Mitophagy regulates mitochondrial network signaling, oxidative stress, and apoptosis during myoblast differentiation

Abstract

Macroautophagy/autophagy is a degradative process essential for various cellular processes. We previously demonstrated that autophagy-deficiency causes myoblast apoptosis and impairs myotube formation. In this study, we continued this work with particular emphasis on mitochondrial remodelling and stress/apoptotic signaling. We found increased (p < 0.05) autophagic (e.g., altered LC3B levels, increased ATG7, decreased SQSTM1) and mitophagic (e.g., BNIP3 upregulation, mitochondrial localized GFP-LC3 puncta, and elevated mitochondrial LC3B-II) signaling during myoblast differentiation. shRNA-mediated knockdown of ATG7 (shAtg7) decreased these autophagic and mitophagic responses, while increasing CASP3 activity and ANXA5/annexin V staining in differentiating myoblasts; ultimately resulting in dramatically impaired myogenesis. Further confirming the importance of mitophagy in these responses, CRISPR-Cas9-mediated knockout of Bnip3 (bnip3^-/-) resulted in increased CASP3 activity and DNA fragmentation as well as impaired myoblast differentiation. In addition, shAtg7 myoblasts displayed greater endoplasmic reticulum (e.g., increased CAPN activity and HSPA) and mitochondrial (e.g., mPTP formation, reduced mitochondrial membrane potential, elevated mitochondrial 4-HNE) stress. shAtg7 and bnip3^-/- myoblasts also displayed altered mitochondria-associated signaling (e.g., PPARGC1A, DNM1L, OPA1) and protein content (e.g., SLC25A4, VDAC1, CYCS). Moreover, shAtg7 myoblasts displayed CYCS and AIFM1 release from mitochondria, and CASP9 activation. Similarly, bnip3^-/- myoblasts had significantly higher CASP9 activation during differentiation. Importantly, administration of a chemical inhibitor of CASP9 (Ac-LEHD-CHO) or dominant-negative CASP9 (ad-DNCASP9) partially recovered differentiation and myogenesis in shAtg7 myoblasts. Together, these data demonstrate an essential role for autophagy in protecting myoblasts from mitochondrial oxidative stress and apoptotic signaling during differentiation, as well as in the regulation of mitochondrial network remodelling and myogenesis. Abbreviations: 3MA: 3-methyladenine; 4-HNE: 4-hydroxynonenal; ACT: actin; AIFM1/AIF: apoptosis-inducing factor, mitochondrion-associated 1; ANXA5: annexin V; ATG7: autophagy related 7; AU: arbitrary units; BAX: BCL2-associated X protein; BCL2: B cell leukemia/lymphoma 2; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; CAPN: calpain; CASP: caspase; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CASP12: caspase 12; CAT: catalase; CQ: chloroquine; CYCS: cytochrome c, somatic; DCF; 2',7'-dichlorofluorescein; DNM1L/DRP1: dynamin 1-like; DM: differentiation media; DMEM: Dulbecco's modified Eagle's medium; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GM: growth media; p-H2AFX: phosphorylated H2A histone family, member X; H2BFM: H2B histone family, member M; HBSS: Hanks balanced salt solution; HSPA/HSP70: heat shock protein family A; JC-1: tetraethylbenzimidazolylcarbocyanine iodide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mPTP: mitochondrial permeability transition pore; MYH: myosin heavy chain; MYOG: myogenin; OPA1: OPA1, mitochondrial dynamin like GTPase; PI: propidium iodide; PINK1: PTEN induced putative kinase 1; PPARGC1A/PGC1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; ROS: reactive oxygen species; SLC25A4/ANT1: solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 4; SOD1: superoxide dismutase 1, soluble; SOD2: superoxide dismutase 2, mitochondrial; SQSTM1/p62: sequestosome 1; VDAC1: voltage-dependent anion channel 1.

Keywords: Apoptosis; autophagy; caspase 9; differentiation; mitochondria; mitophagy; myogenesis; oxidative stress; skeletal muscle.

Figure 1.

Autophagy is upregulated and required during myoblast differentiation. Representative immunoblots (a) and quantitative analysis (b, c) of MAP1LC3B (LC3B-I, LC3B-II, as well as calculated LC3B-II:I ratio) ATG7, BECN1, and BNIP3 during myoblast differentiation. Representative immunoblots (d) and quantitative analysis (e, f) of LC3B-I, LC3B-II, and SQSTM1 in CTRL (Vehicle) and chloroquine (CQ) treated myoblasts during differentiation. Also shown are representative ACT, GAPDH, and ponceau stained loading control blots/membranes. Representative immunoblots (g) of ATG7, LC3B-I, and LC3B-II in SCR and shAtg7 myoblasts and myotubes. Representative immunoblot (h) and quantitative analysis (i) of MYOG in SCR and shAtg7 cells during differentiation. Also shown is a representative ACT loading control blot. Representative images (j) of myotube formation in SCR and shAtg7 cells during differentiation. Cells were stained with DAPI (blue) and MF20 (red) to visualize nuclei and MYH, respectively. Scale bar = 100 µm. Quantitative analysis of the differentiation index (k) and fusion index (l) in SCR and shAtg7 cells during differentiation. *p < 0.05 compared to D0 (within group). ^†p < 0.05 between groups at the same time point.

Figure 2.

Autophagy-deficiency during myoblast differentiation augments apoptotic signaling, ER-stress responses, and cell death. Quantitative analysis of early apoptotic (ANXA5⁺ PI⁻) cells (a), late apoptotic (ANXA5⁺ PI⁺) cells (b), and CASP3 activity (c) in SCR and shAtg7 cells during differentiation. Representative immunoblots (d) and quantitative analysis (e, f, g, h) of BAX, BCL2, and HSPA (as well as calculated BAX:BCL2 ratio) in SCR and shAtg7 cells during differentiation. Quantitative analysis of CAPN activity (i) in SCR and shAtg7 cells during differentiation. *p < 0.05 compared to D0 (within group). ^†p < 0.05 between groups at the same time point.

Figure 3.

Autophagy-deficiency during myoblast differentiation alters cellular and mitochondrial redox status. Representative immunoblots (a) and quantitative analysis of SOD1 (b), CAT (c), and SOD2 (d) in SCR and shAtg7 cells during differentiation. Quantitative analysis of ROS generation (e) in SCR and shAtg7 myoblasts during differentiation. Representative immunoblot (f) and quantitative analysis (g) of mitochondrial 4-HNE content in SCR and shAtg7 cells during differentiation. *p < 0.05 compared to D0 (within group). ^†p < 0.05 between groups at the same time point.

Figure 4.

Mitophagy is upregulated and required during myoblast differentiation. Representative images (a) and quantitative analysis (b) of colocalization (yellow) of LC3 (green) and mitochondria (red) in ad-GFP-LC3 and DsRed-Mito co-transfected SCR and shAtg7 cells during differentiation. Scale bar = 10 µm. Inset shows merged, DsRed-Mito, and GFP-LC3 zoomed images of SCR cells at D1 (SCR-D1). Arrows demonstrate colocalized (yellow) mitochondria (red) and LC3 puncta (green). Representative immunoblots (c) of H2BFM, SOD2, and SOD1 demonstrating nuclear- (Nuc), mitochondria- (Mito), and cytosolic-enriched (Cyto) subcellular fractions. Representative immunoblot (d) and quantitative analysis (e) of LC3B-II in mitochondria-enriched subcellular fractions of SCR and shAtg7 cells during differentiation. Quantitative analysis (f) of the JC-1 red:green fluorescence ratio (where a decrease is indicative of membrane depolarization) in SCR and shAtg7 cells during differentiation. Representative immunoblots (g, h) and quantitative analysis of BNIP3 (i), MYOG (j), and MYH (k) in Scram and bnip3^-/- cells during differentiation. Also shown are representative ACT loading control blots. *p < 0.05 compared to D0 (within group). ^†p < 0.05 between groups at the same time point.

Figure 5.

shAtg7 and bnip3^-/- myoblasts display altered mitochondrial network-related signaling and content. Representative immunoblots (a) and quantitative analysis (b, c, d, e, f, g) of PPARGC1A, DNM1L, OPA1, VDAC1, SLC25A4, and CYCS in SCR and shAtg7 cells during differentiation. Representative immunoblots (h) and quantitative analysis (i, j, k, l, m, n, o) of PPARGC1A, DNM1L, OPA1, VDAC1, SLC25A4, CYCS, and SOD2 in Scram and bnip3^-/- cells during differentiation. Also shown are representative GAPDH and ACT loading control blots. *p < 0.05 compared to D0 (within group). ^†p < 0.05 between groups at the same time point.

Figure 6.

Mitochondria-mediated apoptotic signaling is augmented in shAtg7 and bnip3^-/- myoblasts during differentiation. Quantitative analysis (a) of calcein fluorescence in SCR and shAtg7 cells during differentiation, where a decrease in fluorescence is indicative of an increase in mPTP formation. Representative immunoblots (b) and quantitative analysis (c) of cytosolic CYCS and AIFM1 in SCR and shAtg7 cells during differentiation. Also shown is a representative ponceau stained membrane. Quantitative analysis of CASP9 activity (d) in SCR and shAtg7 cells during differentiation. Representative immunoblot (e) and quantitative analysis (f) of p-H2AFX in Scram and bnip3^-/- cells during differentiation. Also shown is a representative ACT loading control blot. Quantitative analysis of CASP3 (g) and CASP9 (h) activity in Scram and bnip3^-/- cells during differentiation. *p < 0.05 compared to D0 (within group). ^†p < 0.05 between groups at the same time point.

Figure 7.

Attenuation of CASP activity in autophagy-deficient cells partially recovers myoblast differentiation and myogenesis. Quantitative analysis of CASP3 activity (a), representative images of myotube formation (b), representative immunoblot and quantitative analysis of MYH (c, d), as well as quantitative analysis of the differentiation index (e) and the fusion index (f) in SCR, shAtg7, Ac-LEHD-CHO-treated shAtg7, and Ac-DEVD-CHO-treated shAtg7 cells during differentiation. Also shown is a representative ACT control blot. Scale bar = 100 µm. Representative immunoblots (g) and quantitative analysis (h) of MYOG and MYH in shAtg7 cells transfected with a control adenovirus (ad-GFP) or adenovirus expressing dominant-negative CASP9 (ad-DNCASP9) during differentiation. Also shown are representative GAPDH and ponceau control blots/membranes. *p < 0.05 compared to D0 (within group). ^#p < 0.05 compared to all groups at the same time point. ^$p < 0.05 compared to shAtg7 at the same time point. ^†p < 0.05 between groups at the same time point.