Limits...
Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation.

Barbieri E, Battistelli M, Casadei L, Vallorani L, Piccoli G, Guescini M, Gioacchini AM, Polidori E, Zeppa S, Ceccaroli P, Stocchi L, Stocchi V, Falcieri E - J Aging Res (2011)

Bottom Line: These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes.Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process.The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino (PU), Italy.

ABSTRACT
This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

No MeSH data available.


Related in: MedlinePlus

Evaluation of mitochondrial biogenesis during myoblast differentiation. In (a), determination by real-time PCR of mtDNA content expressed as mtDNA/nDNA ratio (COXII/GAPDH), as described in Section 2. In (b), quantitative analyses of PGC-1 α and T-fam by real-time PCR. The amount of each target transcript was related to that of the reference gene (the ribosomal protein S16). Data are expressed as the mean ± SEM of three experiments; all samples were analyzed in triplicate. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey's multiple comparison test. The threshold of significance for the ANOVA and the Tukey's test was fixed at *P ≤ .05; **P ≤ .01.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3100678&req=5

fig3: Evaluation of mitochondrial biogenesis during myoblast differentiation. In (a), determination by real-time PCR of mtDNA content expressed as mtDNA/nDNA ratio (COXII/GAPDH), as described in Section 2. In (b), quantitative analyses of PGC-1 α and T-fam by real-time PCR. The amount of each target transcript was related to that of the reference gene (the ribosomal protein S16). Data are expressed as the mean ± SEM of three experiments; all samples were analyzed in triplicate. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey's multiple comparison test. The threshold of significance for the ANOVA and the Tukey's test was fixed at *P ≤ .05; **P ≤ .01.

Mentions: As shown in Figure 3(a), twenty-four hours after differentiation induction, the relative amount of mtDNA undergoes a 2-fold increment at the intermediate period of differentiation (T = 3) reaching a plateau level at the final stage of maturation (T = 7).


Morphofunctional and Biochemical Approaches for Studying Mitochondrial Changes during Myoblasts Differentiation.

Barbieri E, Battistelli M, Casadei L, Vallorani L, Piccoli G, Guescini M, Gioacchini AM, Polidori E, Zeppa S, Ceccaroli P, Stocchi L, Stocchi V, Falcieri E - J Aging Res (2011)

Evaluation of mitochondrial biogenesis during myoblast differentiation. In (a), determination by real-time PCR of mtDNA content expressed as mtDNA/nDNA ratio (COXII/GAPDH), as described in Section 2. In (b), quantitative analyses of PGC-1 α and T-fam by real-time PCR. The amount of each target transcript was related to that of the reference gene (the ribosomal protein S16). Data are expressed as the mean ± SEM of three experiments; all samples were analyzed in triplicate. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey's multiple comparison test. The threshold of significance for the ANOVA and the Tukey's test was fixed at *P ≤ .05; **P ≤ .01.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3100678&req=5

fig3: Evaluation of mitochondrial biogenesis during myoblast differentiation. In (a), determination by real-time PCR of mtDNA content expressed as mtDNA/nDNA ratio (COXII/GAPDH), as described in Section 2. In (b), quantitative analyses of PGC-1 α and T-fam by real-time PCR. The amount of each target transcript was related to that of the reference gene (the ribosomal protein S16). Data are expressed as the mean ± SEM of three experiments; all samples were analyzed in triplicate. Results from PCR real-time analysis were compared with the ANOVA test, followed by a post hoc test using Tukey's multiple comparison test. The threshold of significance for the ANOVA and the Tukey's test was fixed at *P ≤ .05; **P ≤ .01.
Mentions: As shown in Figure 3(a), twenty-four hours after differentiation induction, the relative amount of mtDNA undergoes a 2-fold increment at the intermediate period of differentiation (T = 3) reaching a plateau level at the final stage of maturation (T = 7).

Bottom Line: These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes.Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process.The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Sciences, University of Urbino Carlo Bo, Via I Maggetti, 26, 61029 Urbino (PU), Italy.

ABSTRACT
This study describes mitochondrial behaviour during the C2C12 myoblast differentiation program and proposes a proteomic approach to mitochondria integrated with classical morphofunctional and biochemical analyses. Mitochondrial ultrastructure variations were determined by transmission electron microscopy; mitochondrial mass and membrane potential were analysed by Mitotracker Green and JC-1 stains and by epifluorescence microscope. Expression of PGC1α, NRF1α, and Tfam genes controlling mitochondrial biogenesis was studied by real-time PCR. The mitochondrial functionality was tested by cytochrome c oxidase activity and COXII expression. Mitochondrial proteomic profile was also performed. These assays showed that mitochondrial biogenesis and activity significantly increase in differentiating myotubes. The proteomic profile identifies 32 differentially expressed proteins, mostly involved in oxidative metabolism, typical of myotubes formation. Other notable proteins, such as superoxide dismutase (MnSOD), a cell protection molecule, and voltage-dependent anion-selective channel protein (VDAC1) involved in the mitochondria-mediated apoptosis, were found to be regulated by the myogenic process. The integration of these approaches represents a helpful tool for studying mitochondrial dynamics, biogenesis, and functionality in comparative surveys on mitochondrial pathogenic or senescent satellite cells.

No MeSH data available.


Related in: MedlinePlus