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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

Myotubes are populated with more tightly coupled mitochondria fortified with increased OXPHOS machinery. Undifferentiated C2C12 myoblasts and differentiated myotubes 6 d PD were examined for qualitative differences in mitochondria. (A) OCRs of isolated mitochondria from myoblasts (Blast Mito) or myotubes (Tube Mito). Palmitoyl carnitine was used as the substrate. Average coupled ratios are presented below. (B) OCRs of intact myoblasts or myotubes using palmitate as the substrate. (C) Western blot analysis detecting OXPHOS complex proteins in mitochondria isolated from myoblasts or myotubes. (D) Quantification of MT-CO1 western blot (*, P < 0.05; Student t test; representative western blot is shown, n=3). GM, growth medium.
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f0009: Myotubes are populated with more tightly coupled mitochondria fortified with increased OXPHOS machinery. Undifferentiated C2C12 myoblasts and differentiated myotubes 6 d PD were examined for qualitative differences in mitochondria. (A) OCRs of isolated mitochondria from myoblasts (Blast Mito) or myotubes (Tube Mito). Palmitoyl carnitine was used as the substrate. Average coupled ratios are presented below. (B) OCRs of intact myoblasts or myotubes using palmitate as the substrate. (C) Western blot analysis detecting OXPHOS complex proteins in mitochondria isolated from myoblasts or myotubes. (D) Quantification of MT-CO1 western blot (*, P < 0.05; Student t test; representative western blot is shown, n=3). GM, growth medium.

Mentions: We and others have observed that myotubes contain a larger population of mitochondria than do undifferentiated myoblasts.7,9 Therefore we wished to determine whether the individual newly-formed mitochondria that reside within these mature, differentiated cells are qualitatively different from those found in primitive, undifferentiated myoblasts. To investigate this, mitochondria were isolated from undifferentiated C2C12 myoblasts and differentiated C2C12 myotubes (6 d PD) and studied using a Seahorse extracellular flux analyzer. Fatty acid oxidation analysis showed that myotube mitochondria had higher state III oxygen consumption rates (OCRs) compared to myoblast mitochondria (Fig. 9A). The addition of the ATP synthase inhibitor oligomycin revealed that the respiratory control ratio was almost 3-fold higher in mitochondria isolated from myotubes compared to those isolated from myoblasts, showing that myotube mitochondria were more tightly coupled. The addition of the uncoupler FCCP showed an enhanced reserve respiratory capacity in myotube mitochondria as well. Interestingly, examining palmitate oxidation in intact cells revealed that myotubes had an elevated basal OCR when exposed to palmitate but myoblasts appeared to be completely unresponsive to fatty acid relative to BSA controls (Fig. 9B). The introduction of oligomycin abolished palmitate-induced OCR elevation in myotubes, bringing down respiration rates close to the levels of BSA controls. Oligomycin also dramatically reduced respiration of myoblasts that were incubated in either BSA or palmitate. These data suggest that myotubes were primarily reliant on fatty acid oxidation, whereas myoblasts utilized glucose oxidation. Western blot analysis also showed that mitochondria isolated from differentiating C2C12s had a higher content of OXPHOS components by 3 d PD (Fig. 9C, D and Fig. S4). In all, these data show that myotubes not only contain more mitochondria, but these mitochondria are qualitatively different, better adapted to supply the energetic requirements of a differentiated muscle cell. Figure 9.


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)

Myotubes are populated with more tightly coupled mitochondria fortified with increased OXPHOS machinery. Undifferentiated C2C12 myoblasts and differentiated myotubes 6 d PD were examined for qualitative differences in mitochondria. (A) OCRs of isolated mitochondria from myoblasts (Blast Mito) or myotubes (Tube Mito). Palmitoyl carnitine was used as the substrate. Average coupled ratios are presented below. (B) OCRs of intact myoblasts or myotubes using palmitate as the substrate. (C) Western blot analysis detecting OXPHOS complex proteins in mitochondria isolated from myoblasts or myotubes. (D) Quantification of MT-CO1 western blot (*, P < 0.05; Student t test; representative western blot is shown, n=3). GM, growth medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0009: Myotubes are populated with more tightly coupled mitochondria fortified with increased OXPHOS machinery. Undifferentiated C2C12 myoblasts and differentiated myotubes 6 d PD were examined for qualitative differences in mitochondria. (A) OCRs of isolated mitochondria from myoblasts (Blast Mito) or myotubes (Tube Mito). Palmitoyl carnitine was used as the substrate. Average coupled ratios are presented below. (B) OCRs of intact myoblasts or myotubes using palmitate as the substrate. (C) Western blot analysis detecting OXPHOS complex proteins in mitochondria isolated from myoblasts or myotubes. (D) Quantification of MT-CO1 western blot (*, P < 0.05; Student t test; representative western blot is shown, n=3). GM, growth medium.
Mentions: We and others have observed that myotubes contain a larger population of mitochondria than do undifferentiated myoblasts.7,9 Therefore we wished to determine whether the individual newly-formed mitochondria that reside within these mature, differentiated cells are qualitatively different from those found in primitive, undifferentiated myoblasts. To investigate this, mitochondria were isolated from undifferentiated C2C12 myoblasts and differentiated C2C12 myotubes (6 d PD) and studied using a Seahorse extracellular flux analyzer. Fatty acid oxidation analysis showed that myotube mitochondria had higher state III oxygen consumption rates (OCRs) compared to myoblast mitochondria (Fig. 9A). The addition of the ATP synthase inhibitor oligomycin revealed that the respiratory control ratio was almost 3-fold higher in mitochondria isolated from myotubes compared to those isolated from myoblasts, showing that myotube mitochondria were more tightly coupled. The addition of the uncoupler FCCP showed an enhanced reserve respiratory capacity in myotube mitochondria as well. Interestingly, examining palmitate oxidation in intact cells revealed that myotubes had an elevated basal OCR when exposed to palmitate but myoblasts appeared to be completely unresponsive to fatty acid relative to BSA controls (Fig. 9B). The introduction of oligomycin abolished palmitate-induced OCR elevation in myotubes, bringing down respiration rates close to the levels of BSA controls. Oligomycin also dramatically reduced respiration of myoblasts that were incubated in either BSA or palmitate. These data suggest that myotubes were primarily reliant on fatty acid oxidation, whereas myoblasts utilized glucose oxidation. Western blot analysis also showed that mitochondria isolated from differentiating C2C12s had a higher content of OXPHOS components by 3 d PD (Fig. 9C, D and Fig. S4). In all, these data show that myotubes not only contain more mitochondria, but these mitochondria are qualitatively different, better adapted to supply the energetic requirements of a differentiated muscle cell. Figure 9.

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