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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

Autophagy is upregulated during myogenic differentiation of C2C12 myoblasts. C2C12 skeletal myoblasts were differentiated for 6 d in low serum differentiation media. (A) Phase contrast microscopy of differentiating C2C12s in growth medium (GM) or 1 d, 3 d, or 6 d PD. Scale bars: 100 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B normalized to ARHGDIA (**, P < 0.01; *****, P < 0.00001; Student t test; representative western blot is shown, n=3).
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f0001: Autophagy is upregulated during myogenic differentiation of C2C12 myoblasts. C2C12 skeletal myoblasts were differentiated for 6 d in low serum differentiation media. (A) Phase contrast microscopy of differentiating C2C12s in growth medium (GM) or 1 d, 3 d, or 6 d PD. Scale bars: 100 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B normalized to ARHGDIA (**, P < 0.01; *****, P < 0.00001; Student t test; representative western blot is shown, n=3).

Mentions: During the course of differentiation, cellular components adapted to support the quiescent stem cell must be remodeled or replaced with elements better suited to support the new functions of the differentiated cell. To investigate whether autophagy is involved in this process of cellular deconstruction, we utilized C2C12 mouse skeletal myoblasts and examined the expression of autophagy markers during differentiation. The C2C12 cell line was originally isolated from the thigh muscle of female C3H mice after crush injury. In full-serum growth media, these cells rapidly proliferate and maintain an undifferentiated myogenic progenitor state; however, when exposed to low-serum differentiation media they undergo growth arrest, differentiating and fusing into mature multi-nucleated myotubes.12,13 As shown in Figure 1A, C2C12 myoblasts grew as small spindle-shaped individual cells when cultured in growth media. As early as 3 d postdifferentiation (3 d PD), many of the cells had become phase-bright fused myotubes. By 6 d PD, the majority of the cells were myotubes that were elongated due to continued fusion. Western blotting revealed that at 6 d PD, the myogenic differentiation marker ACTA1/α-actin was strongly upregulated (Fig. 1B and C). To monitor the activation of autophagy, cell lysates were also probed for MAP1LC3A/LC3A (microtubule-associated protein 1 light chain 3 alpha). In western blots, MAP1LC3A appears as a double band, the top band representing the inactive cytosolic MAP1LC3A-I and the bottom band representing the lipidated active MAP1LC3A-II which is incorporated into the phagophore membrane. An elevation of MAP1LC3A-II and punctate cytoplasmic MAP1LC3A immunostaining are often used as markers of autophagy; an increase in the presence of lysosomal blockade indicates active autophagic flux. Interestingly, MAP1LC3A-II was upregulated early after the introduction of differentiation medium, but was downregulated later in differentiation (Fig. 1B and C). MAP1LC3A immunostaining on differentiating C2C12s recapitulated this result as can be seen by the large number of MAP1LC3A puncta in cells immediately following differentiation and gradual clearance by 6 d PD (Fig. S1). The addition of BAF resulted in the accumulation of MAP1LC3A puncta, indicating intact flux. This phenomenon was recapitulated in transmission electron micrographs (Figs. 4 and 6). These data show that autophagy is a transient phenomenon that is robustly upregulated only during the early steps of differentiation.Figure 1.


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)

Autophagy is upregulated during myogenic differentiation of C2C12 myoblasts. C2C12 skeletal myoblasts were differentiated for 6 d in low serum differentiation media. (A) Phase contrast microscopy of differentiating C2C12s in growth medium (GM) or 1 d, 3 d, or 6 d PD. Scale bars: 100 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B normalized to ARHGDIA (**, P < 0.01; *****, P < 0.00001; Student t test; representative western blot is shown, n=3).
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Related In: Results  -  Collection

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f0001: Autophagy is upregulated during myogenic differentiation of C2C12 myoblasts. C2C12 skeletal myoblasts were differentiated for 6 d in low serum differentiation media. (A) Phase contrast microscopy of differentiating C2C12s in growth medium (GM) or 1 d, 3 d, or 6 d PD. Scale bars: 100 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B normalized to ARHGDIA (**, P < 0.01; *****, P < 0.00001; Student t test; representative western blot is shown, n=3).
Mentions: During the course of differentiation, cellular components adapted to support the quiescent stem cell must be remodeled or replaced with elements better suited to support the new functions of the differentiated cell. To investigate whether autophagy is involved in this process of cellular deconstruction, we utilized C2C12 mouse skeletal myoblasts and examined the expression of autophagy markers during differentiation. The C2C12 cell line was originally isolated from the thigh muscle of female C3H mice after crush injury. In full-serum growth media, these cells rapidly proliferate and maintain an undifferentiated myogenic progenitor state; however, when exposed to low-serum differentiation media they undergo growth arrest, differentiating and fusing into mature multi-nucleated myotubes.12,13 As shown in Figure 1A, C2C12 myoblasts grew as small spindle-shaped individual cells when cultured in growth media. As early as 3 d postdifferentiation (3 d PD), many of the cells had become phase-bright fused myotubes. By 6 d PD, the majority of the cells were myotubes that were elongated due to continued fusion. Western blotting revealed that at 6 d PD, the myogenic differentiation marker ACTA1/α-actin was strongly upregulated (Fig. 1B and C). To monitor the activation of autophagy, cell lysates were also probed for MAP1LC3A/LC3A (microtubule-associated protein 1 light chain 3 alpha). In western blots, MAP1LC3A appears as a double band, the top band representing the inactive cytosolic MAP1LC3A-I and the bottom band representing the lipidated active MAP1LC3A-II which is incorporated into the phagophore membrane. An elevation of MAP1LC3A-II and punctate cytoplasmic MAP1LC3A immunostaining are often used as markers of autophagy; an increase in the presence of lysosomal blockade indicates active autophagic flux. Interestingly, MAP1LC3A-II was upregulated early after the introduction of differentiation medium, but was downregulated later in differentiation (Fig. 1B and C). MAP1LC3A immunostaining on differentiating C2C12s recapitulated this result as can be seen by the large number of MAP1LC3A puncta in cells immediately following differentiation and gradual clearance by 6 d PD (Fig. S1). The addition of BAF resulted in the accumulation of MAP1LC3A puncta, indicating intact flux. This phenomenon was recapitulated in transmission electron micrographs (Figs. 4 and 6). These data show that autophagy is a transient phenomenon that is robustly upregulated only during the early steps of differentiation.Figure 1.

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