1. Genetics and Genomics

Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell

  1. Eran Mick
  2. Denis V Titov
  3. Owen S Skinner
  4. Rohit Sharma
  5. Alexis A Jourdain
  6. Vamsi K Mootha  Is a corresponding author
  1. Howard Hughes Medical Institute and Department of Molecular Biology, Massachusetts General Hospital, United States
  2. Broad Institute, United States
  3. Department of Systems Biology, Harvard Medical School, United States
https://doi.org/10.7554/eLife.49178
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Cite this article
  1. Eran Mick
  2. Denis V Titov
  3. Owen S Skinner
  4. Rohit Sharma
  5. Alexis A Jourdain
  6. Vamsi K Mootha
(2020)
Distinct mitochondrial defects trigger the integrated stress response depending on the metabolic state of the cell
eLife 9:e49178.
https://doi.org/10.7554/eLife.49178
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  3. Download .RIS
5 figures, 1 table and 3 additional files

Figures

Figure 1
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Interrogating the genomic response to mitochondrial dysfunction in proliferating and differentiated muscle cells.

(A) Phase contrast images of proliferating C2C12 myoblasts (left), post-mitotic cells cultured in low-serum media for 24 hr following confluence (middle), and differentiated myotubes (right). Scale …

Figure 2 with 1 supplement
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Mechanistically distinct mitochondrial inhibitors trigger the ISR and depress proliferative gene expression in myoblasts.

(A) PCA of gene expression levels derived from RNA-seq in myoblasts treated with inhibitors for 10 hr. N = 3 (same samples as in Figure 1D). Pier, piericidin; Anti, antimycin; Oligo, oligomycin. …

Figure 2—source data 1

Gene set enrichment analysis.

https://cdn.elifesciences.org/articles/49178/elife-49178-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
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Additional data on transcriptional and protein markers of the ISR following mitochondrial inhibitor treatments in myoblasts.

(A) Heatmap of mean expression fold-changes derived from RNA-seq for upregulated ATF4/DDIT3 target genes most associated with PC1 (jackstraw p-value<0.0001). Atf4 was also included. Data was min-max …

Figure 3 with 1 supplement
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Oxidizing cytosolic NADH/NAD+ is sufficient to ablate ISR activation by complex I inhibition in myoblasts.

(A) Schematic of localization and activity of LbNOX and mitoLbNOX. IMS, intermembrane space. (B) Schematic of localization and activity of NDI1. (C) NADH/NAD+ in extracts of cells expressing …

Figure 3—figure supplement 1
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Additional data on bioenergetics and metabolism in myoblast cell lines.

(A) Oxygen consumption rate (OCR) of control, LbNOX, mitoLbNOX or NDI1 cells sequentially treated with piericidin and antimycin. Mean ± SD, N = 4–5. (B) Peak intensities of glycerol 3-phosphate and …

Figure 4 with 1 supplement
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Complex I inhibition in myoblasts triggers the ISR through the kinase GCN2 due to an aspartate and asparagine deficiency.

(A) Secreted [lactate] following 2 hr piericidin treatment in control (Ctrl), LbNOX (Lb) or NDI1 cells. Data is normalized to DMSO (-) separately for each cell line. Mean ± SD, N = 6 from three …

Figure 4—source data 1

Metabolite profiling data.

https://cdn.elifesciences.org/articles/49178/elife-49178-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
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Additional data on metabolic consequences that trigger the ISR in myoblasts.

(A) Secreted [lactate] following 2 hr inhibitor treatments in control or LbNOX cells. Data is normalized to DMSO separately in each cell line. Mean ± SD, N = 6 from three experiments (expanded …

Figure 5 with 1 supplement
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ATP synthase inhibition triggers the ISR in myotubes in a manner related to inner-membrane hyperpolarization.

(A) PCA of gene expression levels derived from RNA-seq in myotubes treated with inhibitors for 10 hr. N = 3 (same samples as in Figure 1D). Detailed results are provided in Supplementary file 1. (B) …

Figure 5—figure supplement 1
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Additional data on ISR activation in myotubes.

(A) qPCR of Ddit3 following 10 hr inhibitor treatments in C2C12 myoblasts, post-mitotic cells and myotubes. Data is normalized to DMSO separately in each panel. Mean ± SD, N = 3 (myoblast samples …

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or
reference		IdentifiersAdditional
information
Cell line (Mus musculus)C2C12ATCCCRL-1772
Cell line (M. musculus)Ndufa9-knockout C2C12PMID:27622560
Cell line (M. musculus)Primary mouse embryonic fibroblasts (MEF)LonzaM-FB-481
Cell line (Homo sapiens)Primary human skeletal myoblasts (HSkM)Thermo Fisher ScientificA11440
Recombinant DNA reagentDoxycycline-inducible luciferase (pLVX-TRE3G-Luc)Takara Bio (formerly Clontech)Sold as part of 631187
Recombinant DNA reagentDoxycycline-inducible LbNOXPMID:27124460Available without inducible system on Addgene (#75285)
Recombinant DNA reagentDoxycycline-inducible mitoLbNOXPMID:27124460Available without inducible system on Addgene (#74448)
Recombinant DNA reagentDoxycycline-inducible Saccharomyces cerevisiae NDI1PMID:27124460
OtherHuman adenovirus type5 expressing eGFPVector Biolabs1060-HT
OtherHuman adenovirus type5 expressing eGFP and LbNOXVector Biolabs
(PMID:27124460)
OtherHuman adenovirus type5 expressing eGFP and mitoLbNOXVector Biolabs
(PMID:27124460)
OtherDulbecco’s Modified Eagle’s Medium (DMEM), 4 mM L-Glutamine, no glucose, no sodium pyruvateThermo Fisher Scientific11966–025
OtherDialyzed fetal bovine serum (dFBS)Thermo Fisher Scientific26400–044
OtherDialyzed horse serumValley BiomedicalAS3053-DI
OtherHorse serum, New Zealand originThermo Fisher Scientific16050–130
Chemical compound, drugD-(+)-Glucose solutionSigmaG8769Working concentration 10 mM
Chemical compound, drugDoxycycline hyclateSigmaD9891300 ng/ml
Chemical compound, drugPiericidin ASanta CruzSc-2022870.5 μM
Chemical compound, drugAntimycin ASigmaA86740.5 μM
Chemical compound, drugOligomycin ASigma753511 μM
Chemical compound, drugBAM15TimTecST0563880.5 μM (or as indicated)
Chemical compound, drugChloramphenicolSigmaC037820 μg/ml
Chemical compound, drugTunicamycinSigmaT77651 μg/ml
Chemical compound, drugL-Aspartic acidSigmaA925610 mM
Chemical compound, drugL-Asparagine monohydrateSigmaA83810.5 mM
Chemical compound, drugSodium pyruvate solutionThermo Fisher Scientific11360–0701 mM
Chemical compound, drugUridineSigmaU3003200 μM
Chemical compound, drugGCN2iBAcme Bioscience
(PMID:30061420)		0.5 μM
Chemical compound, drugGSK2656157Cayman Chemical173720.25 μM
Chemical compound, drugSodium DL-Lactate-3,3,3-d3CDN IsotopesD6556
Chemical compound, drugSodium pyruvate-13C3Sigma490717
Chemical compound, drugAdenosine-15N5 5′-MonophosphateSigma900382
Chemical compound, drugAdenosine-13C5 5’-DiphosphateToronto Research ChemicalsA281697
Chemical compound, drugAdenosine-13C10 5′-TriphosphateSigma710695
Chemical compound, drugL-Aspartic acid-1,4-13C2Cambridge Isotope LaboratoriesCLM-4455
Chemical compound, drugFormic acid, LC/MS gradeThermo Fisher ScientificA117-50
Chemical compound, drugAmmonium hydrogen carbonate, LC/MS gradeSigma5.33005
Commercial assay or kitTaqMan gene expression master mixThermo Fisher Scientific4369514
Commercial assay or kitUbr3 mouse TaqMan probeThermo Fisher ScientificMm01328979_m1
Commercial assay or kitAtf3 mouse TaqMan probeThermo Fisher ScientificMm00476033_m1
Commercial assay or kitDdit3 mouse TaqMan probeThermo Fisher ScientificMm01135937_g1
Commercial assay or kitAtf4 mouse TaqMan probeThermo Fisher ScientificMm00515325_g1
Commercial assay or kitGdf15 mouse TaqMan probeThermo Fisher ScientificMm00442228_m1
Commercial assay or kitPpp1r15a mouse TaqMan probeThermo Fisher ScientificMm01205601_g1
Commercial assay or kitTrp53inp1 mouse TaqMan probeThermo Fisher ScientificMm00458142_g1
Commercial assay or kitTBP human TaqMan probeThermo Fisher ScientificHs00427620_m1
Commercial assay or kitGDF15 human TaqMan probeThermo Fisher ScientificHs00171132_m1
AntibodyGCN2 (rabbit, polyclonal)Cell Signaling Technology33021:500 dilution
AntibodyPhospho-GCN2 (Thr899) (rabbit, monoclonal)Abcamab758361:500 dilution
AntibodyATF4 (rabbit, monoclonal)Cell Signaling Technology118151:500 dilution
AntibodyeIF2α (mouse, monoclonal)Cell Signaling Technology21031:500 dilution
AntibodyPhospho-eIF2α (Ser51) (rabbit, monoclonal)Cell Signaling Technology35971:500 dilution
AntibodyActin (mouse, monoclonal)SigmaA47001:3000 dilution

Additional files

Supplementary file 1

Analysis of RNA sequencing data (counts, PCA, differential expression).

Related to Figure 2A, D-F, Figure 2—figure supplement 1A, Figure 3F, Figure 5A,I, Figure 5—figure supplement 1D.

https://cdn.elifesciences.org/articles/49178/elife-49178-supp1-v1.xlsx
Supplementary file 2

iRegulon cis-regulatory analysis.

Related to Figure 2B and Figure 3F.

https://cdn.elifesciences.org/articles/49178/elife-49178-supp2-v1.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/49178/elife-49178-transrepform-v1.docx

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