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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

Mitochondrial remodeling is impaired when autophagic flux is blocked. BAF-treated cells were examined to determine if autophagic flux was required for mitochondrial remodeling. (A) C2C12s were pretreated with BAF or vehicle and then stained for TOMM70A (green) and with Hoechst 33342 (blue) and imaged via fluorescence microscopy. Insets are presented at higher magnification below each original image. Exposure times were individually adjusted to bring out detail. Scale bars: 20 µm. (B) Cells in A were analyzed using Image J to measure mitochondrial pixel area/perimeter ratios of individual cells as a measure of network interconnectivity. (***, P<.001; *****, P<.00001; Student t test, n=10) (C) Western blot analysis of whole cell lysates from BAF-treated C2C12s. (D) Quantification of western blots in C. GM, growth medium.
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f0005: Mitochondrial remodeling is impaired when autophagic flux is blocked. BAF-treated cells were examined to determine if autophagic flux was required for mitochondrial remodeling. (A) C2C12s were pretreated with BAF or vehicle and then stained for TOMM70A (green) and with Hoechst 33342 (blue) and imaged via fluorescence microscopy. Insets are presented at higher magnification below each original image. Exposure times were individually adjusted to bring out detail. Scale bars: 20 µm. (B) Cells in A were analyzed using Image J to measure mitochondrial pixel area/perimeter ratios of individual cells as a measure of network interconnectivity. (***, P<.001; *****, P<.00001; Student t test, n=10) (C) Western blot analysis of whole cell lysates from BAF-treated C2C12s. (D) Quantification of western blots in C. GM, growth medium.

Mentions: Earlier, we observed that autophagy was activated during early differentiation accompanied by a transient elevation of DNM1L and fragmentation of the mitochondrial network. To assess the effect of autophagy on mitochondrial remodeling during myogenic differentiation, we pretreated undifferentiated C2C12s with BAF or equivalent volume of vehicle prior to differentiation. Cells were then stained for outer mitochondrial membrane marker TOMM70A/TOM70 (translocase of outer mitochondrial membrane 70 homolog A [S. cerevisiae]) to visualize the mitochondrial network. Vehicle-treated cells underwent the previously observed network remodeling, fragmenting soon after exposure to differentiation media and then fusing into dense filamentous networks as differentiation progressed (Fig. 5A and B). Cells treated with BAF also underwent mitochondrial fragmentation early during differentiation, but failed to reconstitute their networks even by 6 d PD. This aberrant mitochondrial remodeling was accompanied by the expected early upregulation of DNM1L; however the elevated level of DNM1L persisted out to 6 d PD (Fig. 5C and D). The upregulation of OPA1 was also blocked by BAF, consistent with the failure to reconstitute the mitochondrial network. Western blot detection of DNM1L and OPA1 on isolated mitochondria also showed these same trends (Fig. S3). Electron micrographs showed that early in differentiation, cells treated with BAF lacked elongated mitochondria throughout the course of differentiation and also contained a large amount of autophagosomes as is expected from a blockade in autophagic flux (Fig. 6). An abundance of autophagosomes remained until 6 d PD, while instances of elongated mitochondria were rare when compared to non-BAF controls (Fig. 4). While blockade of autophagy prevented differentiation, it did not increase cell death (Fig. S5). The inhibition of differentiation by BAF shows that while the initiation of autophagy is accompanied by fragmentation of mitochondrial networks, intact autophagic flux during early differentiation appears to be required for network reconstruction and myotube formation. Figure 5.


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)

Mitochondrial remodeling is impaired when autophagic flux is blocked. BAF-treated cells were examined to determine if autophagic flux was required for mitochondrial remodeling. (A) C2C12s were pretreated with BAF or vehicle and then stained for TOMM70A (green) and with Hoechst 33342 (blue) and imaged via fluorescence microscopy. Insets are presented at higher magnification below each original image. Exposure times were individually adjusted to bring out detail. Scale bars: 20 µm. (B) Cells in A were analyzed using Image J to measure mitochondrial pixel area/perimeter ratios of individual cells as a measure of network interconnectivity. (***, P<.001; *****, P<.00001; Student t test, n=10) (C) Western blot analysis of whole cell lysates from BAF-treated C2C12s. (D) Quantification of western blots in C. GM, growth medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f0005: Mitochondrial remodeling is impaired when autophagic flux is blocked. BAF-treated cells were examined to determine if autophagic flux was required for mitochondrial remodeling. (A) C2C12s were pretreated with BAF or vehicle and then stained for TOMM70A (green) and with Hoechst 33342 (blue) and imaged via fluorescence microscopy. Insets are presented at higher magnification below each original image. Exposure times were individually adjusted to bring out detail. Scale bars: 20 µm. (B) Cells in A were analyzed using Image J to measure mitochondrial pixel area/perimeter ratios of individual cells as a measure of network interconnectivity. (***, P<.001; *****, P<.00001; Student t test, n=10) (C) Western blot analysis of whole cell lysates from BAF-treated C2C12s. (D) Quantification of western blots in C. GM, growth medium.
Mentions: Earlier, we observed that autophagy was activated during early differentiation accompanied by a transient elevation of DNM1L and fragmentation of the mitochondrial network. To assess the effect of autophagy on mitochondrial remodeling during myogenic differentiation, we pretreated undifferentiated C2C12s with BAF or equivalent volume of vehicle prior to differentiation. Cells were then stained for outer mitochondrial membrane marker TOMM70A/TOM70 (translocase of outer mitochondrial membrane 70 homolog A [S. cerevisiae]) to visualize the mitochondrial network. Vehicle-treated cells underwent the previously observed network remodeling, fragmenting soon after exposure to differentiation media and then fusing into dense filamentous networks as differentiation progressed (Fig. 5A and B). Cells treated with BAF also underwent mitochondrial fragmentation early during differentiation, but failed to reconstitute their networks even by 6 d PD. This aberrant mitochondrial remodeling was accompanied by the expected early upregulation of DNM1L; however the elevated level of DNM1L persisted out to 6 d PD (Fig. 5C and D). The upregulation of OPA1 was also blocked by BAF, consistent with the failure to reconstitute the mitochondrial network. Western blot detection of DNM1L and OPA1 on isolated mitochondria also showed these same trends (Fig. S3). Electron micrographs showed that early in differentiation, cells treated with BAF lacked elongated mitochondria throughout the course of differentiation and also contained a large amount of autophagosomes as is expected from a blockade in autophagic flux (Fig. 6). An abundance of autophagosomes remained until 6 d PD, while instances of elongated mitochondria were rare when compared to non-BAF controls (Fig. 4). While blockade of autophagy prevented differentiation, it did not increase cell death (Fig. S5). The inhibition of differentiation by BAF shows that while the initiation of autophagy is accompanied by fragmentation of mitochondrial networks, intact autophagic flux during early differentiation appears to be required for network reconstruction and myotube formation. Figure 5.

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