Methanobactin reverses acute liver failure in a rat model of Wilson disease

Abstract

In Wilson disease (WD), functional loss of ATPase copper-transporting β (ATP7B) impairs biliary copper excretion, leading to excessive copper accumulation in the liver and fulminant hepatitis. Current US Food and Drug Administration- and European Medicines Agency-approved pharmacological treatments usually fail to restore copper homeostasis in patients with WD who have progressed to acute liver failure, leaving liver transplantation as the only viable treatment option. Here, we investigated the therapeutic utility of methanobactin (MB), a peptide produced by Methylosinus trichosporium OB3b, which has an exceptionally high affinity for copper. We demonstrated that ATP7B-deficient rats recapitulate WD-associated phenotypes, including hepatic copper accumulation, liver damage, and mitochondrial impairment. Short-term treatment of these rats with MB efficiently reversed mitochondrial impairment and liver damage in the acute stages of liver copper accumulation compared with that seen in untreated ATP7B-deficient rats. This beneficial effect was associated with depletion of copper from hepatocyte mitochondria. Moreover, MB treatment prevented hepatocyte death, subsequent liver failure, and death in the rodent model. These results suggest that MB has potential as a therapeutic agent for the treatment of acute WD.

Figure 1. Liver disease in the LPP rat mirrors acute liver failure in WD patients by a devastating mitochondrial copper overload.

(A) H&E staining of liver damage in diseased Atp7b^–/– rats and WD patients with acute liver failure who were either untreated or D-PA treated. Tissue necrosis with resorptive inflammation as well as repair (fibrosis) were detectable (black arrowhead); proliferation of bile ducts (circle), anisokaryosis (black arrow), and several inflammatory infiltrations (white arrow) are marked. Insert shows apoptosis (white asterisk) and nodules with ballooned hepatocytes (black asterisk). Scale bars: 100 μm. (B) Mitochondrial structure impairments in diseased Atp7b^–/– rats (top panels) and in WD patients with acute liver failure who were either untreated (middle panels) or D-PA treated (bottom panels). Vacuolization (asterisks) and cristae dilations (arrows), marked differences in electron densities, and separated inner and outer membranes (arrowheads) could be identified. Bottom panels: Some areas had relatively intact mitochondria (top), and others demonstrated severe structural impairments (bottom). Scale bars: 500 nm. (C) Comparable copper burden in whole-liver homogenates and purified liver mitochondria from Atp7b^–/– rats and untreated WD patients with acute liver failure (patients 1 and 2). Lower total copper content was detected in tissue homogenates and isolated mitochondria from the livers of WD patients who underwent D-PA treatment. Control Atp7b^+/– rats (n = 15); affected Atp7b^–/– rats with markedly elevated copper levels but AST levels below 200 U/l and bilirubin levels below 0.5 mg/dl (n = 13); disease-onset Atp7b^–/– rats with AST levels above 200 U/l, bilirubin levels below 0.5 mg/dl (n = 8); diseased Atp7b^–/– rats with AST levels above 200 U/l and bilirubin levels above 0.5 mg/dl (n = 9). Data were outlier corrected. One-way ANOVA with Tukey’s multiple comparisons test. ****P < 0.0001 versus control; ^####P < 0.0001 versus affected; ^††††P < 0.0001 versus disease onset.

Figure 2. Increasing copper load severely attacks mitochondrial membrane integrity.

(A) Progressive disease in Atp7b^–/– rats was paralleled by a decrease in normally structured mitochondria (Type 1 and Type 2) and an increase in structurally altered mitochondria (Type 3 and Type 4). Scale bars: 500 nm. Control Atp7b^+/– rats (aged 82–89 days): N = 3, n = 561; affected Atp7b^–/– rats (aged 92–93 days): N = 3, n = 372; disease-onset Atp7b^–/– rats (aged 83–93 days): N = 3, n = 575; diseased Atp7b^–/– rats (aged 104–107 days): N = 5, n = 857. (B) Upon calcium- or copper-induced MPT, isolated Atp7b^+/– mitochondria underwent large-amplitude swelling, which was significantly reduced in Atp7b^–/– mitochondria from diseased and disease-onset rats. Control Atp7b^+/– rats: N = 3, n = 6; affected Atp7b^–/– rats: N = 3, n = 6; disease-onset Atp7b^–/– rats: N = 2, n = 4; diseased Atp7b^–/– rats: N = 3, n = 6. (C) Calcium-induced (100 μM) MPT could be efficiently inhibited by Cys-A (5 μM). This blocking effect was severely impaired in mitochondria from diseased and disease-onset Atp7b^–/– rats. A representative comparative MPT measurement of mitochondria isolated from an Atp7b^+/– control rat and from Atp7b^–/– animals at different disease stages is depicted (quantitative analysis is shown in Supplemental Table 2). (D) Atp7b^–/– mitochondria lost their membrane potential (ΔΨ) at earlier time points than did control mitochondria. A representative comparative ΔΨ measurement of mitochondria isolated from an Atp7b^+/– control and Atp7b^–/– animals at different disease stages is depicted (quantitative analysis is shown in Supplemental Table 3). Data in A and B were outlier corrected. P values were determined by 1-way ANOVA with Tukey’s multiple comparisons test. (A) *P < 0.05, **P < 0.01, and ***P < 0.001 versus control. (B) **P < 0.01 and ****P < 0.0001 versus control; ^#P < 0.05, ^##P < 0.01, and ^####P < 0.0001 versus affected; ^††P < 0.01 versus disease onset. N = number of rats, n = number of analyzed mitochondria. FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone.

Figure 3. MB depletes copper from liver mitochondria, hepatocytes, and whole liver.

(A) MB, but not D-PA or TETA, extracted copper from Atp7b^–/– mitochondria (2 mM each, 30-minute incubation, N = 3). (B) MB was less toxic to the mitochondrial respiratory complex IV than TTM (MB: N = 3, n = 9; TTM: N = 1, n = 3). (C) Copper-preloaded HepG2 (N = 3) and WD patient–derived HLCs (1 of 2 preparations) were de-coppered by MB. (–), untreated control; (+), 24-hour treatment with 300–500 μM MB. (D) Dose-dependent MB uptake into HepG2 cells at 2 and 24 hours (N = 3). (E) Toxicity of MB versus TTM in HepG2 (neutral red assay: N = 3, n = 9). CCCP, positive control. (F) MB-treated (500 μM) HepG2 cells showed only intermediate phases of ΔΨ loss (6 hours, 250 μM CCCP). Nuclei, blue; mitochondria with ΔΨ, orange-red; mitochondria without ΔΨ, green. Arrows indicate cells with low ΔΨ (N = 2). Scale bars: 50 μm. (G) Cumulative copper excretion into bile following a 2-hour Atp7b^–/– liver perfusion. Only MB (0.7 μM) forced high copper amounts into bile compared with TTM (0.8 μM), D-PA (2.2 μM), and TETA (1.8 μM). N = 3. Note the different scales for MB (right, blue axis) and buffer control, D-PA, TETA, and TTM (left, black axis). (H) All chelators except TTM transported copper to the perfusate (conditions as in G). N = 2. (I) Only MB markedly reduced liver copper levels during Atp7b^–/– liver perfusion, but not D-PA, TETA, or TTM (conditions as in G). N = 3. One-way ANOVA with Tukey’s multiple comparisons test (A, B, D, E, and I); unpaired t test with Welch’s correction (C). *P < 0.05 and ****P < 0.0001 versus control (A–C and E); ^####P < 0.0001 versus respective MB concentration (B and E). **P < 0.01 versus 0.05 mM MB; ^#P < 0.05 versus 0.1 mM MB; and ^†P < 0.05 versus 0.3 mM MB (D). Co, buffer-treated control.

Figure 4. Acute liver failure is efficiently avoided by a short-term in vivo treatment with MB.

(A) Overt liver damage was reduced in MB-treated but not D-PA– or TETA-treated Atp7b^–/– livers. Scale bars: 100 μm; H&E staining (symbols as in Figure 1 and Supplemental Figure 1). Daily doses were: 150 mg (130 μmol) MB/kg BW; 100 mg (540 μmol) D-PA/kg BW; and 480 mg (2,190 μmol) TETA/kg BW. Three-day MB treatment: 2 experiments; 5-day MB treatment: 5 experiments; D-PA and TETA treatments: 4 experiments. (B) AST values decreased in 2 of 3 and 4 of 6 Atp7b^–/– rats treated for 3 or 5 days with MB, respectively, but not in untreated Atp7b^–/– (N = 6 [3 affected and 3 diseased]) or short-term D-PA– or TETA-treated rats (N = 6 [3 affected and 3 diseased]). Treatment started in rats at 82–90 days of age. (C) Mild reduction of whole-liver and significant reduction of mitochondrial copper level in short-term MB-treated rats (3-day MB treatment, N = 3 [2 affected and 1 disease onset], aged 88–89 days; 5-day MB treatment, N = 5 [5 affected], aged 89–95 days) but not in untreated rats (N = 4 [2 affected, 2 diseased], aged 90–91 days) or D-PA– or TETA-treated rats (N = 4 [3 affected, 1 disease onset], aged 86–89 days). One-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05 versus untreated controls. (D) Massively reduced numbers in mitochondria with severely impaired structure (type 4, arrows) were isolated from MB-treated rats but not from untreated (Figure 2A) or D-PA– or TETA-treated Atp7b^–/– rats (quantification in Supplemental Figure 4A). Scale bars: 1 μm. (E) Treatment of Atp7b^+/– control rats with MB (i.p. once daily on 2 consecutive days; N = 4) did not change whole-liver or mitochondrial copper levels. Untreated control, N = 3. (F) Upon i.p. injection, MB was only detectable in the serum for half an hour, indicating a very short systemic residence time (n = 2). U, untreated.

Figure 5. Short-term MB treatments postpone acute liver failure.

(A) Short-term (5 days) MB-treated Atp7b^–/– rats (animal age at start of treatment: 84–85 days) remained healthy for at least 2 weeks, after which serum AST and bilirubin (not shown) levels rose again. At the time of analysis, 1 animal (Atp7b^–/– rat 1) was still healthy, and 2 animals (Atp7b^–/– rats 2 and 3) were diseased. (B–D) In the order of Atp7b^–/– rats 1 to 3, respectively, mitochondrial copper content was elevated but not the whole-liver copper content (B), the frequency of the typical histological features of overt liver damage increased (C, symbols as in Figure 1 and Supplemental Figure 1), and the severity of mitochondrial structure impairments increased (D, arrows). Scale bars: 100 μm (C) and 500 nm (D).