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Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

Sin J, Andres AM, Taylor DJ, Weston T, Hiraumi Y, Stotland A, Kim BJ, Huang C, Doran KS, Gottlieb RA - Autophagy (2016)

Bottom Line: We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis.Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks.Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: a The Cedars-Sinai Heart Institute and the Barbra Streisand Women's Heart Center Cedars-Sinai Medical Center , Los Angeles , CA , USA.

ABSTRACT
Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

No MeSH data available.


Related in: MedlinePlus

Electron micrographs of differentiating C2C12s. Transmission electron microscopy was performed on differentiating C2C12s to examine alterations in mitochondrial populations. Insets are presented at higher magnification below each original image. Scale bars: 500 nm. GM, growth medium.
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f0004: Electron micrographs of differentiating C2C12s. Transmission electron microscopy was performed on differentiating C2C12s to examine alterations in mitochondrial populations. Insets are presented at higher magnification below each original image. Scale bars: 500 nm. GM, growth medium.

Mentions: As myoblasts differentiate into myotubes, their mitochondria must increase OXPHOS capacity and conform to the rather rigid architecture imposed by the contractile machinery. To visualize alterations in the mitochondrial network, we differentiated C2C12s expressing a mitochondrial matrix-directed DsRed and examined them at various time points during differentiation. As seen in Figure 3A, undifferentiated myoblasts exhibited a sparsely-populated filamentous mitochondrial network. As early as 1 d PD, mitochondrial network fragmentation was observed, giving rise to spherical mitochondria that persisted to 3 d PD. This coincided with a brisk upregulation of the mitochondrial fission protein DNM1L at 1 d PD; DNM1L decreased at 3 d PD and was nearly undetectable by 6 d PD (Fig. 3C and D). At 4 d PD, mitochondrial fusion events led to the formation of a filamentous network concurrent with an increase in OPA1 expression (Fig. 3B, C, and D). We next performed transmission electron microscopy on differentiating cells to examine changes in mitochondrial networks (Fig. 4). In undifferentiated myoblasts, mitochondrial populations were sparse and exhibited primarily elongated morphology. At 1 d PD, numerous autophagosomes were observed and mitochondria were predominantly circular. At 3 d and 6 d PD, fewer autophagosomes were observed and mitochondria were more numerous with more instances of elongation. These data illustrate the dynamic remodeling of the mitochondrial network during the transition from myoblast to myotube.Figure 3.


Mitophagy is required for mitochondrial biogenesis and myogenic differentiation of C2C12 myoblasts.

Sin J, Andres AM, Taylor DJ, Weston T, Hiraumi Y, Stotland A, Kim BJ, Huang C, Doran KS, Gottlieb RA - Autophagy (2016)

Electron micrographs of differentiating C2C12s. Transmission electron microscopy was performed on differentiating C2C12s to examine alterations in mitochondrial populations. Insets are presented at higher magnification below each original image. Scale bars: 500 nm. GM, growth medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836019&req=5

f0004: Electron micrographs of differentiating C2C12s. Transmission electron microscopy was performed on differentiating C2C12s to examine alterations in mitochondrial populations. Insets are presented at higher magnification below each original image. Scale bars: 500 nm. GM, growth medium.
Mentions: As myoblasts differentiate into myotubes, their mitochondria must increase OXPHOS capacity and conform to the rather rigid architecture imposed by the contractile machinery. To visualize alterations in the mitochondrial network, we differentiated C2C12s expressing a mitochondrial matrix-directed DsRed and examined them at various time points during differentiation. As seen in Figure 3A, undifferentiated myoblasts exhibited a sparsely-populated filamentous mitochondrial network. As early as 1 d PD, mitochondrial network fragmentation was observed, giving rise to spherical mitochondria that persisted to 3 d PD. This coincided with a brisk upregulation of the mitochondrial fission protein DNM1L at 1 d PD; DNM1L decreased at 3 d PD and was nearly undetectable by 6 d PD (Fig. 3C and D). At 4 d PD, mitochondrial fusion events led to the formation of a filamentous network concurrent with an increase in OPA1 expression (Fig. 3B, C, and D). We next performed transmission electron microscopy on differentiating cells to examine changes in mitochondrial networks (Fig. 4). In undifferentiated myoblasts, mitochondrial populations were sparse and exhibited primarily elongated morphology. At 1 d PD, numerous autophagosomes were observed and mitochondria were predominantly circular. At 3 d and 6 d PD, fewer autophagosomes were observed and mitochondria were more numerous with more instances of elongation. These data illustrate the dynamic remodeling of the mitochondrial network during the transition from myoblast to myotube.Figure 3.

Bottom Line: We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis.Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks.Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation.

View Article: PubMed Central - PubMed

Affiliation: a The Cedars-Sinai Heart Institute and the Barbra Streisand Women's Heart Center Cedars-Sinai Medical Center , Los Angeles , CA , USA.

ABSTRACT
Myogenesis is a crucial process governing skeletal muscle development and homeostasis. Differentiation of primitive myoblasts into mature myotubes requires a metabolic switch to support the increased energetic demand of contractile muscle. Skeletal myoblasts specifically shift from a highly glycolytic state to relying predominantly on oxidative phosphorylation (OXPHOS) upon differentiation. We have found that this phenomenon requires dramatic remodeling of the mitochondrial network involving both mitochondrial clearance and biogenesis. During early myogenic differentiation, autophagy is robustly upregulated and this coincides with DNM1L/DRP1 (dynamin 1-like)-mediated fragmentation and subsequent removal of mitochondria via SQSTM1 (sequestosome 1)-mediated mitophagy. Mitochondria are then repopulated via PPARGC1A/PGC-1α (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha)-mediated biogenesis. Mitochondrial fusion protein OPA1 (optic atrophy 1 [autosomal dominant]) is then briskly upregulated, resulting in the reformation of mitochondrial networks. The final product is a myotube replete with new mitochondria. Respirometry reveals that the constituents of these newly established mitochondrial networks are better primed for OXPHOS and are more tightly coupled than those in myoblasts. Additionally, we have found that suppressing autophagy with various inhibitors during differentiation interferes with myogenic differentiation. Together these data highlight the integral role of autophagy and mitophagy in myogenic differentiation.

No MeSH data available.


Related in: MedlinePlus