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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.


Confocal microscopy of C2C12 myoblasts (a–d) and late myotubes (e–h), after Mito Tracker (a, b, e, f) and JC-1 (c, d, g, h) staining. Graphs of lower panel show the different fluorescence JC-1 intensity in myoblasts (i) and late myotubes  (j). (a–h): Bar = 20 μm.
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fig2: Confocal microscopy of C2C12 myoblasts (a–d) and late myotubes (e–h), after Mito Tracker (a, b, e, f) and JC-1 (c, d, g, h) staining. Graphs of lower panel show the different fluorescence JC-1 intensity in myoblasts (i) and late myotubes (j). (a–h): Bar = 20 μm.

Mentions: Figure 2 describes mitochondrial characteristics during differentiation, analysed by confocal microscopy, after Mito Tracker green (a–d) and JC-1 (e–h) staining, both specific mitochondrial dyes. The first covalently binds to mitochondrial proteins and is generally considered an available indicator of mitochondrial mass. The second undergoes characteristic fluorescence changes according to the mitochondrial membrane ΔΨ, thus revealing functional mitochondrial alterations. In myoblasts (a, b, c, and d), both fluorescent probes show a perinuclear mitochondrial distribution. Indeed, at initial differentiation stages, numerous mitochondria can be identified as clearly distinguishable single organelles. Moreover, after differentiation induction, mitochondrial mass increased appearing uniform in myotubes (e and f). Mitochondrial membrane potential also increased, highlighted by JC-1 main red staining (g), still more evident in late differentiation condition shown in (h). Graphs of lower panel show the increasing level of red fluorescence JC-1 intensity from myoblasts (i) to late myotubes (j).


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)

Confocal microscopy of C2C12 myoblasts (a–d) and late myotubes (e–h), after Mito Tracker (a, b, e, f) and JC-1 (c, d, g, h) staining. Graphs of lower panel show the different fluorescence JC-1 intensity in myoblasts (i) and late myotubes  (j). (a–h): Bar = 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Confocal microscopy of C2C12 myoblasts (a–d) and late myotubes (e–h), after Mito Tracker (a, b, e, f) and JC-1 (c, d, g, h) staining. Graphs of lower panel show the different fluorescence JC-1 intensity in myoblasts (i) and late myotubes (j). (a–h): Bar = 20 μm.
Mentions: Figure 2 describes mitochondrial characteristics during differentiation, analysed by confocal microscopy, after Mito Tracker green (a–d) and JC-1 (e–h) staining, both specific mitochondrial dyes. The first covalently binds to mitochondrial proteins and is generally considered an available indicator of mitochondrial mass. The second undergoes characteristic fluorescence changes according to the mitochondrial membrane ΔΨ, thus revealing functional mitochondrial alterations. In myoblasts (a, b, c, and d), both fluorescent probes show a perinuclear mitochondrial distribution. Indeed, at initial differentiation stages, numerous mitochondria can be identified as clearly distinguishable single organelles. Moreover, after differentiation induction, mitochondrial mass increased appearing uniform in myotubes (e and f). Mitochondrial membrane potential also increased, highlighted by JC-1 main red staining (g), still more evident in late differentiation condition shown in (h). Graphs of lower panel show the increasing level of red fluorescence JC-1 intensity from myoblasts (i) to late myotubes (j).

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.