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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

Assessment of mitochondrial turnover via MitoTimer. Mitochondrial turnover was examined in differentiating C2C12s expressing MitoTimer. (A) Fluorescent images of MitoTimer-expressing C2C12s throughout differentiation. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Red/green ratios based on images from A. Three fields were analyzed per time point. (C) PE/FITC ratios of differentiating MitoTimer-expressing C2C12s as measured by fluorescence-activated cell sorting. 50,000 events were analyzed per time point. GM, growth medium.
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f0008: Assessment of mitochondrial turnover via MitoTimer. Mitochondrial turnover was examined in differentiating C2C12s expressing MitoTimer. (A) Fluorescent images of MitoTimer-expressing C2C12s throughout differentiation. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Red/green ratios based on images from A. Three fields were analyzed per time point. (C) PE/FITC ratios of differentiating MitoTimer-expressing C2C12s as measured by fluorescence-activated cell sorting. 50,000 events were analyzed per time point. GM, growth medium.

Mentions: To measure mitochondrial turnover dynamics, we utilized MitoTimer-expressing C2C12s (MitoTimer-C2C12s) and examined them throughout the course of differentiation. These cells constitutively express a mitochondria-targeted fluorescent timer protein, which slowly transitions from green to red fluorescence over 48 h.14 Therefore, a decrease in red over green signal indicates mitochondrial biogenesis. Figure 8A shows that mitochondria began mostly green in normal growth media. Upon the introduction of differentiation media, mitochondria progressively shifted to red fluorescence until 3 d PD at which point green signal intensity began to increase again, suggesting that mitochondria were actively importing newly synthesized protein, a characteristic of mitochondrial biogenesis. Ratiometric quantification of red and green fluorescence intensity based on microscopy (Fig. 8B) and fluorescence-activated cell-sorting analysis (Fig. 8C) both revealed that the red/green ratio peaked at 3 d PD and then declined as new (green) MitoTimer protein was incorporated, reflecting mitochondrial biogenesis in the later stages of differentiation.Figure 8.


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)

Assessment of mitochondrial turnover via MitoTimer. Mitochondrial turnover was examined in differentiating C2C12s expressing MitoTimer. (A) Fluorescent images of MitoTimer-expressing C2C12s throughout differentiation. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Red/green ratios based on images from A. Three fields were analyzed per time point. (C) PE/FITC ratios of differentiating MitoTimer-expressing C2C12s as measured by fluorescence-activated cell sorting. 50,000 events were analyzed per time point. GM, growth medium.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f0008: Assessment of mitochondrial turnover via MitoTimer. Mitochondrial turnover was examined in differentiating C2C12s expressing MitoTimer. (A) Fluorescent images of MitoTimer-expressing C2C12s throughout differentiation. All images were collected under identical illumination and exposure parameters. Scale bars: 20 µm. (B) Red/green ratios based on images from A. Three fields were analyzed per time point. (C) PE/FITC ratios of differentiating MitoTimer-expressing C2C12s as measured by fluorescence-activated cell sorting. 50,000 events were analyzed per time point. GM, growth medium.
Mentions: To measure mitochondrial turnover dynamics, we utilized MitoTimer-expressing C2C12s (MitoTimer-C2C12s) and examined them throughout the course of differentiation. These cells constitutively express a mitochondria-targeted fluorescent timer protein, which slowly transitions from green to red fluorescence over 48 h.14 Therefore, a decrease in red over green signal indicates mitochondrial biogenesis. Figure 8A shows that mitochondria began mostly green in normal growth media. Upon the introduction of differentiation media, mitochondria progressively shifted to red fluorescence until 3 d PD at which point green signal intensity began to increase again, suggesting that mitochondria were actively importing newly synthesized protein, a characteristic of mitochondrial biogenesis. Ratiometric quantification of red and green fluorescence intensity based on microscopy (Fig. 8B) and fluorescence-activated cell-sorting analysis (Fig. 8C) both revealed that the red/green ratio peaked at 3 d PD and then declined as new (green) MitoTimer protein was incorporated, reflecting mitochondrial biogenesis in the later stages of differentiation.Figure 8.

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