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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

Myogenic differentiation is accompanied by mitophagy and biogenesis. Differentiating C2C12s were analyzed for mitophagy and biogenesis markers. (A) Differentiating C2C12s immunostained for TOMM70A (green) and imaged via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B. Whole lysate blots were normalized to ARHGDIA. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student t test; representative western blot is shown, n=3). (D) Differentiating C2C12s were immunostained for PPARGC1A (red) as well as with Hoechst 33342 nuclear stain (blue) and examined via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (E) Quantification of the percentage of PPARGC1A+ nuclei over total nuclei (100 nuclei counted per condition). GM, growth medium.
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f0007: Myogenic differentiation is accompanied by mitophagy and biogenesis. Differentiating C2C12s were analyzed for mitophagy and biogenesis markers. (A) Differentiating C2C12s immunostained for TOMM70A (green) and imaged via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B. Whole lysate blots were normalized to ARHGDIA. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student t test; representative western blot is shown, n=3). (D) Differentiating C2C12s were immunostained for PPARGC1A (red) as well as with Hoechst 33342 nuclear stain (blue) and examined via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (E) Quantification of the percentage of PPARGC1A+ nuclei over total nuclei (100 nuclei counted per condition). GM, growth medium.

Mentions: Given that mitochondria undergo an orchestrated program of fission and fusion during myogenic differentiation, we next examined differentiating C2C12s for evidence of mitochondrial clearance and repopulation. Immunofluorescence targeting TOMM70A revealed modest mitochondrial staining when cells were in full growth media. When cells were switched to differentiation media, a marked reduction in TOMM70A immunostaining was observed at 1 d PD (Fig. 7A). TOMM70A expression increased by 3 d PD, and by 6 d PD myotubes contained very dense and brightly stained mitochondrial networks. Western blots of cell lysates from differentiating cells recapitulated the depletion of TOMM70A 1 d PD and the gradual buildup of TOMM70A by 6 d PD (Fig. 7B and C). The initial decline of mitochondrial protein coincided with the previously noted spike in autophagy marker MAP1LC3A-II (Fig. 1B and C) as well as a sustained increase in the ATG12–ATG5 complex. Cellular SQSTM1 levels also began to decline at 3 d PD. Interestingly, mitochondrial SQSTM1 recruitment was significantly elevated at 1 d PD and beyond (Fig. 7B, C and Fig. S4). Together, these data show that mitochondria are targeted for degradation immediately after the differentiation program is set into motion, and the involvement of SQSTM1 as well as upregulation of various autophagy markers suggests that this is an autophagy-mediated process.Figure 7.


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)

Myogenic differentiation is accompanied by mitophagy and biogenesis. Differentiating C2C12s were analyzed for mitophagy and biogenesis markers. (A) Differentiating C2C12s immunostained for TOMM70A (green) and imaged via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B. Whole lysate blots were normalized to ARHGDIA. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student t test; representative western blot is shown, n=3). (D) Differentiating C2C12s were immunostained for PPARGC1A (red) as well as with Hoechst 33342 nuclear stain (blue) and examined via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (E) Quantification of the percentage of PPARGC1A+ nuclei over total nuclei (100 nuclei counted per condition). GM, growth medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f0007: Myogenic differentiation is accompanied by mitophagy and biogenesis. Differentiating C2C12s were analyzed for mitophagy and biogenesis markers. (A) Differentiating C2C12s immunostained for TOMM70A (green) and imaged via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Western blot analysis of whole cell lysates from differentiating C2C12s. (C) Quantification of western blots in B. Whole lysate blots were normalized to ARHGDIA. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student t test; representative western blot is shown, n=3). (D) Differentiating C2C12s were immunostained for PPARGC1A (red) as well as with Hoechst 33342 nuclear stain (blue) and examined via fluorescence microscopy. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (E) Quantification of the percentage of PPARGC1A+ nuclei over total nuclei (100 nuclei counted per condition). GM, growth medium.
Mentions: Given that mitochondria undergo an orchestrated program of fission and fusion during myogenic differentiation, we next examined differentiating C2C12s for evidence of mitochondrial clearance and repopulation. Immunofluorescence targeting TOMM70A revealed modest mitochondrial staining when cells were in full growth media. When cells were switched to differentiation media, a marked reduction in TOMM70A immunostaining was observed at 1 d PD (Fig. 7A). TOMM70A expression increased by 3 d PD, and by 6 d PD myotubes contained very dense and brightly stained mitochondrial networks. Western blots of cell lysates from differentiating cells recapitulated the depletion of TOMM70A 1 d PD and the gradual buildup of TOMM70A by 6 d PD (Fig. 7B and C). The initial decline of mitochondrial protein coincided with the previously noted spike in autophagy marker MAP1LC3A-II (Fig. 1B and C) as well as a sustained increase in the ATG12–ATG5 complex. Cellular SQSTM1 levels also began to decline at 3 d PD. Interestingly, mitochondrial SQSTM1 recruitment was significantly elevated at 1 d PD and beyond (Fig. 7B, C and Fig. S4). Together, these data show that mitochondria are targeted for degradation immediately after the differentiation program is set into motion, and the involvement of SQSTM1 as well as upregulation of various autophagy markers suggests that this is an autophagy-mediated process.Figure 7.

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