INTRODUCTION
Chronic hepatitis B (CHB) affects ~257 million persons worldwide, with a risk of advanced fibrosis (AF), cirrhosis, and HCC. In CHB, patients with AF or cirrhosis (Metavir score, F3‐F4) should undergo surveillance because they are at risk of liver decompensation and HCC when compared to patients with no/mild‐moderate fibrosis (F0‐F2).1–5
Mitochondria play important roles in inflammation, innate immune, and antiviral responses, and both processes require healthy mitochondria.6–10 9
The 16.5‐kbp mitochondrial DNA (mtDNA) is double stranded circular DNA encoding critical subunits of the mitochondrial respiratory chain.11 H and OL is consistent with mutational and instability hot‐spots regions.12
Unlike chromosomal DNA, mtDNA is more vulnerable to oxidative damage, because of its lack of protective histones and its attachment to the mitochondrial inner membrane, a main source of oxygen radicals in the cell.13–15 15,16
Mitochondrial dysfunction and mitochondrial adaptive and protective mechanisms have been described in diverse chronic liver diseases.7 mt UPR), mitophagy, and induction of mitochondrial antioxidant capacities/pathways, all of which maintain mitochondrial homeostasis and ultimately cell survival.7 7 7 7 ] mt UPR involves mitochondrial (HSPD1)/stress‐70 protein, mitochondrial (heat shock protein family A [Hsp70]) member 9 (HSPA9) chaperones and Lon peptidase 1 (LONP1) peptidase, controls the stoichiometric balance between mitochondrial proteins encoded by the nuclear and mitochondrial genomes, ensures mitochondrial quality and proteases, and senses mitochondrial protein misfolding.7
The mitochondrial antioxidants include reduced glutathione (GSH) peroxidases, manganese superoxide dismutase (MnSOD), and peroxiredoxins.7,17 7,18 reactive oxygen species (ROS) and cytokines, but it is inactivated by peroxynitrite.7,18,19 18–20 19
In vitro and in vivo studies have shown that HBV or hepatitis B virus X protein (HBx) cause OS and alter mitochondrial dynamics and homeostasis.7,8,10,21–27 in vitro .
PATIENTS AND METHODS
Patients and controls
One hundred ninety‐one patients including 146 patients with CHB, 12 patients with NASH, 33 patients with CHC, and 24 patients with normal liver (hereafter designed as controls) were enrolled (Table 1 , Figure S1
TABLE 1 -
Characteristics of patients with CHB, CHC, NASH, and controls
CHB
CHC
NASH
Controls
Total no. (n )
146
33
12
24
Sex (M/F)
92/54
14/19
6/6
13/11
Age, years (mean ± SEM)
41 ± 4
48 ± 7b
52 ± 15b
42 ± 10
BMI, kg/m2 (mean ± SEM)
24.5 ± 3.3f
26.7 ± 4.4d
32.9 ± 7.7
23.1 ± 3.7f
ALT, IU/l (mean ± SEM)
111 ± 13
94 ± 41
102 ± 36
74 ± 37
AST, IU/l (mean ± SEM)
74 ± 14
60 ± 23
81 ± 45
51 ± 28
ϒGGT, IU/L (mean ± SEM)
79 ± 19a
133 ± 124
111 ± 103
198 ± 87b
Total cholesterol, mmol/l (mean, range)
4.4 (2.9–6.5)
4.7 (3.4–6.9)
4.6 (2.6–6.4)
3.9 (2.7–5.2)
Triglycerides, mmole/l (mean, range)
1.13e (0.38–3.69)
1.05e (0.47–2.15)
1.98 (0.77–5.50)
1.01e (0.43–1.39)
Glucose, mmole/l (mean ± SEM)
5.0 ± 0.6d ,c
5.5 ± 1.1a ,b
6.0 ± 1.2a ,b
4.7 ± 0.3
Creatinin, μmol/l (mean ± range)
0.65 (0.34–1.34)
0.86a ,b (0.59–1.25)
1.2a ,b ,c (0.80–1.68)
0.66 (0.58–0.82)
Albumin, g/l (mean ± SEM)
45 ± 7
45 ± 3
47 ± 4
43 ± 3
Platelets, 109/l (mean ± SEM)
174 ± 43
225 ± 48b
186 ± 50
271 ± 69
Virology
HBeAg positive, n (%)
77 (53)
—
—
—
HBsAg (mean, log10 IU/ml)
3.8
—
—
—
HBV DNA or HCV RNA, log10 IU/ml (mean, range)
5.4 (2.2–8.2)
5.9 (5.0–7.1)
—
—
Liver histology findings
Necroinflammatory activity
A0 | A1 | A2 | A3
35 | 68 | 30 | 13
2 | 19 | 11 | 1
0 | 1 | 3 | 8
22 | 1 | 0 | 1
Fibrosis stage
F0‐F2 (n )
100
21
0
24
F0 | F1 | F2
26 | 40 | 34
1 |12 |8
0 | 0 | 0
23 | 1 | 0
F3‐F4 (n )
46
12
12
0
F3 | F4
29 | 17
7 | 5
0 |12
0 | 0
Steatosis (n )
≤5% of hepatocytes
118
16
0
21
5–33% of hepatocytes
16
9
1
3
>33–66% of hepatocytes
11
6
6
0
≥66% of hepatocytes
1
2
5
0
Note : For ALT level, the upper limit of normal was 34 IU/l in women and 45 IU/l in men. For AST level, the upper limit of normal was 31 IU/l in women and 35 IU/l in men. For GGT level, the upper limit of normal was 38 UI/l in women and 55 UI/l in men.
a Different from controls, p < 0.05.
b Different from CHB, p < 0.05.
c Different from CHC, p < 0.05.
d Different from NASH, p < 0.05.
e Different from NASH, p < 0.01.
f Different from NASH, p < 0.001; Mann–Whitney U test.
Patients with CHB were eligible if they had been HBsAg‐positive for ≥6 months before inclusion and were excluded if they had evidence of alcohol intake >20 g/d, coinfections, or NASH. Seventy‐seven of 146 (53%) patients with CHB were HBeAg‐positive chronic hepatitis (phase II), and 69 (47%) were HBeAg‐negative chronic hepatitis (phase IV).
Patients with NASH or CHC were older than controls and patients with CHB (p < 0.05; Table 1 ). Patients with NASH also had higher body mass index (BMI) (p < 0.001), blood glucose (p < 0.01), and triglycerides (p < 0.05) than other groups. However, all the group patients and controls had comparable alanine aminotransferase (ALT) and aspartate aminotransferase (AST), but patients with CHB had lesser gamma‐glutamyl transferase (ϒGGT; p < 0.001; Table 1 ).
LB specimens were formalin‐fixed and paraffin‐embedded. Sections (5 μm thick) were stained with hematoxylin and eosin, Mallory trichrome, Picrosirius red, and Perls and analyzed by a pathologist blinded to the clinical and laboratory data.
In patients with CHB and CHC, necroinflammation and fibrosis were assessed according to the Metavir scoring system.3 Tables 1 and 2 ). Necroinflammation was graded as A0 to A3, and steatosis was assessed as the percentage of hepatocytes containing fat droplets (Table 1 ).
TABLE 2 -
Characteristics of patients with CHB and with mtDNA analysis, mRNAs, and/or protein assessment
mtDNA Analysis
mRNA Assessment
Protein Assessment
n = 65
n = 104
n = 44
F0‐F2
F3‐F4
F0‐F2
F3‐F4
F0‐F2
F3‐F4
No. (n )
46
19
76
28
26
18
Sex, M/F (n )
32/14
10/9
61/15
20/8
18/8
10/8
Age, years (mean ± SEM)
42 ± 2
45 ± 5
40 ± 1
45 ± 2
41 ± 2
47 ± 3
BMI, kg/m2 (mean ± SEM)
26.3 ± 2.3
24.5 ± 1.9
25.1 ± 0.6
24.5 ± 0.9
24.9 ± 0.9
23.7 ± 1.4
Serum levels at liver biopsy
ALT, IU/l (mean ± SEM)
109 ± 22
112 ± 15
109 ± 15
121 ± 27
101 ± 21
72 ± 18
AST, IU/l (mean ± SEM)
75 ± 13
83 ± 13
75 ± 9
73 ± 12
82 ± 16
71 ± 19
GGT, IU/l (mean ± SEM)
88 ± 22
80 ± 15
76 ± 20
92 ± 17a
146 ± 53
56 ± 14
Total cholesterol, g/l (mean ± SEM)
0.8 ± 0.3
1.1 ± 0.2
0.9 ± 0.1
0.9 ± 0.2
0.7 ± 0.1
0.8 ± 0.3
Triglycerides, mmole/l (mean ± SEM)
1.0 ± 0.4
0.9 ± 0.3
1.2 ± 0.1
1.6 ± 0.2
1.7 ± 0.3
2.0 ± 0.8
Glucose, mmole/l (mean ± SEM)
4.8 ± 0.3
5.0 ± 0.3
4.9 ± 0.2
4.9 ± 0.3
5.4 ± 0.2
4.9 ± 0.2
HBsAg, log10 IU/ml (mean ± SEM)
4.3 ± 0.2
5.0 ± 0.5
3.7 ± 0.1
4.6 ± 0.6
4.0 ± 0.2
4.8 ± 0.7
HBV DNA, log10 IU/ml (mean ± SD)
4.7 ± 0.3
4.8 ± 0.6
5.5 ± 0.2
5.4 ± 0.4
4.8 ± 0.5
5.6 ± 0.9
Fibrosis stage
F0
12
0
19
0
5
0
F1
21
0
40
0
14
0
F2
13
0
17
0
7
0
F3
0
10
0
17
0
12
F4
0
9
0
11
0
6
Note : RNA, DNA, and proteins were simultaneously obtained in 42 cases (31 with F0‐F2 and 11 with F3‐F4), and both DNA and proteins were obtained in 13 patients. The remaining 91 patients had only mRNA (64 cases), DNA (25 cases), or proteins (4 cases). For ALT level, the upper limit of normal was 34 IU/l in women and 45 IU/l in men. For AST level, the upper limit of normal was 31 IU/l in women and 35 IU/l in men. For GGT level, the upper limit of normal was 38 UI/l in women and 55 UI/l in men.
a Different from F0‐F2, p < 0.01; Mann–Whitney U test.
One hundred patients with CHB had F0‐F2 and 46 had F3‐F4 (Table 1 , Figure S1 Table 1 , Figure S1 Table 1 , Figure S1
The 24 control liver specimens were obtained for diagnostic purposes and comprised percutaneous normal LB specimens obtained from 24 adults with mildly elevated serum ALT activity addressed to Beaujon Hospital (Clichy, France), in whom all causes of liver disease had been ruled out (medication, alcohol, chronic viral hepatitis, autoimmune processes, and metabolic disease; Table 1 ). In these control subjects, LBs were performed percutaneously under local anesthesia.28 Table 1 ).
Study approval
The study was performed in accordance with the guidelines of the 1975 Declaration of Helsinki and the principles of Good Clinical Practice. All patients gave written consent, and the study was approved by the Ethics Committee of Beaujon Hospital (Département de la Recherche Clinique‐Assistance Publique–Hôpitaux de Paris).
Analyses flow chart
Different amounts of DNA, RNAs, and/or proteins were obtained from distinct groups of patients and controls depending on the size of liver fragments that remained available after histological studies (Figure S1 Table 2 ; see Supporting Methods
Detection and sequencing of mtDNA deletions
Liver DNA was isolated as described.15,16 S1
LPCR products were sequenced to determine the exact sizes and boundaries of the detected mtDNA deletions. mtDNA bands were excised from the gel, placed on the filter of a Spin‐X Centrifuge Tube Filter, frozen at −20°C, and recovered by centrifugation. One hundred nanograms of the recovered product were then subjected to a single primer PCR sequencing reaction using the BigDye Terminator v3.1 Cycle Sequencing Kit and either primer C or D (Table S1
Slot blotting and Southern blotting hybridization
Slot blotting was used to quantitate mtDNA and nuclear DNA (nDNA). Hepatic DNA (100 ng) samples from controls and patients with CHB or NASH were blotted onto a Hybond‐N1 nylon membrane and hybridized with a 6080‐bp probe that was generated by LPCR and αP32 dCTP‐labeled by random priming. Membranes were stripped and hybridized with a human C0 t‐1 nDNA probe, as described, and29
Proportions of mtDNA in its supercoiled, circular, and 16,569‐bp linear forms were analyzed by Southern blotting using a human mtDNA specific‐radiolabeled probe.29 32 ‐labeled 6080‐bp human mtDNA probe. The mtDNA forms were quantitated by densitometry of autoradiographs.
Real‐time RT‐qPCR assessment for liver mRNA levels, liver adenosine triphosphate determination, and western blotting analysis
RT‐qPCR, liver adenosine triphosphate (ATP) determination and western blotting were performed also in our patients and controls (see the Supporting Methods in vitro (Supporting Methods
Statistical analysis
Data are expressed as mean ± SD. Categorical variables are expressed as absolute and relative frequencies. Statistical analyses were performed using GraphPad Prism software (version 9.0.1; GraphPad Software Inc., La Jplla, CA). The Mann–Whitney U test was used to assess differences between means. The chi‐square (κ 2 ) or chi‐square corrected for continuity (κc 2 ) tests were used to assess differences between frequencies. The Pearson method was used to assess the correlation between numerical variables. Patients with CHB, CHC, or NASH were compared to controls. Patients with F3‐F4 were compared to those with F0‐F2 within each group of patients and between these groups. The n values for each experiment are indicated in the figure legends. Significance was set up at p < 0.05.
RESULTS
mtDNA deletions in patients with CHB or NASH
mtDNA deletions were examined by LPCR and characterized by sequencing. In addition to the expected 6080‐bp fragment amplified from undeleted mtDNA, the hepatic DNA from patients with CHB and patients with NASH gave amplification products of smaller lengths corresponding to diverse mtDNA deletions (Figure 1A,B ). To determine the exact position of these rearrangements, these mtDNA bands were sequenced (Figure S2
FIGURE 1: Liver mtDNA levels and mtDNA deletions. (A,B) Representative gels of LPCR detection of mtDNA deletions in controls, patients with CHB, and patients with NASH. In patients with CHB, primers A‐B gave the 6080‐bp product amplified from undeleted mtDNA and 3629‐, 1199‐, 1103‐, or 695‐bp products from deleted mtDNA (exact sizes were determined from the subsequently characterized 5385‐, 4977‐, 4881‐, and 2451‐bp mtDNA deletions in these patients) (A). In patients with NASH, the fragments of 1103‐bp corresponding to the 4977‐bp deletion was obtained (B). (C,D,E) Representative slot blottings (C,D) and mtDNA/nDNA ratios (mean ± SEM) from 65 patients with CHB (46 F0‐F2 and 19 F3‐F4) and 12 patients with NASH are expressed as percentages of 24 control values (E). M, 1 Kb DNA Ladder Plus. ### Different from control subjects, p < 0.001; ***Different from F0‐F2 or from patients with NASH; p < 0.001, Mann–Whitney U test. Ctrl, controls.
Four different 5385‐, 4977‐, 4881‐, and 2451‐bp mtDNA deletions, all flanked by direct repeats, were observed in these patients (Figure S2A
The 4977‐ and 5385‐bp deletions start within the gene for subunit 8 of ATP synthase (MT‐ATP8), and the 4881‐bp deletion starts within the gene for subunit 3 of cytochrome c oxidase (MT‐CO3, complex IV; Figure S2A,B S2B
The 2451‐bp deletion starts within subunit 3 of the NADH dehydrogenase gene and affected the genes for subunits 4, 4L, and 5 of complex I and tRNA‐Tyr, tRNAs‐Arg, tRNA‐His, tRNA‐Ser, and tRNA‐Leu (Figure S2A,B
The common 4977‐bp deletion was observed in 46 (71%) of 65 patients with CHB and in 7 (58%) of the 12 patients with NASH and cirrhosis, but only in 5 (21%) of the 24 controls (Table S3 S3
Considering all the four deletions, 56 patients with CHB (86%), but only 5 of 24 controls (19%), carried at least one mtDNA deletion (κ 2 = 34.68, p < 0.005; Table S3 κ 2 = 20.04, p < 0.005), and 1 patient harbored two distinct deletions. Two of the patients with F3‐F4 had two concomitant deletions, and 2 exhibited three concurrent deletions (Figure 1A ).
Compared to controls, prevalence of the 4977‐bp deletion was significantly higher in patients with NASH and cirrhosis. Whereas 7 (58%) of 12 patients with NASH and cirrhosis exhibited the 4977‐bp deletion, only 5 of 24 controls (19%) carried this common deletion (kc 2 = 5.06, p < 0.025; Table S3
Similarly, the difference in prevalence of the 4977‐bp deletion persisted when patients with CHB and F3‐F4 were compared to patients with NASH and cirrhosis. Whereas 18 of 19 (95%) patients with CHB and F3‐F4 exhibited at least one mtDNA deletion, only 7 of 12 (58%) patients with NASH and cirrhosis carried a single mtDNA deletion (k 2 = 6.93, p < 0.005; Table S3
The 5 patients with CHB exhibiting two or three different mtDNA deletions were those with the highest viral loads (HBV DNA: 6.0, 6.6, 7.2, 7.9, and 8.2 log10 IU/ml). All 7 patients with NASH and with the 4977‐bp deletion had massive steatosis (5 with 80% and 2 with 70% of fat‐containing hepatocytes).
mtDNA levels in patients with CHB and patients with NASH
mtDNA levels were analyzed by slot blotting. nDNA levels were not modified when slot blottings were hybridized with the human C0 t‐1 nDNA probe (Figure 1C,D ). We thus used the mtDNA/nDNA hybridization ratio to quantitate mtDNA changes (Figure 1E ). The mtDNA/nDNA ratio significantly declined in patients with CHB to 43% of control values (p < 0.001; Figure 1E ). Compared to controls, the mtDNA/nDNA ratio significantly decreased by 55% in patients with CHB and F0‐F2 (p < 0.001) and by 65% in patients with CHB and AF or cirrhosis (p < 0.001; Figure 1E ).
Similarly, the mtDNA/nDNA ratio significantly declined to 53% of control values in patients with NASH and cirrhosis (p < 0.001; Figure 1E ). The mtDNA/nDNA ratio was also different when patients with CHB and F3‐F4 were compared to patients with NASH and cirrhosis (35.37 ± 11.55 and 53.41 ± 11.55, respectively; p < 0.001).
Patients with CHB and highest viral loads (HBV DNA >5 log10 IU/ml) all had the lowest mtDNA levels (mtDNA/nDNA ratio <0.3). Similarly, patients with NASH and >66% of steatosis had minimal mtDNA levels (mtDNA/nDNA ratios <0.5). Quantitative PCR confirmed mtDNA depletion in patients with CHB (Figure S3A
We looked also for mtDNA strand breaks, topology, and changes in the main mtDNA forms in patients with CHB and controls (Figure S3B,C Supporting Results
Exacerbated decreases in mtDNA‐encoded cytochrome c oxidase subunits 1 and 2 in patients with CHB, CHC, or NASH and F3‐F4 as compared to those with F0‐F2
Compared to control livers (n = 8), mtDNA‐encoded cytochrome c oxidase subunit 1 (MT‐CO1) and mtDNA‐encoded cytochrome c oxidase subunit 2 (MT‐CO2) mRNA decreased significantly by 83% and 82% in patients with CHB and F3‐F4 (n = 21), 70% and 61% in patients with CHC and F3‐F4 (n = 12), and finally 65% and 59% in patients with NASH and cirrhosis (n = 12; p < 0.001; Figure 2A,C ). Although significant, these decreases were, to a lesser extent, in patients with F0‐F2 as compared to controls (Figure 2A,C ).
FIGURE 2: Liver mRNA and protein levels for mtDNA‐encoded MT‐CO1 and MT‐CO2. (A,B) MT‐CO1 mRNA and protein levels. MT‐CO1 mRNA levels were compared in patients with CHB and F3‐F4 (n = 21) or F0‐F2 (n = 66), patients with CHC and F3‐F4 (n = 12) or F0‐F2 (n = 12), patients with NASH and cirrhosis (n = 12), and controls (n = 8) (A). MT‐CO1 and β‐Actin protein levels were assessed in patients with CHB and F3‐F4 (n = 11) or F0‐F2 (n = 23), patients with NASH and cirrhosis (n = 7), and controls (n = 7) (B). (C,D) MT‐CO2 mRNA and protein levels. MT‐CO2 mRNA levels were compared in patients with CHB and F3‐F4 (n = 21) or F0‐F2 (n = 66), patients with CHC and F3‐F4 (n = 12) or F0‐F2 (n = 21), patients with NASH and cirrhosis (n = 12), and controls (n = 8) (C). MT‐CO2 and β‐Actin protein levels were assessed in patients with CHB and F3‐F4 (n = 21) or F0‐F2 (n = 11), patients with NASH and cirrhosis (n = 7), and controls (n = 7) (D). Representative gels are shown. ##,### Different from control subjects, ## p < 0.01, ### p < 0.01. *,**,*** Different from F0‐F2 or from patients with NASH; *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney U test. Ctrl, controls.
MT‐CO1 and MT‐CO2 mRNA levels were then compared between F3‐F4 and F0‐F2 in patients with CHB or CHC. In patients with CHB and F3‐F4 (n = 21), MT‐CO1 mRNA (0.17 ± 0.11; n = 21) and MT‐CO2 mRNA (0.18 ± 0.10; n = 21) decreased, respectively, by 53% and by 56% (p < 0.001) as compared to patients with F0‐F2 (0.32 ± 0.24, n = 66 and 0.32 ± 0.22, n = 66; Figure 2A,B ). In patients with CHC, MT‐CO1 (F0‐F2, n = 21; 0.61 ± 0.32 and F3‐F4, n = 12; 0.30 ± 0.18) and MT‐CO2 (F0‐F2, n = 21; 0.64 ± 0.27 and F3‐F4, n = 12; 0.39 ± 0.23) mRNA expression significantly decreased by 50% and 40% (p < 0.001), respectively, in patients with F3‐F4 (n = 12) compared to those with F0‐F2 (n = 21; Figure 2A,C ).
We also observed a significant decrease (p < 0.001) in the expression of MT‐CO2 mRNA, but not MT‐CO1 mRNA, in patients with CHB and F3‐F4 when compared to patients with CHC and F3‐F4 and a significant decrease in the expression of both MT‐CO1 and MT‐CO2 (p < 0.05) when patients with CHB and F3‐F4 were compared to patients with NASH and cirrhosis (Figure 2A,C ).
At the protein levels, the MT‐CO1/β‐Actin ratio significantly decreased in patients with CHB and F0‐F2 (1.49 ± 1.01, n = 23; p < 0.01) or F3‐F4 (0.81 ± 0.43, n = 11; p < 0.001) as compared to controls (2.56 ± 0.72, n = 7; Figure 2B ). Compared to controls (n = 7), the MT‐CO1/β‐Actin and MT‐CO2/β‐Actin ratios were significantly decreased in patients with NASH and cirrhosis (1.45 ± 0.62 and 0.41 ± 0.32, respectively, n = 7; p < 0.01; Figure 2B,D ).
Again, the decrease in the MT‐CO1/β‐Actin (p < 0.01) and MT‐CO2/β‐Actin (p < 0.001) ratios was worst in patients with CHB and F3‐F4 (n = 11, 0.81 ± 0.43 and n = 11, 0.55 ± 0.23) than patients with CHB and F0‐F2 (1.49 ± 1.01, n = 23 and 1.07 ± 0.35, n = 21). MT‐CO1/β‐Actin significantly decreased in CHB and F3‐F4 as compared to those with NASH and cirrhosis (1.45 ± 0.61, n = 7; p < 0.05; Figure 2B ).
However, nDNA‐encoded cytochrome c oxidase subunit 4I1 (COX4I1) mRNA levels were unchanged in CHB and CHC whatever the degree of fibrosis, but significantly decreased in patients with NASH and cirrhosis as compared to control subjects (p < 0.001; Figure S4A p < 0.001) or with CHC and F3‐F4 (p < 0.001; Figure S4A
Altered mt UPR in patients with CHB, patients with CHC, and patients with NASH
LONP1 degrades protein aggregates attributable to misfolding/disassembling of a surplus of mitochondrial proteins, whereas HSPA9 and HSPD1 allow respiratory chain complex assembly.7
Although expression of the mt UPR markers was altered in both patients with F0‐F2 or with F3‐F4, this alteration was more accentuated in patients with AF or cirrhosis associated with CHB or NASH (Figure 3 ).
FIGURE 3: Evaluation of the mtUPR and mitophagy markers. (A) LONP1 mRNA levels were compared in patients with CHB and F3‐F4 (n = 18) or F0‐F2 (n = 66), patients with CHC and F3‐F4 (n = 11) or F0‐F2 (n = 21), 12 patients with NASH and F4, and 8 controls. (B) LONP1 protein levels were compared in patients with CHB and F3‐F4 (n = 7) or F0‐F2 (n = 6), 7 patients with NASH and F4, and 7 controls. (C) HSPA9 mRNA levels were compared in patients with CHB and F3‐F4 (n = 20) or F0‐F2 (n = 66), patients with CHC and F3‐F4 (n = 12) or F0‐F2 (n =21), 12 patients with NASH and F4, and 8 controls. (D) HSPA9 protein levels were compared in patients with CHB and F3‐F4 (n = 10) or F0‐F2 (n = 17), 7 patients with NASH and F4, and 7 controls. (E) HSPD1 mRNA levels were compared in patients with CHB and F3‐F4 (n = 20) or F0‐F2 (n = 66), patients with CHC and F3‐F4 (n = 11) or F0‐F2 (n = 20), 12 patients with NASH and F4, and 8 controls. #,##,### Different from controls (p < 0.05, 0.01, 001); *,**,*** F3‐F4 compared to F0‐F2 within the same or between etiologies, *p < 0.05, **p < 0.01, ***p < 0.001, Mann–Whitney U test. Ctrl, controls.
LONP1 mRNA was induced in patients with CHB and with CHC whatever the degree of fibrosis, but this induction failed in patients with NASH and cirrhosis (Figure 3A ). Compared to controls (n = 8), LONP1 mRNA levels significantly increased by 120% in patients with CHB and F0‐F2 (2.20 ± 0.83, n = 66; p < 0.001), 65% in patients with CHB and F3‐F4 (1.65 ± 0.56, n = 18; p < 0.05), and 140% in patients with CHC and F3‐F4 (2.40 ± 1.03, n = 11; p < 0.05), but significantly decreased in patients with NASH and cirrhosis (0.18 ± 0.13, n = 12; p < 0.001; Figure 3A ). Western blotting confirmed the induction of the LONP1 gene at the protein levels in patients with CHB and F0‐F2 and, to a lesser extent, in F3‐F4 (controls, n = 7; 0.23 ± 0.13 vs. CHB F0‐F2, n = 7; 0.81 ± 0.29, p < 0.01 and vs. CHB F3‐F4, n = 6; 0.52 ± 0.10, p < 0.01; Figure 3B ).
Contrary to LONP1 mRNA, HSPA9 mRNA levels instead decreased significantly in patients with CHB or with NASH, but not in CHC, whereas HSPD1 mRNA significantly decreased in all these groups as compared to control subjects (Figure 3C,E ).
In patients with CHB, LONP1 mRNAs levels significantly decreased in AF or cirrhosis (F3‐F4, n = 20; 1.65 ± 0.56) as compared to those with F0‐F2 (n = 66; 2.20 ± 0.83, p < 0.05; Figure 3A ). Similarly, LONP1/β‐Actin significantly decreased by 1.3‐fold (F3‐F4, n = 6; 0.52 ± 0.10 vs. F0‐F2, n = 7; 0.82 ± 0.29, p < 0.05; Figure 3B ).
Similarly, relative HSPA9 mRNA expression decreased significantly by 36% in patients with CHB and F3‐F4 as compared to patients with CHB and F0‐F2 (F3‐F4, n = 20; 0.37 ± 0.19 and F0‐F2, n = 66; 0.57 ± 0.23, p < 0.001; Figure 3C ). This difference was confirmed by western blotting with a 1.7‐fold significant decrease in patients with CHB and F3‐F4 (n = 10; 0.57 ± 0.33) compared to those with F0‐F2 (n = 10; 0.98 ± 0.25, p < 0.01; Figure 3D ).
As compared to patients with CHB and F0‐F2, HSPD1 mRNAs levels significantly decreased in patients with CHB and AF or cirrhosis (F3‐F4, n = 20; 0.37 ± 0.17 and F0‐F2, n = 66; 0.53 ± 0.24; p < 0.05; Figure 3E ).
These differences in HSPA9 and HSPD1 mRNA expression were not observed between F3‐F4 and F0‐F2 within the group of patients with CHC (Figure 3C,E ).
We finally compared patients with AF or cirrhosis within groups of CHB, CHC, and NASH. As compared to patients with CHB and F3‐F4 or with CHC and F3‐F4, LONP1 and HSPA9 mRNAs significantly decreased in patients with NASH and cirrhosis (n = 12; 0.18 ± 0.13, p < 0.001 and n = 12; 0.30 ± 0.20, p < 0.001, respectively; Figure 3A,C ).
Mitophagy markers were induced in patients with CHB, CHC, or NASH, but this induction was altered during fibrosis progression in CHB
Expression of mitophagy markers was induced in patients with F0‐F2 or F3‐F4 whatever the etiology, but to a lesser extent in CHB and NASH patients with AF or cirrhosis (Figure 4 ). Compared to controls (n = 8; 1.00 ± 0.38), PRKN mRNA increased significantly by 144‐fold in patients with F0‐F2 (n = 66; 144.0 ± 54.3, p < 0.001) and by 45‐fold in patients with F3‐F4 (n = 20; 44.7 ± 20.2, p < 0.001) in the group of patients with CHB (Figure 4A ). In patients with CHC, PRKN mRNA increased significantly by 35‐fold in patients with F0‐F2 (n = 18; 35.5 ± 25.1, p < 0.001) and by 115‐fold in patients with F3‐F4 (n = 12; 115.0 ± 64.2, p < 0.001) as compared to controls (Figure 4A ). PRKN mRNA also increased significantly by 20‐fold in patients NASH and cirrhosis (n = 12; 20.3 ± 11.4, p < 0.001) as compared to controls (Figure 4A ). Compared to controls, induction of PRKN protein was confirmed in patients with CHB (p < 0.001) and in patients with NASH (p < 0.01; Figure 4B ).
FIGURE 4: Expression of PRKN and PINK1 mRNA and proteins.(A) PRKN mRNA levels were compared in patients with CHB and F3‐F4 (n = 20) or F0‐F2 (n = 66), patients with CHC and F3‐F4 (n = 21) or F0‐F2 (n = 12), patients with NASH and F4 (n = 12), and controls (n = 8).(B) PRKN protein levels were compared in patients with CHB and F3‐F4 (n = 7) or F0‐F2 (n = 12), patients with NASH and F4 (n = 7), and controls (n = 7). (C) PINK1 mRNA levels were compared in patients with CHB and F3‐F4 (n = 18) or F0‐F2 (n = 60), patients with CHC and F3‐F4 (n = 12) or F0‐F2 (n = 19), patients with NASH and F4 fibrosis (n = 12), and controls (n = 8). (D) PINK1 protein levels were compared in patients with CHB and F3‐F4 (n = 7) or F0‐F2 (n = 16), patients with NASH and F4 (n = 7), and controls (n = 7). #,##,### Different from controls (p < 0.05, 0.01, 0.001); *,**,*** F3‐F4 compared to F0‐F2 within the same or between etiologies, *p < 0.05, **p < 0.01, ***p < 0,001, Mann–Whitney U test. Ctrl, controls.
Similarly, PINK1 mRNA increased significantly by 4‐fold in patients with F0‐F2 (n = 60; 4.03 ± 1.12, p < 0.001) and by 1.9‐fold in patients with F3‐F4 (n = 18; 1.94 ± 0.53, p < 0.001) in the group of CHB as compared to controls (n = 8; 1.00 ± 0.50; Figure 4C ). In patients with CHC, PINK1 mRNA increased significantly by 7‐fold whatever the fibrosis stage (F0‐F2, n = 19; 7.20 ± 3.25, p < 0.001 and F3‐F4, n = 12; 7.11 ± 3.44, p < 0.001) as compared to control values (Figure 4C ). PRKN and PINK1 mRNAs also increased significantly, by 20‐ and 1.3‐fold, respectively, in patients with NASH and cirrhosis (n = 12; 20.3 ± 11.4, p < 0.001 and n = 12; 1.32 ± 0.75, p < 0.001) as compared to controls (Figure 4C ). Induction of PINK1 protein was confirmed in patients with CHB and F0‐F2 (n = 16; 0.88 ± 0.36, p < 0.001) and F3‐F4 (n = 7; 0.71 ± 0.27, p < 0.01; Figure 4D ).
In the group of patients with CHB, PRKN as well as PINK1 mRNAs significantly decreased, respectively, by 3.2‐fold (p < 0.001) and 2.1‐fold (p < 0.001) in patients with F3‐F4 as compared to patients with F0‐F2 (Figure 4A,C ). PRKN protein expression also significantly decreased by 1.3‐fold in patients with CHB and F3‐F4 as compared to those with F0‐F2 (p < 0.05; Figure 4B ).
In the group of patients with CHC, a 3.2‐fold increase in PRKN mRNA expression was observed in F3‐F4 (n = 12; 115.0 ± 64.2) when compared to F0‐F2 (n = 19; 35.5 ± 25.1, p < 0.001), whereas PINK1 mRNA remains unchanged whatever the fibrosis stage (Figure 4A,C ).
When we compared AF or cirrhosis between these three groups, we noticed that PRKN and PINK1 mRNA expression significantly collapsed in patients with NASH and cirrhosis as compared to patients with CHB and F3‐F4 (p < 0.001) or with CHC and F3‐F4 (p < 0.001 and p < 0.01, respectively; Figure 4A,C ).
In addition to the above analyzed markers for mitochondrial function, mitophagy, and mt UPR, we also investigated mRNA and/or protein levels for the mitochondrial biogenesis markers, PPARGC1α and TFAM (Figure S5 S5 Supporting Results
Decreased liver ATP levels and MT‐ATP8 in patients with CHB
Liver ATP levels were 62% of control values in patients with CHB (0.5 ± 0.1 and 0.2 ± 0.1 μmole/mg of protein; p < 0.05; Figure 5A ). A significant decrease of liver ATP was observed in patients with CHB and F3‐F4 when compared to those with F0‐F2 (0.1 ± 0.1 and 0.4 ± 0.2; p < 0.01; Figure 5A ). Compared to control livers, expression of MT‐ATP8 protein significantly decreased by 58% (p < 0.01) in patients with CHB whatever the degree of fibrosis (Figure 5B ).
FIGURE 5: Liver ATP, MT‐ATP8, iNOS, and MnSOD protein levels and liver mRNA of TNFα and IL6. (A) ATP levels were measured in four pools of 30‐μg liver homogenates each obtained from 3 controls and 10 pools of 30‐μg liver homogenates each obtained from 3 patients with CHB (5 pool F0‐F2, 5 pool F3‐F4). (B) Western blotting analysis for the MT‐ATP8 subunit. MT‐ATP8/β‐Actin protein ratios (mean ± SEM) for 30 patients with CHB are expressed as the percentage of the mean of 12 control values. (C,D) Western blotting analysis for iNOS and MnSOD proteins and corresponding iNOS/β‐actin and MnSOD/β‐actin ratios from 5 controls, 5 F0‐F2, and 5 F3‐F4. Relative mRNA expression for TNFα (E) and IL6 (F) were determined by RT‐qPCR in 6 patients with CHB F3‐F4 and in 10 F0‐F2 using specific primers. Different from controls, *p < 0.05; **p < 0.01. # Different from F0‐F2, p < 0.05, Mann–Whitney U test.
Increased iNOS protein but unchanged MnSOD protein expression in patients with CHB and F3‐F4 when compared to patients with CHB and F0‐F2
Compared to controls, iNOS protein, but not Manganese superoxide dismutase (MnSOD), significantly increased in patients with CHB (Figure 5C,D ). iNOS was also significantly induced in patients with F3‐F4 compared to patients with F0‐F2 (Figure 5C ) whereas MnSOD remained unchanged in these two groups (Figure 5D ).
Increased expression of liver TNFα and IL6 mRNAs in patients with CHB and F3‐F4
Liver TNFα mRNAs were 1.72 ± 0.20 and 0.99 ± 0.20 (p < 0.05) and liver IL6 mRNAs were 7.82 ± 0.90 and 1.14 ± 0.26 (p < 0.05) as assessed in 6 patients with CHB and F3‐4 and 10 with F0‐F2, respectively (Figure 5E,F ). Liver IL1β mRNAs were comparable in both groups (1.04 ± 0.40 in F3‐F4 and 1.24 ± 0.51 in F0‐F2).
Similarly, expression of the mitochondrial fusion marker, MFN1, and the fission marker, OPA1, was altered in patients with CHB and F3‐F4 (Figure S4 Supporting Results
Mitochondrial stress was also examined in HepG2 cells replicating HBV or expressing wild‐type or mutated HBx proteins in vitro (Figures S6–S8 S4 S5 Supporting Results
DISCUSSION
The present study investigated the incidence of mtDNA damage and/or alterations in mitochondrial function and homeostasis in patients with CHB, CHC, and NASH and in healthy control subjects. We also compared patients with AF or cirrhosis (F3‐F4) to patients with minimal/mild‐moderate fibrosis (F0‐F2). The study also explored in vitro the possible mechanisms involved in HBV‐induced mitochondrial stress.
Four mtDNA deletions (5385‐, 4977‐, 4881‐, and 2451‐bp) associated with mtDNA depletion and mtDNA strand breaks were identified in patients with CHB (Figure 1 , Figures S2 S3 Figure 1B ). Expression of mtDNA‐encoded MT‐CO1, MT‐CO2, and MT‐ATP8 as well as liver ATP levels significantly decreased in patients with CHB when compared to healthy controls (Figures 2 and 6 ). Expression of mtDNA‐encoded MT‐CO1 and MT‐CO2 also decreased in patients with NASH or CHC as compared to controls (Figure 2 ). Expression of the markers for mt UPR, mitophagy, and mitochondrial biogenesis all significantly altered in patients with AF or cirrhosis compared to patients with F0‐F2 (Figures 3–6 ).
FIGURE 6: Suggested mechanisms for the role of mtDNA damage and mitochondrial stress in the progression of fibrosis. By depressing liver antioxidant capacities and partially blocking electron flow within the mitochondrial respiratory chain, HBV proteins or lipoperoxidation products malondialdehyde (MDA) and 4‐hydroxynonenal (4‐HNE) increase mitochondrial ROS formation, depress mitochondrial antioxidants, and favor hepatic OS. ROS and these lipotoxic intermediates then cause mtDNA damage. The replication of damaged mtDNA molecules leads to progressive accumulation of mtDNA deletions, resulting in profound mitochondrial dysfunction, which further increases ROS production and further mtDNA damage accumulation in a vicious cycle. ROS and mDAMPs also activate the proliferation and dedifferentiation of HSCs into myofibroblasts secreting collagens and fibrosis develops. In some patients, the mitochondrial resilience involving adaptive mechanisms (mtUPR, biogenesis, fusion, fission, and mitophagy) might maintain or restore mitochondrial homeostasis and limit ROS formation and liver inflammation. The fibrosis in this case stabilizes or regresses. However, in other patients, the altered mitochondrial mtUPR in combination with mitophagic burden lead to significant accumulation of extensively damaged mitochondria within infected or fat‐engorged hepatocytes. Leakage of mtDNA and other mDAMPs in the cytosol could activate autocrine inflammatory responses. Alternatively, hepatocyte necrosis may lead to mDAMP release in the extracellular medium, leading to an activation of paracrine inflammatory responses in the neighboring hepatocytes, Kupffer cells, and resident macrophages. High ROS and inflammatory cytokines in this case could sustain mitochondrial stress, which may favor the progression of fibrosis.
The 4977‐ and 5385‐bp deletions, but not 4881‐ and 2451‐bp, have been reported. The common 4977‐bp mtDNA deletion is associated with normal age.13,14 15,16 30 31,32 33 34
In patients with CHB, the higher prevalence and diversity of these mtDNA deletions were associated with AF or cirrhosis (Table S3 S3 S3
Subsequently, expression of mtDNA‐encoded MT‐CO1 and MT‐CO2 was significantly decreased in F3‐F4 when compared to F0‐F2 (Figure 2 ). Expression of these mtDNA‐encoded genes might be interpreted as the consequence of mtDNA deletions and lower mtDNA content detected in the present study, but also as a consequence of several other mtDNA point mutations including those described in the mtDNA regulatory sequence D‐Loop in HBV‐infected patients.31,32,35 11
The mitophagy markers, PRKN and PINK1, as well as mt UPR markers HSPD1, HSPA9, and LONP1 or biogenesis marker TFAM were all down‐regulated in patients with F3‐F4 when compared to patients with F0‐F2 (Figures 3 and 4 , Figure S5 Figure 5 ). In addition to impaired mtDNA replication, mtDNA depletion might also be caused by mitophagy‐mediated clearance of excessively damaged mitochondria.7 Figure 1 , Figure S3 Figure 4 ).
Important possible mechanisms on the role of lipid peroxidation, mtDNA damage, and associated mitochondrial dysfunction in the development of liver steatosis in NASH and CHC were discussed (see the Supporting Discussion
HBV and HBx block electron flow within the respiratory chain and depressed mitochondrial antioxidant capacities, thus increasing ROS formation within mitochondria.7,8,22,36,37 16,29,38,39 S2A 38,40
HBx has also been shown to interact with the mitochondrial voltage‐dependent anion channel, consistent with its role in decreasing mitochondrial membrane potential and thus its increasing of the intracellular levels of ROS.41 41 41 S8 S4 S5 42 4 ‐induced mitochondrial dysfunction and OS resulting in reduced fibrosis.42
Our data in vitro confirmed that HBV and HBx protein both induced a mitochondrial superoxide release (Figure S8A S4 17,19,20 17 43,44 2 O2 ‐scaveging peroxisomal catalase, leading to high hydrogen peroxide accumulation within this organelle, which may account for further hepatocellular damage through the lipotoxicity of lipid peroxidation by‐products.45 S8A S4 Figure 5 , Figure S7
Peroxynitrite and/or peroxynitrite‐generated reactive intermediates can damage mtDNA.18–20 18–20 S5
Peroxynitrite also damages proteins, causing nitrotyrosine formation, tyrosine oxidation, and loss of function.17,18 S7 17,18 7
MnSOD is another target for ROS‐ and peroxynitrite‐mediated inactivation.18 S7
Thus, several redundant mechanisms may be involved in OS after HBV infection, including TNFα, respiratory chain impairment, decreased MnSOD activity, and induction of iNOS.
To preserve mitochondrial homeostasis, hepatocytes respond by inducing mitochondrial antioxidative capacities such as MnSOD, increasing mitochondrial fission to allow the mitophagy degradation of dysfunctional mitochondria, and increasing mitochondrial fusion and biogenesis to restore mtDNA levels and mitochondrial mass.7,46 mt UPR mechanisms through mitochondrial HSPA9 and HSPD1 to allow a correct folding of the mtDNA‐ and nDNA‐encoded mitochondrial proteins.7 7 mt UPR, PINK1, and PRKN involved in the mitophagy process (Figures 3 and 4 ) as well as TOMM20, MFN1, and OPA1 involved, respectively, in mitochondrial import, fusion, and fission (Figure S4 Figure 4 ).
Whatever the etiology, the above discussed mechanisms of oxidative mtDNA damage, mitochondrial dysfunction, and altered mitochondrial dynamics might all favor the development of fibrosis in several ways. First, the preexisting (inherited, acquired, or age‐related) mtDNA deletions might aggravate the effects of HBV in CHB and the effects of lipoperoxidation products in NASH on mitochondrial function, which, in turn, might accelerate the development and progression of liver fibrosis. Patients with mtDNA deletions in the present study have developed hepatic fibrosis to a different extent. Those subjects, however, with multiple mtDNA deletions (and possibly many other mtDNA mutations) may produce more oxygen radicals and poorly eliminate ROS because of the altered mitochondrial antioxidant capacities.7,47,48 7 Table 1 ). Unlike in the NASH group, where patients with mtDNA deletions were significantly older, the age of patients with mtDNA deletions and CHB was not different from the age of all other patients without the deletions in the present study. Similarly, the age of patients with CHB patients and AF was not different from the age of all other patients without mtDNA deletions. Thus, these observations excluded the effect of age on mtDNA in CHB and likely supported direct damaging effects of HBV on mtDNA in AF. Second, one cannot exclude that steatosis itself likely causes mtDNA damage and alters mitochondrial homeostasis. Steatosis and ROS favor the accumulation of lipid peroxidation products known to cause mtDNA damage, which may then favor the development of fibrosis. Lipohydroperoxides lead to mtDNA adducts and single‐strand breaks and favor slipped mispairing of repeated sequences during replication of damaged mtDNA molecules, resulting in impaired oxidative phosphorylation, even though the percentage of mtDNA affected with these deletions is usually low.13,22,49 7 Table 1 ) and other studies.50–52 7,9,53,54 7,9,55–57 7,55–61 9 62,63
Although the present study has several strengths (relevant knowledges for the understanding of the pathophysiology and mechanisms of chronic liver diseases), it also has several main limitations: First of all, there was our inability to simultaneously investigate all of the addressed parameters (RNA, DNA, and proteins) and characterize the intrahepatic HBV biomarkers (covalently closed circular DNA [cccDNA], 3.5‐kb mRNAs, and the cccDNA transcriptional activity) in all samples because of the limited sizes of available biopsies. Second, the heterogeneity of different cell types (hepatocytes, inflammatory cells, and HSCs) has not been addressed. Third, the role of HBV‐induced mitochondrial dysfunction in fibrosis progression needs further explorations. To this end, it will be of great interest to treat in vitro HSCs with purified mtDNA (as an mDAMP) or to coculture HBV‐infected hepatocytes with HSCs and monitor their differentiation into collagen‐secreting myofibroblasts and their expression of profibrogenic factors/markers. Studying mitochondrial stress in an HBV‐infected liver humanized mouse model treated or not with CCl4 would allow to explore the role of these mitochondrial lesions in the progression of fibrosis. Finally, experiments in other relevant models relying on HBV infection (with wild‐type and HBx mutant viruses) are needed to confirm whether the mitochondrial alterations observed in our transfection system are induced by HBV replication and/or by other HBV proteins in addition to HBx.
We propose that mtDNA damage resulted in the disturbance of mitochondrial homeostasis, function, and dynamics in patients with CHB and patients with NASH. Suppressed mt UPR in combination with altered mitophagy leads to an overall significant mtDNA damage accumulation that subsequently increases mitophagic burden, leading to mDAMP release, and subsequent expression of inflammatory and profibrogenic mediators (Figure 6 ). All these mitochondrial alterations likely play important roles in triggering/exacerbating inflammatory and profibrotic responses that promote the development and progression of fibrosis (Figure 6 ).
Our observation that HBV‐ and HBx‐induced mtDNA damage and mitochondrial dysfunction is preventable in vitro raises the hope that the dire prognosis of AF or cirrhosis could be therapeutically improved or slowed in vivo . Further investigations of the modulation of these potential mitochondrial biomarkers in large cohorts of patients with other liver diseases are urgently needed to confirm the feasibility of these markers for therapeutic and diagnosis purposes.
AUTHORS CONTRIBUTIONS
Concept, setup, and design (Abdellah Mansouri, Tarik Asselah); acquisition and analysis of data (Dimitri Loureiro, Abdellah Mansouri, Issam Tout, Cheikh Mohamed Bed, Morgane Roinard, Stéphanie Narguet, Ahmad Sleiman, and Tarik Asselah); writing original and final drafts (Abdellah Mansouri, Tarik Asselah, and Dimitri Loureiro); all authors were involved in critical review of the manuscript and approved the final version of the manuscript.
FUNDING INFORMATION
This work was funded by INSERM and Université de Paris Cité.
CONFLICT OF INTEREST
Nothing to report.
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