The mitochondrial negative regulator MCJ is a therapeutic target for acetaminophen-induced liver injury

Abstract

Acetaminophen (APAP) is the active component of many medications used to treat pain and fever worldwide. Its overuse provokes liver injury and it is the second most common cause of liver failure. Mitochondrial dysfunction contributes to APAP-induced liver injury but the mechanism by which APAP causes hepatocyte toxicity is not completely understood. Therefore, we lack efficient therapeutic strategies to treat this pathology. Here we show that APAP interferes with the formation of mitochondrial respiratory supercomplexes via the mitochondrial negative regulator MCJ, and leads to decreased production of ATP and increased generation of ROS. In vivo treatment with an inhibitor of MCJ expression protects liver from acetaminophen-induced liver injury at a time when N-acetylcysteine, the standard therapy, has no efficacy. We also show elevated levels of MCJ in the liver of patients with acetaminophen overdose. We suggest that MCJ may represent a therapeutic target to prevent and rescue liver injury caused by acetaminophen.

Fig. 1

MCJ expression determines the susceptibility of hepatocytes to APAP toxicity. a, b Cell death in WT and MCJ KO hepatocytes using TUNEL assay (a) and Trypan blue staining (b) after treatment with APAP for 9 and 6 h, respectively. c JNK activation was evaluated by western blotting in WT and MCJ KO hepatocytes after APAP exposure. d, e Cell death in MCJ KO hepatocytes and MCJ KO hepatocytes transfected with MCJ (MCJ KO-MCJ) using TUNEL assay (d) and Trypan blue staining (e) after treatment with APAP for 9 and 6 h, respectively. f, g Cell death in WT hepatocytes and WT hepatocytes transfected with siMCJ (siMCJ) using TUNEL assay (f) and Trypan blue staining (g) after treatment with APAP for 9 and 6 h, respectively. h–j Mitochondrial ROS in WT and MCJ KO hepatocytes (h), MCJ KO hepatocytes, and MCJ KO hepatocytes transfected with MCJ (i), WT hepatocytes, and WT hepatocytes transfected with siMCJ (j) MCJ KO-MCJ upon 6 h of APAP treatment using MitoSOX staining. Values are represented as mean ± SEM. *P < 0.05 (Student’s t test) (MCJ KO vs. WT, MCJ KO-MCJ vs. MCJ KO, and siMCJ vs. WT). Triplicates were used for experimental condition

Fig. 2

MCJ deletion protects against APAP hepatotoxicity in vivo. WT (n = 6) and MCJ KO mice (n = 6) were treated with APAP 360 mg/kg for 48 h. Liver damage was evaluated by a hematoxylin and eosin (H&E) and b TUNEL staining in liver sections. c, d Serum alanine aminotransferase (ALT) levels in WT and MCJ KO fed animals (c) and starved animals at 24 h after APAP (d). e Inflammation assessed by F4/80 staining in liver. f Total GSH levels measured by HPLC-MS in WT and MCJ KO livers after 6 h of APAP treatment (n = 5). g GSSG/GSH levels measured by HPLC-MS in WT and MCJ KO livers. h JNK activation by western blotting in WT and MCJ KO liver extracts after 1 h of APAP treatment (n = 5). i ROS in vivo measured by dihydroethidium (DHE) staining in liver sections. Scale bar corresponds to 100 μm. Values are represented as mean ± SEM. *P < 0.05 (Student’s t test) (MCJ KO vs. WT)

Fig. 3

MCJ liver-specific silencing prevents against APAP-induced liver injury in vivo. a–f WT mice were treated with APAP 360 mg/kg and 6 h after control (Control) (n = 6) or a MCJ-specific siRNA (siMCJ) (n = 6) were intravenously injected. a MCJ silencing was evaluated by western blotting, b liver necrosis was assessed by H&E staining, c ROS in vivo measured by DHE staining in liver sections, d inflammation assessed by F4/80 staining in liver, e relative mRNA expression of different inflammatory genes, f serum ALT levels in Control and siMCJ-treated animals. g–k WT mice were treated with APAP 360 mg/kg and 24 h after control (Control) (n = 6) or a MCJ-specific siRNA (siMCJ) (n = 6) were intravenously injected. g Liver necrosis was assessed by H&E staining, h ROS in vivo measured by DHE staining in liver sections, i inflammation assessed by F4/80 staining in liver, j serum ALT, and k serine aminotransferase (AST) levels in Control and siMCJ-treated animals. Scale bar corresponds to 100 μm. Values are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test) (siMCJ vs. Control)

Fig. 4

Lack of MCJ counteracts APAP toxicity in hepatocytes by maintaining mitochondrial function and ATP levels. Triplicates were used for experimental condition. a Total ATP levels in WT and MCJ KO primary hepatocytes treated with APAP for 9 h. b Oxygen consumption rate (OCR) and c mitochondrial ATP production using the Mitostress assay at basal conditions and in the presence of APAP in WT and MCJ KO primary hepatocytes. d ATP levels in mitochondria isolated from WT and MCJ KO livers after 24 h of APAP administration. e–g WT mice were administered with APAP 360 mg/kg (n = 12), 24 h later mice (n = 4) received siMCJ and 12 h later mice were harvested and livers were used to examine e MCJ silencing by western blotting, f liver necrosis by H&E staining, and g PCNA expression by immunohistochemistry. Scale bar corresponds to 100 μm. Values are represented as mean ± SEM. *P < 0.05, ***P < 0.001 (Student’s t test) (APAP vs. Control, MCJ KO vs. WT, and siMCJ vs. Control)

Fig. 5

Loss of MCJ prevents APAP-induced inhibition of respiratory supercomplexes and ROS generation in liver. a Complex I OCR in WT and MCJ KO liver mitochondria at basal conditions (n = 3). b Complex I OCR in WT and MCJ KO liver mitochondria after APAP treatment (n = 3). c Mitochondrial ROS in WT and MCJ KO hepatocytes treated with APAP (6 h) and Rotenone (complex I inhibitor) (3 h) using MitoSOX dye. BN-PAGE of digitonin-solubilized mitochondrial extracts from WT and MCJ KO hepatocytes d, e livers treated with APAP transferred into a membrane and immunoblotted for NDUFA9 protein. Immunoreactivity within the supercomplex (SC) region and the monomeric complex I (CI) is shown. f Cell death was evaluated using TUNEL in WT and MCJ KO hepatocytes treated with APAP (9 h) and Rotenone (3 h). Values are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test) (MCJ KO vs. WT and APAP + Rotenone vs. APAP). Triplicates were used for experimental condition

Fig. 6

MCJ expression is increased in human DILI. a–c Human samples from healthy control subjects (Healthy) (n = 7) and from patients with drug-induced liver injury (DILI) (n = 21). a MCJ expression in liver was determined by immunohistochemistry and b quantified. c Correlation between MCJ expression and drug grade of mitochondrial liability in DILI patients. d, e Human samples from healthy control subjects (Healthy) (n = 7) and from patients with acetaminophen-induced liver injury (APAP) (n = 24). a MCJ expression in liver was determined by immunohistochemistry and b quantified. Scale bar corresponds to 100 μm. Values are represented as mean ± SEM. *P < 0.05 (Student’s t test) (DILI vs. Healthy, mitochondrial hazardous vs. non-mitochondrial hazardous, and APAP vs. Healthy)