Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt
- PMID: 27154400
- PMCID: PMC4931557
- DOI: 10.15252/embr.201642077
Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt
Abstract
The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPR(mt)) in Caenorhabditis elegans Far less is known about mammalian UPR(mt) signaling, although similar roles were assumed for central players, including CLPP To better understand the mammalian UPR(mt) signaling, we deleted CLPP in hearts of DARS2-deficient animals that show robust induction of UPR(mt) due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPR(mt) in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPR(mt) signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases.
Keywords: CLPP; DARS2; cardiomyopathy; mitochondrial translation; mitochondrial unfolded protein response.
© 2016 The Authors.
Figures
![Figure EV1](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g003.gif)
- A
Breeding scheme to generate WT, ClpP KO, Dars2 KO, and DKO animals. Note that ClpP KO and Dars2 KO animals are heterozygous for Dars2 and ClpP, respectively. Allele nomenclature: wild type (+), floxed (L), and transgene (T).
- B–D
Heart weight (B), body weight (C), and heart‐to‐body weight ratio (D) of Dars2 KO mice that are homozygous for the ClpP allele (n = 10–14). Bars represent mean ± SEM (Student's t‐test, ****P < 0.0001).
![Figure EV2](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g005.gif)
- A, B
Western blot analysis of mitochondrial chaperones and proteases and the autophagy marker p62 in hearts from 6‐week‐old mice: (A) Dars2 KO with heterozygous vs. homozygous ClpP background and (B) ClpP KO with heterozygous vs. homozygous Dars2 background. HSC70 and Ponceau S staining serve as loading controls.
- C
Western blot analysis of mitochondrial chaperones, proteases, and maintenance markers in isolated heart mitochondria.
![Figure 1](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g002.gif)
- A
Kaplan–Meier survival curves (n = 12–16).
- B
Heart gross morphology.
- C–E
(C) Body weight, (D) heart weight, and (E) heart‐to‐body weight ratio (n = 28–40).
- F
Relative expression levels of cardiac hypertrophy markers (Nppa and Nppb) (n = 5).
- G
Enzyme histochemical double staining for COX and SDH activities (n = 4).
- H
Assessment of cardiac fibrosis by Masson's trichrome staining (n = 4). White scale bars, 100 μm; black scale bars, 1 mm.
![Figure 2](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g004.gif)
- A, B
(A) Western blot analysis and (B) relative quantification of UPRmt markers in heart extracts. HSC70 is used as a loading control (CTRL) (n = 3).
- C, D
Relative expression levels normalized to WT of (C) established UPRmt markers and (D) mitochondrial chaperones, proteases, and TFAM in 3‐ to 4‐week‐old mice (n = 3–4).
- E
Western blot analysis of cellular stress markers. HSC70 is used as a loading control (CTRL) (n = 3).
- F
Relative expression levels of transcription factors involved in UPRmt and ISR (left) and Fgf21 (right) (n = 5).
![Figure EV3](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g007.gif)
- A, B
Relative expression levels normalized to WT of (A) established UPRmt markers and (B) mitochondrial chaperones, proteases, and TFAM in 6‐week‐old mice (n = 6).
- C, D
Relative expression levels normalized to WT of (C) established UPRmt markers and (D) mitochondrial chaperones, proteases, and TFAM upon control or SPG7 knockdown in both wild‐type and ClpP KO HEK293T cells (n = 6).
- E
Relative quantification of cellular stress markers. HSC70 is used as a loading control (CTRL) (see also Fig 2E).
![Figure 3](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g006.gif)
- A, B
(A) Western blot analysis of individual OXPHOS subunits in heart extracts. HSC70 and calnexin are used as loading controls (n = 3). (B) BN‐PAGE and subsequent Western blot analysis for OXPHOS complexes. Antibodies against individual OXPHOS subunits (on the right) were used to detect OXPHOS complexes (on the left).
- C, D
In‐gel activity of complexes I (C) and IV (D) performed after BN‐PAGE.
- E
Oxygen consumption rates in intact heart mitochondria in the presence of pyruvate–glutamate–malate (C I) and pyruvate–glutamate–malate + succinate (C I + C II) as substrates. State 3 (substrates + ADP), State 4 (+oligomycin), uncoupled (+FCCP). (n = 3). Bars represent mean ± SEM (Student's t‐test, *P < 0.05, **P < 0.01, ***P < 0.001).
![Figure EV4](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g009.gif)
- A
Western blot analysis of individual OXPHOS subunits in isolated mitochondria. Antibodies against individual OXPHOS subunits are depicted on the left. SDHA and VDAC serve as loading controls (n = 3).
- B, C
BN‐PAGE and subsequent Western blot analysis of OXPHOS complexes in isolated mitochondria: (B) Dars2 KO with Clpp +/+ vs. Clpp +/− vs. Clpp −/− background and (C) ClpP KO with Dars2 +/+ vs. Dars2 +/− background. Antibodies against individual OXPHOS subunits (on the left) were used to detect OXPHOS complexes (on the right).
![Figure 4](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g008.gif)
Representative gel of the in organello translation analysis of heart mitochondria. De novo synthetized proteins are isolated after labeling with 35S‐Met (1‐h pulse) or after cold chase (3‐h chase). Positions of individual proteins are indicated on the left. Position of full‐length proteins (proficient) and low molecular weight polypeptides (abortive) is indicated on the right. Note that in WT and ClpP KO, low molecular weight polypeptides include ATP8/ND4L proteins.
Relative overall protein synthesis and turnover rate.
Western blot analysis of DARS2 levels. HSC70 is used as a loading control (CTRL).
Relative levels of the individual de novo synthesized OXPHOS subunits in DKO mitochondria normalized to the corresponding Dars2 KO polypeptide level.
Relative levels of protein synthesis of proficient and abortive polypeptides.
Quantification of the turnover rate of full‐length (proficient) de novo synthesized proteins.
![Figure EV5](https://cdn.statically.io/img/www.ncbi.nlm.nih.gov/pmc/articles/instance/4931557/bin/EMBR-17-0953-g010.gif)
- A, B
Mitochondrial translation rate assessed by in organello 35S‐Met pulse labeling for 10, 30, and 60 min in isolated heart mitochondria of WT and ClpP KO. Autoradiograph and representative part of Coomassie brilliant blue‐stained gel (A) and corresponding relative de novo synthesis rate curves (B) (n = 3).
- C
Representative part of Coomassie brilliant blue‐stained gel used as loading control for Fig 4A.
- D
Representative densitometric analysis de novo synthesized proteins in WT, ClpP KO, Dars2 KO, and DKO heart mitochondria after labeling as in Fig 4A.
- E
Relative expression levels of mtDNA‐encoded OXPHOS subunits in ClpP KO, Dars2 KO, and DKO hearts normalized to WT (n = 5).
- F
Western blot analysis of turnover rate of individual OXPHOS subunits (on the left) of OXPHOS complexes (on the right). WT and ClpP KO mouse embryonic fibroblasts are grown in the presence of cycloheximide (CHX), and proteins are isolated after the indicated time points.
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References
-
- Haynes CM, Petrova K, Benedetti C, Yang Y, Ron D (2007) ClpP mediates activation of a mitochondrial unfolded protein response in C. elegans . Dev Cell 13: 467–480 - PubMed
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