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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.


Related in: MedlinePlus

Undifferentiated (a, b, c), early differentiation (d, e, f), intermediate differentiation (g, h, i) and late differentiation stages (j, k, l), are indicated by RM (a, d, g, j), Giemsa staining (b, e, h, k), and TEM (c, f, i, l). Mitochondrial morphology is further detailed by the correspondent insets, showing TEM analysis of isolated mitochondria. C2C12 cell differentiation morphological progression is evident, as well as mitochondrial behaviour in the various stages. (a, b, d, e, g, h, j, k): Bar = 20 μm; (c, f, i, l): Bar = 0.5 μm; insets, Bar = 0.1 μm.
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fig1: Undifferentiated (a, b, c), early differentiation (d, e, f), intermediate differentiation (g, h, i) and late differentiation stages (j, k, l), are indicated by RM (a, d, g, j), Giemsa staining (b, e, h, k), and TEM (c, f, i, l). Mitochondrial morphology is further detailed by the correspondent insets, showing TEM analysis of isolated mitochondria. C2C12 cell differentiation morphological progression is evident, as well as mitochondrial behaviour in the various stages. (a, b, d, e, g, h, j, k): Bar = 20 μm; (c, f, i, l): Bar = 0.5 μm; insets, Bar = 0.1 μm.

Mentions: The monolayer organization, as directly analysed at RM and by means of Giemsa staining, deeply changes from undifferentiated myoblasts to myotubes. In the undifferentiated condition (Figures 1(a), 1(b), and 1(c)), myoblasts appear as fusiform or star-shaped cells, mostly flattened and closely adherent to the substrate. At the initial differentiation stage (Figures 1(d), 1(e), and 1(f)), intercellular spaces disappear, cells progressively align, and, occasionally, elongate. Four days after differentiation induction (Figures 1(g), 1(h), and 1(i)), early myotubes, with 2 or more centrally located nuclei, appear (T = 4, fusion index = 38 ± 3.4%). The late differentiation condition (7 days) is characterized by the presence of highly structured myotubes (Figures 1(j), 1(k), and 1(l)). These are 100–600 μm syncytia and contain even more than 20 nuclei, mainly centrally located or, occasionally, aligned in parallel rows (T = 7, fusion index  84.6 ± 6%).


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)

Undifferentiated (a, b, c), early differentiation (d, e, f), intermediate differentiation (g, h, i) and late differentiation stages (j, k, l), are indicated by RM (a, d, g, j), Giemsa staining (b, e, h, k), and TEM (c, f, i, l). Mitochondrial morphology is further detailed by the correspondent insets, showing TEM analysis of isolated mitochondria. C2C12 cell differentiation morphological progression is evident, as well as mitochondrial behaviour in the various stages. (a, b, d, e, g, h, j, k): Bar = 20 μm; (c, f, i, l): Bar = 0.5 μm; insets, Bar = 0.1 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3100678&req=5

fig1: Undifferentiated (a, b, c), early differentiation (d, e, f), intermediate differentiation (g, h, i) and late differentiation stages (j, k, l), are indicated by RM (a, d, g, j), Giemsa staining (b, e, h, k), and TEM (c, f, i, l). Mitochondrial morphology is further detailed by the correspondent insets, showing TEM analysis of isolated mitochondria. C2C12 cell differentiation morphological progression is evident, as well as mitochondrial behaviour in the various stages. (a, b, d, e, g, h, j, k): Bar = 20 μm; (c, f, i, l): Bar = 0.5 μm; insets, Bar = 0.1 μm.
Mentions: The monolayer organization, as directly analysed at RM and by means of Giemsa staining, deeply changes from undifferentiated myoblasts to myotubes. In the undifferentiated condition (Figures 1(a), 1(b), and 1(c)), myoblasts appear as fusiform or star-shaped cells, mostly flattened and closely adherent to the substrate. At the initial differentiation stage (Figures 1(d), 1(e), and 1(f)), intercellular spaces disappear, cells progressively align, and, occasionally, elongate. Four days after differentiation induction (Figures 1(g), 1(h), and 1(i)), early myotubes, with 2 or more centrally located nuclei, appear (T = 4, fusion index = 38 ± 3.4%). The late differentiation condition (7 days) is characterized by the presence of highly structured myotubes (Figures 1(j), 1(k), and 1(l)). These are 100–600 μm syncytia and contain even more than 20 nuclei, mainly centrally located or, occasionally, aligned in parallel rows (T = 7, fusion index  84.6 ± 6%).

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