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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

Blocking autophagy prevents myogenic differentiation. C2C12 cells were pretreated with autophagy-inhibiting agents and were subsequently differentiated. (A, C, and E) Phase contrast microscopy of differentiating C2C12s pretreated with either siRNA targeting Atg5 (A), BAF (C), or siRNA targeting Sqstm1 prior to differentiation (E). Scale bars: 100 µm. (B, D, and F) Western blot analysis of whole cell lysates from siAtg5 (B), BAF (D), or siSqstm1 (F)-treated cells. GM, growth medium.
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f0002: Blocking autophagy prevents myogenic differentiation. C2C12 cells were pretreated with autophagy-inhibiting agents and were subsequently differentiated. (A, C, and E) Phase contrast microscopy of differentiating C2C12s pretreated with either siRNA targeting Atg5 (A), BAF (C), or siRNA targeting Sqstm1 prior to differentiation (E). Scale bars: 100 µm. (B, D, and F) Western blot analysis of whole cell lysates from siAtg5 (B), BAF (D), or siSqstm1 (F)-treated cells. GM, growth medium.

Mentions: To determine if autophagy is crucial for myogenic differentiation, we pretreated myoblasts with autophagy inhibitors targeting various stages of the process. These inhibitors were well-tolerated, and did not substantially increase cell death (Fig. S5). Phase contrast imaging showed that C2C12s treated with siRNA targeting Atg5, BAF, or siRNA targeting Sqstm1 (Fig. 2A, C, and E, respectively) did not develop myotube morphology but rather maintained a primitive fibroblast-like shape throughout the differentiation time course. Western blots revealed that the myotube marker ACTA1 was robustly expressed at 6 d PD by cells in differentiation media with vehicle only, but this was either delayed or completely inhibited by treatment with autophagy inhibitors (Fig. 2B, D, and F). Similar effects were seen when cells were treated with 3-methyladenine (3-MA) (Fig. S2). These data illustrate that disruption of autophagy, whether at the initiation, cargo trafficking, or lysosomal fusion steps, impairs myogenic differentiation.Figure 2.


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)

Blocking autophagy prevents myogenic differentiation. C2C12 cells were pretreated with autophagy-inhibiting agents and were subsequently differentiated. (A, C, and E) Phase contrast microscopy of differentiating C2C12s pretreated with either siRNA targeting Atg5 (A), BAF (C), or siRNA targeting Sqstm1 prior to differentiation (E). Scale bars: 100 µm. (B, D, and F) Western blot analysis of whole cell lysates from siAtg5 (B), BAF (D), or siSqstm1 (F)-treated cells. GM, growth medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0002: Blocking autophagy prevents myogenic differentiation. C2C12 cells were pretreated with autophagy-inhibiting agents and were subsequently differentiated. (A, C, and E) Phase contrast microscopy of differentiating C2C12s pretreated with either siRNA targeting Atg5 (A), BAF (C), or siRNA targeting Sqstm1 prior to differentiation (E). Scale bars: 100 µm. (B, D, and F) Western blot analysis of whole cell lysates from siAtg5 (B), BAF (D), or siSqstm1 (F)-treated cells. GM, growth medium.
Mentions: To determine if autophagy is crucial for myogenic differentiation, we pretreated myoblasts with autophagy inhibitors targeting various stages of the process. These inhibitors were well-tolerated, and did not substantially increase cell death (Fig. S5). Phase contrast imaging showed that C2C12s treated with siRNA targeting Atg5, BAF, or siRNA targeting Sqstm1 (Fig. 2A, C, and E, respectively) did not develop myotube morphology but rather maintained a primitive fibroblast-like shape throughout the differentiation time course. Western blots revealed that the myotube marker ACTA1 was robustly expressed at 6 d PD by cells in differentiation media with vehicle only, but this was either delayed or completely inhibited by treatment with autophagy inhibitors (Fig. 2B, D, and F). Similar effects were seen when cells were treated with 3-methyladenine (3-MA) (Fig. S2). These data illustrate that disruption of autophagy, whether at the initiation, cargo trafficking, or lysosomal fusion steps, impairs myogenic differentiation.Figure 2.

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