Down-regulation of the mitochondrial matrix peptidase ClpP in muscle cells causes mitochondrial dysfunction and decreases cell proliferation

Abstract

The caseinolytic peptidase P (ClpP) is the endopeptidase component of the mitochondrial matrix ATP-dependent ClpXP protease. ClpP degrades unfolded proteins to maintain mitochondrial protein homeostasis and is involved in the initiation of the mitochondrial unfolded protein response (UPR(mt)). Outside of an integral role in the UPR(mt), the cellular function of ClpP is not well characterized in mammalian cells. To investigate the role of ClpP in mitochondrial function, we generated C2C12 muscle cells that are deficient in ClpP using siRNA or stable knockdown using lentiviral transduction. Reduction of ClpP levels by ~70% in C2C12 muscle cells resulted in a number of mitochondrial alterations including reduced mitochondrial respiration and reduced oxygen consumption rate in response to electron transport chain (ETC) complex I and II substrates. The reduction in ClpP altered mitochondrial morphology, changed the expression level of mitochondrial fission protein Drp1 and blunted UPR(mt) induction. In addition, ClpP deficient cells showed increased generation of reactive oxygen species (ROS) and decreased membrane potential. At the cellular level, reduction of ClpP impaired myoblast differentiation, cell proliferation and elevated phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) suggesting an inhibition of translation. Our study is the first to define the effects of ClpP deficiency on mitochondrial function in muscle cells in vitro. In addition, we have uncovered novel effects of ClpP on mitochondrial morphology, cell proliferation and protein translation pathways in muscle cells.

Keywords: ClpP; ClpX; Mitochondrial fission/fusion; Mitochondrial unfolded protein response; Reactive oxygen species; Respiration.

Fig. 1

Decrease in ClpP levels inhibits Hsp60 induction in response to DOX. (A) Immunoblots showing the expression level of ClpP, ClpX, Lon and HSp60 in control and ClpP KD cells. β-Tubulin was used as a loading control. (B) Quantification of ClpP, ClpX, Lon and HSp60 to β-tubulin is represented graphically. White and gray bars represent control and ClpP KD cells, respectively. (C) Control and ClpP KD cells were treated with DMSO (DOX-) or DOX (DOX+) (15 mg/ml) for 48 h and changes in the expression level of ClpP and Hsp60 were assessed by western blotting. Quantification of Hsp60 to β-tubulin is represented graphically on the right panel. Error bars represent±SEM from three independent experiments, ***p<0.001, **p<0.01.

Fig. 2

A decrease in ClpP level alters mitochondrial morphology. (A) Electron micrographs of control and ClpP KD cells (magnification 25,000x). White arrows indicate mitochondria. (B) Quantification of mitochondrial area (left panel) and distribution of mitochondria based on size (right panel) in control (open bars) and ClpP KD cells (gray bars) using Image J software (left panel). (C) Tom20 immunofluorescence images of control and ClpP KD cells by confocal microscopy. Scale bar=10 μM. (D) Immunoblots showing expression level of Mfn1, Mfn2, OPA1, Fis1, Drp1 and ClpP in control and ClpP KD cells (left panel). β-Tubulin was used as a loading control. Quantification of protein levels normalized to β-Tubulin is shown in the right panel. All the experiments were repeated three times and error bar in graphs represent±SEM from three independent experiments, ***p<0.001.

Fig. 3

Mitochondrial respiration is decreased and glycolysis is increased in ClpP KD cells. (A) Cellular bioenergetics in control and ClpP KD cells were measured using the Seahorse Bioscience XF24 Extracellular Flux Analyzer mitostress assay. Control (open bars) and ClpP KD cells (gray bars) are represented graphically. (B) Glycolysis in control and ClpP KD cells were measured using the Seahorse Bioscience XF24 Extracellular Flux Analyzer glycolysis stress assay. Control (open bars) and ClpP KD cells (gray bars) are represented graphically. OCR obtained from each well is normalized with total protein concentration in the well. Error bar in graphs represent±SEM from three independent experiments. ***p<0.001, **p<0.01, *p<0.05.

Fig. 4

ETC complex activities are lower and ROS generation is higher in ClpP KD cells. (A) Activities of complexes I and II were measured using the Seahorse Bioscience XF24 Extracellular Flux Analyzer. Glutamate/malate (complex I substrate) or succinate (complex II substrate) were injected at a concentration 10 mM and changes in OCR was measured in control (open bars) and ClpP KD (gray bars). (B) Immunoblots of complex I (20 kDa subunit), complex I (30 kDa subunit), complex IV-4, complex II (70 kDa subunit), complex III subunit, complex IV-4, complex IV-1 and complex V (56.5 kDa subunit) and β-tubulin (loading control) in control and ClpP KD cells (left panel). Quantification of protein expression to β-tubulin is shown in the right panel. (C) H₂O₂ production in control (open bars) and ClpP KD (gray bars) cells with complex I-linked substrates (glutamate/malate) and complex II-linked substrate (succinate+rotenone). (D) Aconitase activity represented in percentage in control and ClpP KD cells. Error bars represent±SEM obtained from three independent experiments. ***p<0.001, **p<0.01, *p<0.05.

Fig. 5

Reduction in mitochondrial membrane potential in ClpP KD cells. (A) Staining of mitochondria in control (left) and ClpP KD (right) cells using mitotracker. Magnification (20x). (B) Mitochondrial membrane potential measured using JC-1 in control (open bar) and ClpP KD (gray bar) cells (20,000 cells). **p<0.01. The experiments were repeated at least three times and the data represents±SEM obtained from three independent experiments. **p<0.01.

Fig. 6

A decrease in ClpP alters cell morphology, reduces cell proliferation and attenuates protein translation. (A) β-actin immunostaining of control (left) and ClpP KD (right) cells. Magnification 20x. (B) Cell number of control and ClpP KD cells on days 1, 2 and 3 after seeding equal number of cells. (C) Immunoblots of MHC, myogenin, ClpP, PGC1-α and β-tubulin in control and ClpP KD cells at differentiation days 1–4 (left panel). Quantification of proteins to β-tubulin is shown in the right panel. (D) Immunoblots showing expression level of P-eIF2α (Ser51) and eIF2α in control and ClpP KD cells. Quantification of P-eIF2α/eIF2α is shown in the right panel. (D) Immunoblots showing expression level of GCN2, Bip and PKR in control and ClpP KD cells. β-tubulin was used a loading control. Quantification of GCN2, Bip and PKR to β-tubulin is shown in the right panel. The experiments were repeated three times and the data represents±SEM obtained from the experiments. ***p<0.001, **p<0.01, *p<0.05.

Fig. 7

A decrease in ClpP attenuates protein translation. (A) Immunoblots showing expression level of P-eIF2α (Ser51) and eIF2α in control and ClpP KD cells. Quantification of P-eIF2α/eIF2α is shown in the right panel. (B) Immunoblots showing expression level of GCN2, Bip and PKR in control and ClpP KD cells. β-Tubulin was used a loading control. Quantification of GCN2, Bip and PKR to β-tubulin is shown in the right panel. The data represents±SEM from three independent experiments. *p<0.05.