reperfusion injury via phosphoglycerate mutase family member 5-mediated mitochondrial quality control

Accepted Manuscript Heme oxygenase-1 protects liver against ischemia/reperfusion injury via phosphoglycerate mutase family member 5-mediated mitochondrial quality control

Jeong-Min Hong, Sun-Mee Lee PII: DOI: Reference:

S0024-3205(18)30122-X doi:10.1016/j.lfs.2018.03.017 LFS 15593

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Life Sciences

Received date: Revised date: Accepted date:

12 January 2018 28 February 2018 7 March 2018

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Heme oxygenase-1 protects liver against ischemia/reperfusion injury via phosphoglycerate mutase family member 5-mediated mitochondrial quality control

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Jeong-Min Hong, Sun-Mee Lee*

School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, Republic of

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Corresponding author

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*

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Korea

Sun-Mee Lee, Ph.D.

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Sungkyunkwan University

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School of Pharmacy

300 Cheoncheon-dong, Jangan-gu

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Suwon, Gyeonggi-do 440-746, Republic of Korea Fax: +82 31 292 8800

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Tel: +82 31 290 7712

E-mail: [email protected]

Title length and word count Title: 150/150 characters including spaces Abstract: 246/250

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Introduction: 500/500 Materials and methods: 1715 Results: 1111 Discussion: 1303/1500

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Conclusion: 66/150

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Figure/Table count

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Figure: 7

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Table: 1

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Abbreviations Alanine aminotransferase, ALT; dynamin-related protein 1, Drp1; ethylenediaminetetraacetic acid, EDTA; glutamate dehydrogenase, GDH; hematoxylin&eosin, H&E; heme oxygenase-1, HO-1;

hypoxia/reoxygenation,

H/R;

interleukin,

IL;

ischemia/reperfusion,

I/R;

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lipopolysaccharide, LPS; malondialdehyde, MDA; mitochondrial division inhibitor, Mdivi-1; mitofusin 2, MFN2; mitochondrial DNA, mtDNA; nuclear factor erythroid 2-related factor 2,

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Nrf2; nuclear respiratory factor 1, NRF-1; one-way analysis of variance, ANOVA; optic

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atrophy 1, OPA1; phosphoglycerate mutase family member, PGAM; peroxisome proliferatoractivated receptor-gamma coactivator 1α, PGC1α; PTEN-induced putative kinase 1, PINK1;

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quality control, QC; reactive oxygen species, ROS; standard error of the mean, S.E.M.;

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transmission electron microscopy, TEM; mitochondrial transcription factor A, TFAM; Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling,

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TUNEL; zinc protoporphyrin, ZnPP.

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Abstract Aims: Heme oxygenase-1 (HO-1), an endogenous cytoprotective enzyme, is reported that can be localized in mitochondria under stress, contributing to preserve mitochondrial function. Mitochondrial quality control (QC) is essential to cellular health and recovery linked with

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redox homeostasis. Recent studies reported that phosphoglycerate mutase family member (PGAM) 5, a mitochondria-resident phosphatase, plays critical role in mitochondrial

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homeostasis. Therefore, we aim to investigate cytoprotective mechanisms of HO-1 in I/R-

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induced hepatic injury focusing on mitochondrial QC associated with PGAM5 signaling. Main methods: Mice were subjected to 60 min of hepatic ischemia followed by 6 h

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reperfusion and were pretreated twice with hemin (HO-1 inducer, 30 mg/kg) or zinc

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protoporphyrin (ZnPP; HO-1 inhibitor, 10 mg/kg) 16 and 3 h before ischemia. Key findings: I/R increased hepatic and mitochondrial HO activity, which was augmented by

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hemin. I/R-induced hepatocellular and mitochondrial damages were attenuated by hemin and

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augmented by ZnPP. Meanwhile, I/R increased mitochondrial biogenesis, as evidenced by increased mitochondrial DNA contents and mitochondrial transcription factor A protein expression. Hemin augmented these results. I/R impaired mitophagy, as indicated by

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decreases in Parkin protein expression and the number of mitophagic vacuoles. These

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changes were attenuated by hemin. Hemin attenuated the I/R-induced increase in mitochondrial fission-related protein, dynamin-related protein 1, and the decrease in PGAM5 protein expression. Furthermore, PGAM5 siRNA abolished the effect of HO-1 on mitochondrial QC in HepG2 cells subjected to hypoxia/reoxygenation. Significance: Our findings suggest that HO-1 protects against I/R-induced hepatic injury via regulation of mitochondrial QC by PGAM5 signaling. Keywords: Heme oxygenase-1; Ischemia/reperfusion; Mitochondrial quality control; Phosphoglycerate mutase family member 5

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1. Introduction Hepatic ischemia/reperfusion (I/R) injury commonly occurs in many clinical conditions including hypovolemic shock, liver transplantation and liver surgery performed for trauma and cancer. Accumulating evidence suggests that mitochondria are major contributor to

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injury during I/R, because mitochondrial dysfunction leads to bioenergetic failure, ROS production and cell death [1]. Nonetheless, the precise molecular mechanisms behind I/R-

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induced hepatic injury have remained unclear.

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Mitochondria are dynamic organelles that continuously undergo changes in their number and morphology in order to respond to changes in the intracellular environment and to

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maintain their function. Mitochondrial quality control (QC), collectively encompassing

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mitochondrial biogenesis, mitophagy, and mitochondrial dynamics, is increasingly being recognized as essential for the recovery of ischemic damage linked to redox homeostasis [2].

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Hypobaric hypoxia resulted in imbalance of mitochondrial fission and fusion in rat brain

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hippocampus [3]. Mitochondrial division inhibitor (Mdivi-1) attenuated heart I/R injury by inhibiting mitochondrial fission and cell death [4]. In cultured hepatocytes, a potent sirtuin1 activator, SRT1720, protected against H/R-induced mitochondrial damage by restoration of

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mitochondrial biogenesis and enhancement of autophagy [5]. Moreover, mitophagy and

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mitochondrial biogenesis occurred in atrial tissue of patients undergoing cardiac surgery, suggesting that mitochondrial QC may be important target for I/R stress [6] Heme oxygenase-1 (HO-1), an inducible endogenous cytoprotective enzyme, is activated during various cellular stress, including inflammation, hypoxia or hyperthermia, and it has been reported to play crucial role in redox homeostasis maintenance [7]. HO-1 induction protected against hepatic I/R injury by inhibiting oxidative damage and inflammatory cytokine production [8]. Recent studies have reported that oxidative stress may increase translocation of HO-1 to mitochondria, suggesting a relationship between HO-1 and

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mitochondrial function. Translocation of HO-1 to mitochondria attenuated indomethacininduced gastric mucosal injury and mitochondrial oxidative stress [9]. Hull et al. [10] observed that HO-1 protected against doxorubicin-induced cardiac toxicity by regulating mitochondrial QC in mice. Furthermore, cilostazol, a potent inhibitor of type III

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phosphodiesterase, reduced tissue injury in a rodent model of hepatic I/R by HO-1-dependent mitochondrial biogenesis activation [11]. However, there is no information available on the

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effect of HO-1 on integrative mitochondrial QC during hepatic I/R.

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Phosphoglycerate mutase (PGAM) is an evolutionarily conserved enzyme of intermediary metabolism that converts 3-phosphoglycerate to 2-phosphoglycerate in glycolysis. PGAM5 is

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localized in mitochondria and displays activity as a mitochondrial serine/threonine protein

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phosphatase [12]. PGAM5 has been implicated in a diverse cellular activities related to control of signal transduction pathways including antioxidant response, cell death and

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mitophagy. It has been reported that PGAM5 can regulate levels of the nuclear factor

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erythroid 2-related factor 2 (Nrf2) transcription factor and thereby control cellular defenses against oxidative stress through interaction with Keap1 to the outer membrane of mitochondria [13]. PGAM5 protected cardiomyocyte from apoptosis via regulating Keap1-

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mediated degradation of anti-apoptosis protein Bcl-xL after I/R [14]. PGAM5 activation

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protected heart and brain tissues against I/R injury by promoting mitophagy [15]. Therefore, in this study, we aimed to investigate the protective mechanisms of HO-1 against hepatic I/R injury in particular focusing on mitochondrial biogenesis, mitophagy, and mitochondrial dynamics associated with PGAM5 signaling.

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2. Materials and methods 2.1. Animals Male C57BL/6 mice (21-23 g; Daehan Biolink Co., Ltd., Eumsung, Korea) were housed at Sungkyunkwan University in accordance with the Principles of Laboratory Animal Care

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and were allowed to acclimate to the laboratory animal facility with a temperature of 25 ±

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1°C, humidity of 55 ± 5% and a 12 h light/dark cycle for at least 1 week prior to the initiation

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of experiments. All experiments were performed in compliance with the guidelines of the National Institutes of Health (NIH publication No. 86-23, revised 1985) and the

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Sungkyunkwan University Animal Care Committee.

2.2. Liver I/R procedure

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Mice were fasted for 18 prior to experiment but were provided with tap water ad libitum. Mice were anaesthetized using an intraperitoneal administration of ketamine (100 mg/kg) and xylazine (10 mg/kg). The anesthetized mice abdomens were transversely incised. Complete

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ischemia of the median and left lobes of the liver was performed by clamping the branches of

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the portal triad with a micro serrefine clip (Fine Science Tools Inc., Vancouver, Canada). The right lobes remained perfused to prevent venous congestion of the intestine. After 60 min of ischemia, the clamp was removed to allow reperfusion. Body temperature was maintained at 36°C using heating pads throughout the experiment. At 6 h of reperfusion, mice were killed with overdose of ketamine and xylazine and blood and liver tissues were collected. Serum and liver tissues were frozen in liquid nitrogen and stored at -80°C for a period before they were used in later analysis; part of the left lobe was used for histological staining.

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2.3. Drug treatment Hemin (30 mg/kg; Sigma-Aldrich, St Louis,. MO, USA) and zinc protoporphyrin (ZnPP, 10 mg/kg; Sigma-Aldrich) were prepared under dark condition. Drugs were dissolved in 1 ml

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0.2 M NaOH, adjusted to pH 7.4 with 1 M HCl and finally diluted with saline (vehicle) to

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their final volume. Mice were pretreated twice with hemin or with ZnPP 16 and 3 h before

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ischemia. The dose and injection time of hemin and ZnPP treatment were based on earlier reports [16, 17] and our preliminary studies. Mice were randomly divided into six groups

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(n=6-8, each group): (a) vehicle-treated sham; (b) hemin-treated sham; (c) ZnPP-treated sham; (d) vehicle-treated I/R (I/R); (e) hemin-treated I/R (Hemin + I/R) and (f) ZnPP-treated I/R

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(ZnPP + I/R);. Because there were no significant differences in any of the parameters among vehicle-, hemin-, or ZnPP-treated sham groups, the results of groups (a), (b), and (c) were

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pooled and were collectively referred to as sham.

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2.4. Isolation of liver microsomal fractions

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According to the method described by Maines [18], the liver tissues homogenized in buffer containing 50 mM Tris, 1.15% KCl, 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 7.4 were centrifuged at 9 000 g for 10 min at 4 °C. After centrifugation, microsomal fractions were obtained by further centrifuging the supernatant at 105 000 g for 60 min at 4°C. Finally, the microsomal fractions were resuspended in 0.1 M potassium phosphate buffer at pH 7.4 and used for HO activity analysis.

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2.5. Isolation of liver mitochondrial fractions Liver tissues were homogenized on ice using Teflon pestle homogenizer in medium containing 250 mM sucrose, 5 mM HEPES, and 1 mM EDTA at pH 7.2 and centrifuged at

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600 g for 10 min at 4°C. Liver mitochondrial fractions were isolated using a differential

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centrifugation method based on a previous protocol [19]. Protein concentration was

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determined using the BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA).

2.6. HO enzyme activity

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Microsomal and mitochondrial HO activities were measured in both whole liver microsomal and mitochondrial fraction using the spectrophotometric determination of

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bilirubin formation, in accordance with the method described by Maines [18].

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2.7. Serum alanine aminotransferase (ALT) activity

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Serum ALT activity was analyzed at 37°C using a ChemiLab ALT assay kit (IVDLab Co., Ltd., Uiwang, Korea).

2.8. Serum interleukin (IL)-6 and IL-1β levels The serum levels of IL-6 and IL-1β were quantified using a commercial mouse IL-6 and IL-1β enzyme-linked immunosorbent assay kits (BD Biosciences, San Diego, CA, USA), respectively, in accordance with the manufacturer’s guidelines.

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2.9. Histological analysis Fresh liver tissues were sampled from a portion of the left lobe and fixed instantly in 10% neutral buffered formalin (Sigma-Aldrich) at room temperature, embedded in paraffin, and

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then cut with a microtome into 5 μm serial slices. The sections were stained with

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hematoxylin&eosin (H&E) and were assessed in a blind manner at ⅹ 200 magnification

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using an optical microscope (Olympus Optical CO., Tokyo, Japan). The stained sections were examined in randomly chosen histological fields at 200 x magnification and ideally evaluated by two

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different and blinded pathologist using the system devised by Suzuki et al. [20]. In this classification, three liver injury indices sinusoidal congestion (score: 0-4), hepatocyte death (score: 0-4), and

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ballooning degeneration (score: 0-4) are graded for total score of 0-12. No congestion, cell death and ballooning is given a score of 0, while severe congestion/ballooning and >60% lobular cell death is

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given a value of 12.

2.10. Transmission electron microscopy (TEM)

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For fixation, liver tissues were immersed in 2.5% glutaraldehyde and 4%

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paraformaldehyde in 100 mM sodium phosphate buffer at pH 7.2 then washed with 100 mM Na cacodylate at pH 7.4. The post fixation process was performed in 2% osmium tetroxide, and rewashed. After being dehydrated in a graded series of ethanol and propylene oxide, the samples were embedded in epoxy resin (Taab 812 Resin; Marivac Industries, Montreal, QC, Canada). The double contrast method for ultrathin sections (60-70 nm) using uranyl acetate and lead citrate was used as a contrasting technique for electron microscopy. Images were viewed using a Hitachi 7600 TEM (Hitachi High-Technologies America, Inc., Schaumburg, IL, USA).

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2.11. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay Apoptotic cells were detected in situ by TUNEL staining with a commercially available

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kit (Apoptosis Detection kit, Takara Bio Inc., Shiga, Japan). The percentage of TUNEL-

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positive cells was measured by an automated digital image analyzer (iSolution FL ver. 9.1,

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IMT i-solution Inc. Vancouver, BC, Canada).

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2.12. Serum glutamate dehydrogenase (GDH) activity

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Using the method of Ellis and Goldberg [21], serum GDH activity was determined and

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analyzed by monitoring spectrophotometrically at 340 nm for 6 min.

2.13. Mitochondrial Lipid peroxidation and glutathione content

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According to method described by Buege and Aust [22], the level of malondialdehyde (MDA) in liver mitochondria was analyzed by measuring the level of substances that were

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reacted with thiobarbituric acid at 535 nm. The total glutathione level in liver mitochondria was analyzed following the method described by Tietze [23]. Using the same method, the GSSG level was assessed in the presence of 2-vinylpyridine. The level of GSH was calculated as the difference between total glutathione and GSSG levels.

2.14. MtDNA copy number

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To extraction total mtDNA from liver tissues, a DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) was used in accordance with the manufacturer’s guidelines. The mtDNA copy number was measured using a real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) with a thermocycler (Lightcycler® Nano, Roche Applied Science,

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Mannheim, Germany) and a SYBR Green detection system (Roche Applied Science). The primers used for DNA amplification were 5’-ACGCTTCCGTTACGATCAAC-3’ (sense) and

and

5’-AGCCATGTACGTAGCCATCC-3’

(sense)

and

5’-

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(ND1)

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5’ACTCCCGCTGTAAAAATTGG-3’ (antisense) for mitochondrial NADH dehydrogenase 1

GCTGTGGTGGTGAAGCTGTA-3’ (antisense) for β-actin. The level of mitochondrial ND1

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indicates the total amount of mtDNA. Real-time RT-PCR was conducted with initial

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denaturation at 94°C for 5 min and final extension at 72°C for 7 min. The cycling conditions were as follows: 45 cycles of 30 s at 94°C, 30 s at 52°C and 30 s at 72°C for ND1, and 45 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C for β-actin. The expression levels of

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mRNA were normalized to β-actin mRNA level, and are relative to the average of all ΔCt-

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values in each sample using the cycle threshold (Ct) method.

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2.15. Western blot analysis

Samples of protein (12-16 μg) from both liver homogenates and mitochondrial fractions were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) using the Semi-Dry Trans-Blot Cell (Bio-rad Laboratories, Hercules, CA, USA). Bands on the Western blot were immunologically detected using specific antibodies against COX IV, mitofusin2 (MFN2), nuclear respiratory factor 1 (NRF-1), PTEN-induced putative kinase 1 (PINK1), Parkin (1:2500 dilution, Abcam, Cambridge, MA,

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USA), dynamin-related protein 1 (Drp1), peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC1α) (1:2500 dilution, Santa Cruz Biotechnology, CA, USA), caspase-3, GDH (1:2500 dilution, Cell signaling Co., MA, USA) mitochondrial transcription factor A (TFAM), PGAM5 (1:4000 dilution, Abcam), and β-actin (1:5000 dilution, Sigma-Aldrich).

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The immunoreactive bands were visualized using West-Q Pico ECL Solution (GenDEPOT, Barker, TX, USA) and evaluated by densitometric analysis using TotalLab TL 120 software

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(Nonlinear Dynamics Ltd., Newcastle, UK).

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2.16. In vitro assay

Previous studies have demonstrated that this H/R model shows similar features with in

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vivo I/R conditions and has been widely used to study mechanisms of I/R injury in cells [24]. For H/R, HepG2 cells were purchased from the American Type Culture Collection (Manassas,

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VA, USA). The cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin

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and maintained at 37°C in an atmosphere of 5% CO2. The cells from passage numbers 10-20 were used. Briefly, the plates (5 ⅹ 105 cells/well) were placed into a modular incubator

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chamber (Billups-Rothenburg, Del Mar, CA, USA) and maintained at 37°C with 5% CO2 and 95% N2. After incubation under hypoxic conditions for 12 h, the cells were moved to 95% air

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and 5% CO2 for 3 h reoxygenation. The cells were pretreated with hemin (25 μM for 3 h) and ZnPP (10 μM for 1 h) and subjected to H/R. The dose and the time of hemin and ZnPP treatment were based on our previous reports [25].

2.17. siRNA silencing of PGAM5 Non-specific control siRNA and PGAM5 siRNA were purchased from Bioneer (Daejeon,

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Korea). HepG2 cells were transfected with PGAM5 siRNA using LipofectamineTM RNAiMAX (Invitrogen, Carlsbad, CA, USA) for 48 h. The cells were harvested to determine PGAM5 protein expression and for further analysis.

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2.18. Statistical analysis

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All results are presented as the mean ± standard error of the mean (S.E.M.). One-way

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analysis of variance (ANOVA) was used to examine the overall statistical differences. Differences between the groups were considered statistically significant at a P < 0.05 with the

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appropriate Bonferroni correction made for multiple comparisons.

3. Results

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3.1. HO activity and HO-1 protein expression during hepatic I/R The hepatic microsomal HO activity in the sham group was 74.2 ± 3.43 pmol/min/mg

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protein. After 6 h of reperfusion, hepatic microsomal HO activity significantly increased to 119.5 ± 10.2 pmol/min/mg protein compared with that of the sham group. The increase in

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HO-1 activity was augmented by hemin and inhibited by ZnPP. The mitochondrial HO activity in the sham group was 96.7 ± 5.08 pmol/min/mg protein. After reperfusion, mitochondrial HO activity significantly increased to 148.4 ± 10.6 pmol/min/mg compared with that of the sham group and this increase was augmented by hemin and inhibited by ZnPP (Table 1). Similar to HO activity, the level of both hepatic and mitochondrial levels of HO-1 protein expression significantly increased after reperfusion. This increase was augmented by hemin and attenuated by ZnPP (data not shown).

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3.2. Effect of HO-1 on hepatocellular damage during hepatic I/R

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Serum ALT activity was 31.2 ± 2.67 U/L in the sham group. However, serum ALT activity significantly increased to 8247.3 ± 635.1 U/L after reperfusion. This increase was attenuated

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by hemin and augmented by ZnPP. The serum levels of IL-6 and IL-1β were 14.9 ± 0.50

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pg/ml and 33.2 ± 1.35 pg/ml in the sham group, respectively. After reperfusion, the levels of IL-6 and IL-1β significantly increased to 578.6 ± 17.9 pg/ml and 177.7 ± 28.0 pg/ml

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compared with those of the sham group, respectively. These increases were attenuated by hemin (Fig.2A-C). Histological analysis performed with H&E staining showed normal liver

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architecture in the sham group (Suzuki score: 0.0 ± 0.0). In the I/R group, the liver sections

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showed multiple areas of hepatocellular death and inflammatory cell infiltration as well as moderate sinusoidal congestion (Suzuki score: 7.3 ± 0.4). Hemin ameliorated these changes

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whereas ZnPP resulted in more severe pathological changes (Fig. 2D). The percentage of TUNEL-positive cells in the liver tissue significantly increased after reperfusion compared

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with that of the sham group. Hemin attenuated this change and ZnPP augmented this increase (Fig. 2E). In the I/R group, the level of cleaved caspase-3 protein expression significantly increased compared with that of the sham group. This increase was attenuated by hemin (Fig. 2F).

3.3. Effect of HO-1 on oxidative mitochondrial damage during hepatic I/R

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The serum GDH activity was 7.60 ± 3.04 U/L in the sham group. After reperfusion, serum GDH activity significantly increased to 183.4 ± 21.8 U/L compared with that of the sham group. This increase was attenuated by hemin and augmented by ZnPP (Fig. 3A). The level of MDA in liver mitochondria was 0.18 ± 0.01 nmol/mg protein in the sham group. However,

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the level of MDA significantly increased to 0.47 ± 0.04 nmol/mg protein after reperfusion. This increase was attenuated by hemin and augmented by ZnPP (Fig. 3B). The GSH/GSSG

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ratio in liver mitochondria was 10.7 ± 0.68 in the sham group. After reperfusion, the

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GSH/GSSG ratio markedly decreased to 5.07 ± 0.30 compared with that of the sham group.

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This decrease was attenuated by hemin and augmented by ZnPP (Fig. 3C).

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3.4. Effect of HO-1on mitochondrial biogenesis during hepatic I/R

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I/R did not affect the levels of PGC1α and NRF-1 protein expression compared with

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those of the sham group whereas I/R significantly increased the level of TFAM protein expression compared with that of the sham group. Hemin significantly increased the levels NRF-1 and TFAM protein expression compared with those of the I/R group whereas ZnPP

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significantly decreased the levels of PGC1α protein expression (Fig. 4A). Hepatic mtDNA

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copy number significantly increased 2.1-fold compared with that of the sham group after reperfusion. This increase was augmented by hemin and attenuated by ZnPP (Fig. 4B).

3.5. Effect of HO-1 on mitophagy during hepatic I/R After reperfusion, the hepatic level of PINK1 protein expression significantly increased and the hepatic level of Parkin protein expression significantly decreased compared with

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those of the sham group. These changes were attenuated by hemin (Fig. 5A). I/R also significantly increased the level of mitochondrial PINK1 protein expression and significantly decreased the level of mitochondrial Parkin protein expression compared with those of the sham group. These changes were attenuated by hemin (Fig. 5B). These results were

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confirmed with TEM analysis. Liver tissue after hemin treatment showed a higher number of

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autophagic vacuoles engulfing mitochondria compared with I/R group (Fig. 5C).

3.6. Effect of HO-1on mitochondrial dynamics during hepatic I/R

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After reperfusion, the protein expression level of Drp1, a key component of mitochondrial

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fission, significantly increased compared with that of the sham group. This increase was attenuated by hemin (Fig. 6A). The protein expression level of MFN2 regulating

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mitochondrial fusion significantly decreased after reperfusion compared with that of the sham

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group. This decrease was neither affected by hemin nor by ZnPP (Fig. 6B).

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in vitro H/R

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3.7. Effect of PGAM5 on HO-1-mediated mitochondrial QC regulation during in vivo I/R and

Hepatic I/R significantly decreased the level of PGAM5 protein expression compared with that of the sham group. This decrease was attenuated by hemin (Fig. 7A). We also observed the levels of HO-1 and PGAM5 protein expression in H/R. H/R significantly increased the level of HO-1 protein expression compared with that of the control group and this increase was augmented by hemin (Fig. 7B). In the H/R group, the level of PGAM5 protein expression significantly decreased compared with that of the control group and this

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decrease was attenuated by hemin (Fig. 7C). To investigate the involvement of PGAM5 in HO-1-mediated mitochondrial QC regultion, we used PGAM5 gene silencing methods in H/R model. PGAM5 siRNA significantly decreased the level of PGAM5 protein expression in HepG2 cells (Fig. 7D). The level of GDH protein expression significantly decreased

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compared with that of the control group in HepG2 cells subjected to H/R. This decrease was attenuated by hemin, which was abolished by PGAM5 siRNA. (Fig. 7E). Furthermore,

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similar to results of in vivo I/R, H/R did not affect the levels of PGC1α and NRF-1 protein

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expression. However, H/R significantly increased the level of TFAM protein expression compared with that of the control group. Hemin increased the levels of PGC1α and NRF-1

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and augmented H/R-induced TFAM protein expression compared with those of the H/R

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group. PGAM5 siRNA abolished effect of hemin on the levels of PGC1α, NRF-1 and TFAM protein expression in H/R. H/R significantly increased the level of PINK1 protein expression

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and significantly decreased the level of Parkin protein expression compared with those of the

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control group. These changes were attenuated by hemin, which were abolished by PGAM5 siRNA. After H/R, the level of Drp1 protein expression significantly increased compared with that of the control group. Hemin attenuated this increase and PGAM5 siRNA abolished

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effect of hemin in H/R (Fig. 7F).

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4. Discussion HO-1, an endoplasmic reticulum resident protein, can redistribute to mitochondria in response to stress, which contributes to preserve mitochondrial function [9]. Indeed, increased mitochondrial HO activity prevented cigarette smoke-induced cell death in lung

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epithelial cells [26]. Furthermore, in lipopolysaccharide (LPS)-treated rat liver, mitochondrial

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HO-1 protein expression increased mitochondrial respiratory chain activity and reduced ROS production via reduction in mitochondrial nitric oxide synthase activity [27]. In the present

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study, hepatic I/R increased HO enzyme activity and HO-1 protein expression in liver

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mitochondria similar to those in whole liver tissue. Mitochondria are both the source of intracellular ROS and the target of their damaging effects [28]. I/R injury is closely

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associated with mitochondrial dysfunction. GDH, a mitochondrial matrix enzyme, has been used as a marker of mitochondrial membrane integrity. Frederiks et al. [29] demonstrated

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histologically and electron microscopically that increased serum level of GDH indicates the

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presence of cell necrosis in ischemic rat liver. Mitochondrial membrane lipid peroxidation linked to the redox state is an important mechanism leading to mitochondrial dysfunction

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such as mitochondrial membrane potential collapse, and swelling [30]. In this study, I/R induced significant oxidative mitochondrial damage, as evidenced by increased serum GDH

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activity, lipid peroxidation, and decreased GSH/GSSG ratio. Furthermore, I/R induced hepatocellular damage and inflammation, as demonstrated by increased serum ALT activity and inflammatory cytokine levels. Hemin attenuated both the mitochondrial and hepatocellular damages and ZnPP augmented these changes. Collectively, our results suggest that overexpression of HO-1 prevents mitochondrial damage, which is responsible for protection of the liver against I/R injury. Many studies have suggested that irreversible mitochondrial damage following I/R

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induces cell death leading to tissue damage [31]. Mitochondria may shift the balance away from irreversible cellular injury towards recovery via maintenance of mitochondrial quality control [32]. Mitochondrial biogenesis is required for maintenance of mitochondrial number and mass, which is tightly regulated by PGC1α/ NRF-1/ TFAM signaling. PGC1α leads to

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stimulation of transcription factor NRF-1 that subsequently activates the synthesis of TFAM, a final effector activating mtDNA [33]. Lee et al. [34] reported that the increase in mtDNA

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copy number in response to oxidative stress compensates for damaged mitochondria by

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harboring an impaired respiratory chain in human lung fibroblast cell line. Knockdown of manganese-dependent superoxide dismutase stimulated mitochondrial biogenesis which

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conferred resistance to acute oxidative stress following I/R in kidney [35]. However, in a rat

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model of I/R-induced pulmonary capillary injury, oxidative stress suppressed mitochondrial biogenesis by reducing PGC1α mRNA expression [36]. These differences in results may be

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due to the use of different animal models, the inflammatory response to the stress and the

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perspective on the determination of mitochondrial biogenesis. Recently, cilostazol treatment induced Nrf2 and HO-1-dependent mitochondrial biogenesis, which protected against liver injury following I/R. [11]. In the present study, hepatic I/R increased mtDNA copy number

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and TFAM protein expression. Interestingly, hemin augmented the I/R-induced increases in

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mtDNA copy number and TFAM protein expression and increased the levels of PGC1α and NRF-1 protein expression. These results indicate that HO-1 induction enhances mitochondrial biogenesis during I/R. The importance of cross-regulation is highlighted by studies indicating that disruption of anabolic-catabolic balance between mitochondrial biogenesis and mitophagy can delay post injury recovery [37]. Mitophagy, a selective autophagy of dysfunctional mitochondria, regulates the rate of mitochondrial turnover. One of the best studied mechanisms for

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mitophagy in mammalian cells is the PINK1-Parkin pathway. When mitochondria depolarize under stress conditions, PINK1 stabilized on the mitochondrial outer membrane acts as a marker of damaged mitochondria and recruits Parkin to mitochondria leading to mitophagy [38]. In a mouse model of cigarette smoke-exposed lung, decreased Parkin translocation to

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damaged mitochondria impaired mitophagy, which was associated with increased ROS and DNA damage [39]. TEMPOL, a superoxide scavenger, restored mitophagy and resulted in

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protection of the aging myocardium [40]. Overexpression of Parkin in isolated cardiac

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myocytes reduced hypoxia-mediated cell death by promoting the removal of damaged mitochondria [41]. In the present study, we observed that I/R increased mitochondrial protein

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expression level of PINK1, indicating that hepatic I/R damages mitochondria. In contrast, I/R

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decreased the level of mitochondrial Parkin protein expression. These changes were attenuated by hemin. TEM images clearly revealed that the number of autophagic vacuoles

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engulfing mitochondria was increased in the ischemic livers of hemin-treated mice,

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suggesting that HO-1 restores impaired mitophagy during I/R. Mitochondria form networks resulting from balance between fission and fusion, which

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allows homeostatic adjustment of mitochondrial quality through mitochondrial biogenesis and mitophagy [42]. The cytoplasmic Drp1 protein plays a key role in mitochondrial fission,

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which facilitates the segregation of damaged compartments for mitophagy, and in final step of mitochondrial biogenesis for generation of daughter mitochondria [43, 44]. However, excessive fission generates dysfunctional mitochondria and causes cell death. MFN-mediated mitochondrial fusion enables mitochondrial repair by diluting the damaged components into healthy mitochondria and it is essential for the maintenance of mtDNA [45]. Cerebral I/R induced mitochondrial fragmentation by increasing mitochondrial fission and decreasing mitochondrial fusion and subsequent mitochondrial damage [46]. The inhibition of

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mitochondrial fission by mdivi-1 protected heart subjected to I/R [4]. Heart-specific MFN2deficient mice displayed autophagy defects, mitochondrial dysfunction and cell death [47]. Most recently, Yu et al. [48] reported that the HO-1 ameliorated the excessive mitochondrial fission caused by LPS and reduced the endotoxin-induced lung injury in in vitro and in vivo

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models. In doxorubicin-treated mice, cardiac specific HO-1 overexpressing mice inhibited mitochondrial fission and increased mitochondrial fusion [10]. In the present study, hepatic

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I/R induced an imbalance of mitochondrial dynamics towards fission, as evidenced by the

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increased level of Drp1 and the decreased level of MFN2. Increased fission was attenuated by hemin. Collectively, our results suggest that HO-1 reduces mitochondrial fission during I/R.

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integrative mitochondrial QC during I/R.

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To the best our knowledge, this is the first study demonstrating that HO-1 regulates

PGAM5, a member of PGAM family, lacks a similar enzymatic function of PGAM and

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instead acts as a Ser/Thr protein phosphatase. In human embryonic kidney cells, PGAM5

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dephosphorylated mitogen-activated protein kinase kinase kinase and activated JNK and p38 kinases [12]. Recent in vitro and in vivo studies have shown that PGAM5 is critical for

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mitochondrial homeostasis. Ectopic expression of PGAM5 in fibroblast-like cells resulted in perinuclear aggregation or small fragmentation of mitochondria, indicating that PGAM5 is a

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novel regulator of mitochondrial morphology and distribution [13]. PGAM5 protected PINK1 from mitochondrial inner membrane protease-mediated PINK1 degradation in response to the loss of mitochondrial membrane potential after CCCP exposure in human embryonic kidney cells [49]. Knockdown of PGAM5 caused the defective mitochondrial translocation of Parkin and exhibited defective mitophagy in mouse embryonic fibroblasts [50]. In contrast, PGAM5 induced necroptosis and Drp1-Ser637 dephosphorylation-mediated mitochondrial fission, which led to concanavalin A-induced liver injury [51]. Under fasting and cold stress

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conditions, PGAM5 deficiency failed to maintain proper mitochondrial integrity, but it showed resistance to metabolic stresses in mice [52]. Meanwhile, Claude et al. [53] suggested that a ternary complex containing Nrf2, Keap1 and PGAM5 might contribute to initiate mitochondrial biogenesis following oxidative stress by activating HO-1. In our study, hepatic

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I/R significantly decreased the level of PGAM5 protein expression, which was attenuated by hemin. After H/R, PGAM5 and HO-1 protein expressions showed similar patterns compared

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with those in I/R. To clarify the involvement of PGAM5 in HO-1-mediated mitochondrial QC

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regulation, we used PGAM5 gene silencing by siRNA in H/R model. PGAM5 siRNA abolished the effect of hemin on mitochondrial biogenesis, mitophagy and mitochondrial

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fission, as demonstrated by the decreased levels of PGC1α, NRF-1, TFAM and Parkin protein

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expression and the increased levels of PINK1 and Drp1 protein expression. Moreover, PGAM5 siRNA abolished the protective effect of HO-1 on mitochondrial damage, as

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evidenced by decreased level of GDH protein expression in H/R. These results suggest that

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HO-1 regulates mitochondrial QC collectively via activation of PGAM5 signaling. Elucidation of PGAM5 signaling in mitochondrial QC and its regulation by HO-1 are novel

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5. Conclusion

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finding in the current study.

In conclusion, these results suggest that upregulation of HO-1 protects the liver against I/R injury by enhancing mitochondrial biogenesis, recovering impaired mitophagy and suppressing mitochondrial fission. Moreover, our findings suggest that HO-1 regulates mitochondrial QC via activation of PGAM5 signaling. These findings highlight potential of PGAM5 as a therapeutic target for mitochondrial QC and suggest HO-1 as a possible regulator of mitochondrial QC in ischemic diseases.

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Acknowledgements This research was supported by the Mid-Career Researcher Program through an NRF grant funded by the Ministry of Education, Science, and Technology (MEST) in Korea (NRF2016R1A2B4009880). J.-M.H. received ‘Global Ph.D. Fellowship Program’ support (NRF-

Conflict of interest

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The authors declare no conflicts of interest.

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2016H1A2A1909480) from NRF funded by the MEST in Korea.

Authors’ contributions

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Jeong-Min Hong designed experiments, performed experiments, analyzed data, and wrote

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the manuscript. Sun-Mee Lee designed experiments, analyzed data, contributed reagents or

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other essential material, and wrote the manuscript.

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Figure legends Fig. 1. Schematic presentation of the experimental procedures. Protocol 1: Mice were subjected to 1 h of hepatic ischemia followed by 6 h of reperfusion. Protocol 2: Effects of Hemin or ZnPP on hepatocellular damage and mitochondrial QC during hepatic I/R. Hemin

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or ZnPP was administered 16 h and 3 h prior to ischemia. Mice were sacrificed 6 h after

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reperfusion.

Fig. 2. HO-1 protects hepatocellular damage during I/R. (A) Serum ALT activity. Mean ±

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S.E.M. (n = 6-8), **p < 0.01 vs. sham, ##p < 0.01 vs. I/R. (B) Serum IL-6 level. Mean ± S.E.M.

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(n = 6-8), **p < 0.01 vs. sham, ##p < 0.01 vs. I/R. (C) Serum IL-1β level. Mean ± S.E.M. (n = 6-8), **p < 0.01 vs. sham, ##p < 0.01 vs. I/R. (D) Representative histological images of liver

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section stained with H&E at 6 h of reperfusion. Original magnification × 200. Black arrows

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indicate necrotic areas and white arrows indicate inflammatory cell infiltration. Histological lesions were graded using Suzuki score. Mean ± S.E.M. (n = 6-8), **p < 0.01 vs. sham, ##p < 0.01 vs. I/R. (E) Representative liver section images of TUNEL staining at 6 h of reperfusion.

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Original magnification × 200. Mean ± S.E.M. (n = 6-8), **p < 0.01 vs. sham, #p < 0.05, ##p <

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0.01 vs. I/R. (F) Western blot analysis of cleaved caspase-3 protein expression level. Mean ± S.E.M. (n = 7), **p < 0.01 vs. sham, ##p < 0.01 vs. I/R.

Fig. 3. HO-1 protects mitochondrial oxidative damage during hepatic I/R. (A) Serum GDH activity. Mean ± S.E.M. (n = 6-8), **p < 0.01 vs. sham, #p < 0.05, ##p < 0.01 vs. I/R. (B) MDA level in liver mitochondria. Mean ± S.E.M. (n = 7), *p < 0.05, **p < 0.01 vs. sham. #p < 0.05,

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##

p < 0.01 vs. I/R. (C) Mitochondrial GSH/GSSG ratio. Mean ± S.E.M. (n = 7), **p < 0.01 vs.

sham. ##p < 0.01 vs. I/R.

Fig. 4. HO-1 increases mitochondrial biogenesis during hepatic I/R. (A) Western blot analysis

##

p < 0.01 vs. I/R. (B) Real-time RT-PCR analysis of mtDNA

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< 0.01 vs. sham, #p < 0.05,

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of PGC1α, NRF-1 and TFAM protein expression levels. Mean ± S.E.M. (n = 7), *p < 0.05, **p

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copy number. Mean ± S.E.M. (n = 7), *p < 0.05, **p < 0.01 vs. sham, ##p < 0.01 vs. I/R.

Fig. 5. HO-1 restores impaired mitophagy during hepatic I/R. (A) Western blot analysis of

0.01 vs. sham, #p < 0.05,

##

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hepatic PINK1 and Parkin protein expression levels. Mean ± S.E.M. (n = 7), *p < 0.05, **p < p < 0.01 vs. I/R. (B) Western blot analysis of mitochondrial

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PINK1 and Parkin protein expression levels. Mean ± S.E.M. (n = 7), *p < 0.05, **p < 0.01 vs.

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sham, #p < 0.05, ##p < 0.01 vs. I/R. (C) Representative TEM images of mitophagic vacuoles (white arrows) and autophagic vacuoles (black arrows); M, mitochondria. Mean ± S.E.M. (n

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= 6-8), **p < 0.01 vs. sham, ##p < 0.01 vs. I/R.

Fig. 6. HO-1 regulates mitochondrial dynamics during hepatic I/R. (A) Western blot analysis of Drp1 protein expression level. Mean ± S.E.M. (n = 7), *p < 0.05, **p < 0.01 vs. sham, #p < 0.05 vs. I/R. (B) Western blot analysis of MFN2 protein expression level. Mean ± S.E.M. (n = 7), *p < 0.05, **p < 0.01 vs. sham.

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Fig. 7. HO-1 regulates mitochondrial QC through PGAM5 signaling. (A) Western blot analysis of PGAM5 protein expression level during hepatic I/R. Mean ± S.E.M. (n = 7), **p < 0.01 vs. sham, #p < 0.05 vs. I/R. (B-C) Western blot analysis of HO-1 and PGAM5 protein expression levels in HepG2 cells subjected to H/R. Mean ± S.E.M. of three independent

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experiments. *p < 0.05, **p < 0.01 vs. control, #p < 0.05, ##p < 0.01 vs. H/R. (D) Western blot analysis of PGAM5 protein expression to confirm the efficacy of PGAM5 siRNA. **p < 0.01

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vs. control. (E) Western blot analysis of GDH protein expression level in HepG2 cells

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subjected to H/R. Mean ± S.E.M. of three independent experiments. *p < 0.05, **p < 0.01 vs. control, #p < 0.05 vs. H/R, &&p < 0.01 vs. Hemin + H/R. (F) Western blot analysis of PGC1α,

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NRF-1, TFAM, PINK1, Parkin and Drp1 protein expression levels in HepG2 cells subjected

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to H/R. Mean ± S.E.M. of three independent experiments. *p < 0.05, **p < 0.01 vs. control, #p

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< 0.05, ##p < 0.01 vs. H/R, &p < 0.05, &&p < 0.01 vs. Hemin + H/R.

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Table 1 Effect of pretreatment with hemin or ZnPP on HO activity in both the liver microsome and mitochondria fractions during hepatic I/R. HO activity in the liver microsome

HO activity in the liver mitochondria

(pmol/min/mg protein)

(pmol/min/mg protein)

Sham

74.2 ± 3.43

96.7 ± 5.08

I/R

119.5 ± 10.2**

Hemin + I/R

156.1 ± 9.47**, #

ZnPP + I/R

77.2 ± 5.67#

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Group

148.4 ±10.6*

196.6 ± 11.6**, # 102.2 ± 10.1#

Results are presented as mean ± S.E.M of 6-8 animals per group. *p < 0.05,

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sham. #p < 0.05 vs. I/R.

**

p < 0.01 vs.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7