. (D) Quantification of blots from three experiments similarly performed as shown in panel C. The relative signal intensity for pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01 and **P < 0.05. (E) C2C12 myoblasts were treated with 2.5 μM SB203580 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pp38, p38, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (F) Quantification of three blots, similarly performed as shown in panel E. The signal intensity of pp38, myogenic proteins such as MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (G) C2C12 myoblasts were treated with 1 μM LY294002 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pAkt, Akt, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (H) Quantification of three blots from experiments similarly performed as shown in panel G. The signal intensity of pp38, MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (I) C2C12 cells from replica experiments as shown in panel E and G were immunostained for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. (J) Quantification of myotube formation from data shown in panel I. Data from three independent experiments were presented as the means ± 1 SD. Asterisks indicate significant difference from the control. *P < 0.01, **P < 0.05.
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pone.0175271.g002: EC induces activation of p38MAPK and Akt in a dose-dependent manner.(A) C2C12 myoblasts were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to p-p38MAPK (pp38), p38MAPK (p38), p-Akt (pAkt) and Akt. The experiment was repeated three times with similar results. (B) Quantification of blots from three experiments similarly performed as shown in panel A. The relative signal intensity of pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01. (C) Primary mouse MEFs were treated with EC and differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies to pp38, p38, pAkt and Akt. (D) Quantification of blots from three experiments similarly performed as shown in panel C. The relative signal intensity for pp38 and pAkt proteins to total p38 and Akt proteins, respectively was determined. The values from DMSO-treated control cells were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. Significant difference from control, *P < 0.01 and **P < 0.05. (E) C2C12 myoblasts were treated with 2.5 μM SB203580 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pp38, p38, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (F) Quantification of three blots, similarly performed as shown in panel E. The signal intensity of pp38, myogenic proteins such as MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (G) C2C12 myoblasts were treated with 1 μM LY294002 for 30 min prior to the treatment with EC, and then differentiated in DM for 2 days. Cell lysates were subjected to immunoblotting with antibodies against pAkt, Akt, MHC, MyoD, Myogenin and pan-Cadherin as a loading control. (H) Quantification of three blots from experiments similarly performed as shown in panel G. The signal intensity of pp38, MHC, MyoD and Myogenin was quantified, and the relative values were normalized to p38 and pan-Cadherin, respectively. The values of control sample were set to 1.0. Values represent the means of triplicate determinations ± 1 SD. *p < 0.01, **p <0.05. NS, not significant. (I) C2C12 cells from replica experiments as shown in panel E and G were immunostained for MHC expression (red) and DAPI to visualize nuclei (blue) to reveal myotube formation. (J) Quantification of myotube formation from data shown in panel I. Data from three independent experiments were presented as the means ± 1 SD. Asterisks indicate significant difference from the control. *P < 0.01, **P < 0.05.
Mentions: It has been well documented that two promyogenic kinases, p38MAPK and Akt play essential roles in myoblast differentiation [17, 18]. To assess the molecular regulatory pathways of EC-mediated myoblast differentiation, C2C12 myoblasts were treated with the indicated concentration of EC and assessed for the activation status of p38MAPK and Akt by using antibodies recognizing the active phosphorylated form of p38MAPK (pp38) or Akt (pAkt) by immunoblotting. The treatment of EC increased the levels of pp38 and pAkt in a dose-dependent manner, whereas the level of total proteins stayed relatively constant (Fig 2A and 2B). In agreement with EC-treated myoblasts, primary MEFs treated with EC displayed enhanced levels of pp38 and pAkt (Fig 2C and 2D). Especially, the effective concentration of EC to activate p38MAPK in primary MEFs appears to range from 1 to 5 μM.

View Article: PubMed Central - PubMed

ABSTRACT

Prevention of age-associated reduction in muscle mass and function is required to manage a healthy life. Supplemental (-)-Epicatechin (EC) appears to act as a potential regulator for muscle growth and strength. However, its cellular and molecular mechanisms as a potential muscle growth agent have not been studied well. In the current study, we investigated a role of EC in differentiation of muscle progenitors to gain the molecular insight into how EC regulates muscle growth. EC enhanced myogenic differentiation in a dose-dependent manner through stimulation of promyogenic signaling pathways, p38MAPK and Akt. EC treatment elevated MyoD activity by enhancing its heterodimerization with E protein. Consistently, EC also positively regulated myogenic conversion and differentiation of fibroblasts. In conclusion, EC has a potential as a therapeutic or nutraceutical remedy to treat degenerative muscle diseases or age-related muscle weakness.

No MeSH data available.