Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by...

12
Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells LEI SUN, 1 MEI ZHAO, 1 YANG YANG, 1 RUN-QING XUE, 1 XIAO-JIANG YU, 1 JIAN-KANG LIU, 2 AND WEI-JIN ZANG 1 * 1 Departmentof Pharmacology, Xian Jiaotong University Health Science Center, Xian, Shaanxi, P.R. China 2 Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xian Jiaotong University, Xian, Shaanxi, P.R. China Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement of mitophagy in ACh-elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN-induced kinase 1 (PINK1) and Parkin translocation were examined using western blot and confocal uorescence microscopy. Mitochondrial membrane potential and reactive oxygen species (ROS) were detected with uorescence staining. We found that H/R-treated cells exhibited reduced levels of PINK1 and Parkin in mitochondria, accompanied with decreased autophagy ux (reduced LC3-II/LC3-I and increased p62). Conversely, ACh increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTracker Green-labeled mitochondria further conrmed ACh-induced mitochondrial translocation of Parkin, which was reversed by M 2 receptor antagonist methoctramine and M 2 receptor siRNA, suggesting ACh could induce mitophagy by M 2 receptor after H/R. Mitophagy inhibitor 3-methaladenine abolished ACh-induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP and membrane potential depletion, increased ROS overproduction, and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated the protective effects of ACh against ATP loss and oxidative stress due to mitochondrial-dependent injury. Taken together, ACh promoted mitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M 2 receptor, which may provide benecial targets in the preservation of cardiac homeostasis against H/R injury. J. Cell. Physiol. 231: 11711181, 2016. ß 2015 Wiley Periodicals, Inc. Myocardial ischemia/reperfusion (I/R) injury is a complex process which causes damage to proteins, DNA and plasma membrane, leading to cell death and a decreased cardiac output (Hausenloy and Yellon, 2013; Go et al., 2014). Mitochondrial dysfunction has been emerged as a critical pathophysiological factor of I/R injury (Walters et al., 2012). As important gate- keepers of life and death, damaged mitochondria are prone to release hazardous materials, particularly high levels of reactive oxygen species (ROS), Ca 2þ and cytochrome c to the cytosol, and thereby trigger mitochondria-dependent apoptosis (Ong and Gustafsson, 2012; Lopez-Armada et al., 2013). Especially cardiomyocytes, which are post-mitotic and rely heavily on energy production by mitochondria, are more sensitive to mitochondrial dysfunction (Liang et al., 2013). Thus, elimination of dysfunctional mitochondria is imperative to cell survival in cardiomyocytes. Autophagy is an evolutionarily conserved process through which the cytoplasmic components and organelles are degraded and recycled in lysosomes (Kroemer et al., 2010). In cardiomyocytes, autophagy involves predominantly in a pro-survival pathway (Przyklenk et al., 2012). Concurrent mitochondrial elimination and autophagy in many organs has led to the proposal that autophagy is the main regulator of mitochondrial turnover (Zhang, 2013; Shimizu et al., 2014). Recently, mitochondrial autophagy (termed mitophagy) is found to eliminate dysfunctional mitochondria and regulate mitochondrial population (Ashraand Schwarz, 2013; Hammerling and Gustafsson, 2014). Previous studies have demonstrated that impaired mitophagy exists in a number of neurodegenerative diseases (de Vries and Przedborski, 2013; Lu et al., 2013). Moreover, PINK1/Parkin-mediated mitophagy has been reported to prevent cellular damage and participate in preconditioning (Huang et al., 2011). Accordingly, mitophagy may have a therapeutic potential for mitochondria-associated diseases such as I/R injury. Conicts of interest: None. Lei Sun and Mei Zhao contributed equally to this work. Contract grant sponsor: National Natural Science Foundation of China; Contract grant numbers: 81473203, 81120108002. Contract grant sponsor: Specialized Research Fund; Contract grant number: 20130201130008. *Correspondence to: Wei-Jin Zang, Department of Pharmacology, Xian Jiaotong University Health Center, No. 76 Yanta West Road, Xian, 710061 Shaanxi, P.R. China E-mail: [email protected] Manuscript Received: 7 March 2015 Manuscript Accepted: 12 October 2015 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 14 October 2015. DOI: 10.1002/jcp.25215 ORIGINAL RESEARCH ARTICLE 1171 Journal of Journal of Cellular Physiology Cellular Physiology © 2015 WILEY PERIODICALS, INC.

Transcript of Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by...

Page 1: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

Acetylcholine AttenuatesHypoxia/Reoxygenation Injury byInducing Mitophagy ThroughPINK1/Parkin Signal Pathway inH9c2 CellsLEI SUN,1 MEI ZHAO,1 YANG YANG,1 RUN-QING XUE,1 XIAO-JIANG YU,1

JIAN-KANG LIU,2 AND WEI-JIN ZANG1*1Departmentof Pharmacology, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi, P.R. China2Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an

Jiaotong University, Xi’an, Shaanxi, P.R. China

Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement ofmitophagy in ACh-elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected tohypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN-induced kinase 1 (PINK1) and Parkintranslocation were examined using western blot and confocal fluorescence microscopy. Mitochondrial membrane potential and reactiveoxygen species (ROS) were detected with fluorescence staining. We found that H/R-treated cells exhibited reduced levels of PINK1 andParkin in mitochondria, accompanied with decreased autophagy flux (reduced LC3-II/LC3-I and increased p62). Conversely, AChincreased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTrackerGreen-labeled mitochondria further confirmed ACh-induced mitochondrial translocation of Parkin, which was reversed by M2 receptorantagonist methoctramine and M2 receptor siRNA, suggesting ACh could induce mitophagy by M2 receptor after H/R. Mitophagy inhibitor3-methaladenine abolished ACh-induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP andmembrane potential depletion, increased ROS overproduction, and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated theprotective effects of ACh against ATP loss and oxidative stress due to mitochondrial-dependent injury. Taken together, ACh promotedmitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M2 receptor, which may provide beneficial targetsin the preservation of cardiac homeostasis against H/R injury.J. Cell. Physiol. 231: 1171–1181, 2016. � 2015 Wiley Periodicals, Inc.

Myocardial ischemia/reperfusion (I/R) injury is a complexprocess which causes damage to proteins, DNA and plasmamembrane, leading to cell death and a decreased cardiac output(Hausenloy and Yellon, 2013; Go et al., 2014). Mitochondrialdysfunction has been emerged as a critical pathophysiologicalfactor of I/R injury (Walters et al., 2012). As important gate-keepers of life and death, damaged mitochondria are prone torelease hazardous materials, particularly high levels of reactiveoxygen species (ROS), Ca2þ and cytochrome c to the cytosol,and thereby trigger mitochondria-dependent apoptosis (Ongand Gustafsson, 2012; Lopez-Armada et al., 2013). Especiallycardiomyocytes, which are post-mitotic and rely heavily onenergy production by mitochondria, are more sensitive tomitochondrial dysfunction (Liang et al., 2013). Thus, eliminationof dysfunctional mitochondria is imperative to cell survival incardiomyocytes.

Autophagy is an evolutionarily conserved process throughwhich the cytoplasmic components and organelles aredegraded and recycled in lysosomes (Kroemer et al., 2010). Incardiomyocytes, autophagy involves predominantly in apro-survival pathway (Przyklenk et al., 2012). Concurrentmitochondrial elimination and autophagy in many organs hasled to the proposal that autophagy is the main regulator ofmitochondrial turnover (Zhang, 2013; Shimizu et al., 2014).Recently, mitochondrial autophagy (termed mitophagy) isfound to eliminate dysfunctional mitochondria and regulatemitochondrial population (Ashrafi and Schwarz, 2013;Hammerling and Gustafsson, 2014). Previous studies have

demonstrated that impaired mitophagy exists in a number ofneurodegenerative diseases (de Vries and Przedborski, 2013;Lu et al., 2013). Moreover, PINK1/Parkin-mediated mitophagyhas been reported to prevent cellular damage and participate inpreconditioning (Huang et al., 2011). Accordingly, mitophagymay have a therapeutic potential for mitochondria-associateddiseases such as I/R injury.

Conflicts of interest: None.

Lei Sun and Mei Zhao contributed equally to this work.

Contract grant sponsor: National Natural Science Foundation ofChina;Contract grant numbers: 81473203, 81120108002.Contract grant sponsor: Specialized Research Fund;Contract grant number: 20130201130008.

*Correspondence to: Wei-Jin Zang, Department of Pharmacology,Xi’an Jiaotong University Health Center, No. 76 YantaWest Road,Xi’an, 710061 Shaanxi, P.R. ChinaE-mail: [email protected]

Manuscript Received: 7 March 2015Manuscript Accepted: 12 October 2015

Accepted manuscript online in Wiley Online Library(wileyonlinelibrary.com): 14 October 2015.DOI: 10.1002/jcp.25215

ORIGINAL RESEARCH ARTICLE 1171J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

© 2 0 1 5 W I L E Y P E R I O D I C A L S , I N C .

Page 2: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

The imbalance of autonomic nervous system plays a vitalrole in the heart diseases (Manfrini et al., 2008; De andSchwartz, 2011; Schwartz and De, 2011). Therapeuticstrategies targeting enhancing vagal activity have the advantagesto reduce injury and enhance recovery of myocardial functionin both animal studies and human clinical practice (Hiraki et al.,2012; Kong et al., 2012; Mastitskaya et al., 2012). Althoughacetylcholine (ACh) has been reported to decrease oxidativestress and improve mitochondrial function, mechanisms ofACh-mediated protection are not fully understood (Miao et al.,2014; Sun et al., 2013, 2014). Our recent study hasdemonstrated a role of autophagy in ACh-elicitedcardioprotection against I/R injury (Zhao et al., 2013).However, little information is available concerning whethermitophagy is involved in ACh-induced cardioprotection.Therefore, we examined the protective effects of ACh incardiomyocytes against H/R-induced defective mitophagy, witha focus on PINK1/Parkin pathway.

Materials and MethodsCell culture and treatment

An original clonal cell line derived from embryonic rat heart H9c2was obtained from ATCC (CRL-1446, Manassas, VA). Cells weregrown in DMEM (Hyclone, China) containing 10% FBS (Hyclone,China) and 1% penicillin/streptomycin (Sigma, St. Louis, MO) at37°C in humidified incubator with 95% atmosphere and 5% CO2.

After serum starvation, H9c2 cells were transferred into a hypoxicincubator containing 1% O2, 5% CO2, and 94% N2 withischemia-mimetic solution (in mM: NaCl, 135; KCl, 8; MgCl2,0.5; NaH2PO4, 0.33; HEPES, 5.0; CaCl2, 1.8; Naþ-lactate, 20; pH6.8; Lu et al., 2005). For reoxygenation, cells subjected to hypoxiawere incubated with fresh DMEM and rapidly transferred into anormoxic incubator. ACh (10�8�10�5M or 10�6M) with orwithout 3-methyladenine (autophagy inhibitor, 3-MA, 5mM) ormethoctramine (M2 receptor antagonist, METH, 10�6M) wereadministered at the onset of reoxygenation.

RNA interference and gene transfection

siRNA oligonucleotides were synthesized by GenePharma(GenePharma, Shanghai, China). The sequences were as follows:negative control siRNA: 50-UUC UCC GAA CGU GUC ACGUTT-30; M2 receptor siRNA: 50-GUGCUC AUCAAUACUUUCUTT-30; PINK1 siRNA: 50-GCCCAUCCAUCUAAGUUCUTT-30, Parkin siRNA: 50-CAA GGA AGC AUA CCA UGA ATT-30.siRNAs were then transfected into cells using Lipofectamine 2000(Invitrogen, Carlsbad, CA) according to the manufacturer’sprotocol with 100 nM of each siRNA. The transfection complex(siRNA and the transfection reagent mixture) were added to themedium and mixed gently by rocking the media. After 6 h, the cellculture mediumwas changed back to DMEM containing serum andincubated at 37°C for 48 h. The cells were then used for thefollowing experiments.

Fig. 1. H/R caused cell injury and defective mitophagy during different reoxygenation periods. (A) H/R reduced cell viability. (B and C) H/Rincreased the level of cleaved-caspase 3. (D–H) H/R led to impaired autophagy and mitophagy, appearing as decreased levels of LC3-II/LC3-I,accumulation of p62, and suppressive mitochondrial translocation of PINK1 and Parkin by western blot analysis. �P< 0.05 and ��P< 0.01versus control group. (n¼ 6).

JOURNAL OF CELLULAR PHYSIOLOGY

1172 S U N E T A L .

Page 3: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

Cell viability assay

Cell viability was determined using the conventional MTT assay.While grown in 96-well microplates at a density of 20,000 cells perwell, cells were treated with hypoxia/reoxygenation (H/R) and/orACh. Then MTT solution (Sigma) was added to DMEM with a finalconcentration of 0.5mg/ml for 4 h at 37°C. Absorbancemeasurements were read at a wave length of 450 nm. The opticaldensity value was reported as the ratio to control group (set as100%).

Cell fractionation and mitochondrial isolation

Cells were homogenized in RIPA buffer (Beyotime, Nanjing,China) and the lysates were further centrifuged at 12,000 rpmfor 15min at 4°C, then the supernatants were collected andstored at �80 °C. For cytosolic and mitochondrial proteinsextraction, an isolation kit (Beyotime, Nanjing, China) wasused based on manufacturer’s instruction as follows. Cellswere rinsed with pre-cooling PBS and gently lysed with ahypotonic buffer on ice. The lysate was disrupted byforcefully passing cells in a microfuge tube and then centrifugedat 600g for 10min at 4°C to eliminate the nuclear fraction andunbroken cells. The supernatant was further centrifuged at11,000g for 10min at 4°C, the pellet was collected as themitochondria-enriched fraction, from which the mitochondrial

proteins were further resuspended in mitochondrial lysisbuffer. The remaining supernatant was then centrifuged 12,000gfor 10min at 4°C as cytosolic proteins. Protein concentrationswere determined using NanoPhotometer

1

P-330 (Implen,Germany).

Western blot analysis

Equal amount of protein from each sample were separated usingSDS-PAGE and electrotransferred on to polyvinylidene difluoridemembrane (Roche diagnostics, Indianapolis, IN). After blocking,blots were probed with primary antibodies against LC3 (1:1000dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST,USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707),Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST,USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S),GAPDH (1:5000 dilution, Sinopept, China, 41301M), and SubunitIV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA,4850) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibody for 30min at 37°C. Then, theproteins were visualized by chemiluminescence exposure toX-ray film. GAPDH and COX IV were served as the loadingcontrol of cytosolic and mitochondrial fractions, respectively(Yu et al., 2006).

Fig. 2. ACh induced cytoprotective mitophagy following H/R. (A) ACh (10-8–10-5–M) increased cell viability in concentration-dependentmanner. (B and C) ACh reduced the level of cleaved-caspase 3. (D–H) ACh promoted autophagy and mitophagy, appearing as increased levelsof LC3-II/LC3-I, decreased p62, and mitochondrial translocation of PINK1 and Parkin by western blot analysis. �P< 0.05 and ��P< 0.01 versuscontrol group. #P< 0.05 and ##P< 0.01 versus H/R group. (n¼ 6).

JOURNAL OF CELLULAR PHYSIOLOGY

A C h I N D U C E D P I N K 1 / P A R K I N - D E P E N D E N T M I T O P H A G Y 1173

Page 4: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

Measurement of caspase 3 activity

Caspase-3 activity was determined using the caspase-3 activityassay kit (Bestbio, Shanghai, China). Cells were harvested andextracted on ice in lysis buffer for 15min. After centrifugation at500g for 5min, supernatant samples containing 50mg proteinwere incubated with reaction buffer. and caspase 3 substrate(containing 40mmol/L N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide)by the volume proportion of 9:1 at 37°C for 2 h away from light.The OD values were measured at 405 nm with aspectrophotometer.

Determination of ROS

H9c2 cells were incubated with 10 mM 20,70-dichlorofluorescindiacetate (DCFH-DA, Beyotime) for 30min at 37°C in thedark, then washed with pre-warmed serum-free DMEM atleast three times. The fluorescence of the cells was observedwith confocal scanning microscope (Leica, Heidelberg,Germany).

Mitochondrial membrane potentials assay

Mitochondrial membrane potential was assessed using the5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-benzimidazolecarbocyanide iodine (JC-1, Beyotime, Nanjing,China). H9c2 cells were incubated with JC-1 staining solution(10mg/ml) for 20min at 37°C in the dark and rinsed twice withbuffer (provided as part of the kit and pre-cooled at 4°C). JC-1fluorescence was measured by confocal scanning microscopeunder single excitation (488 nm) and dual-emission (530 and590 nm). The ratio of red and green fluorescence intensitiesreflects the changes in mitochondrial membrane potential.

Assessment of ATP content

ATP content was determined by bioluminescent assay kit (Beyotime,Nanjing, China) according to the manufacturer’s protocol. Briefly,cells were homogenized in lysis buffer and centrifuged at 12,000g for10min at 4°C. The supernatant was added to detection reagent(provided in the kit) and the ATP content was determined by a

Fig. 3. M2 receptor antagonist METH suppressed ACh-activated mitophagy. (A–E) Expressions of autophagy/mitophagy markers includingLC3, p62, PINK1, and Parkin. (F and G) Co-localization of Parkin and MitoTracker Green-labeled mitochondria. Fluorescent images werecaptured by confocal microscopy and columns represent the Manders overlap coefficient of Parkin and mitochondria. At least 62 cells fromthree independent experiments for each group were included. Scale bar: 50mm. �P< 0.05 and ��P< 0.01 versus control group. #P< 0.05 versusH/R group. &P< 0.05 versus H/RþACh group.

JOURNAL OF CELLULAR PHYSIOLOGY

1174 S U N E T A L .

Page 5: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

multi-mode microplate reader with luminescence luminometer(FLUOstar Omega, BMG Labtech, Germany).

Transmission electron microscopy

H9c2 cells were harvested and fixed with fresh 2.5%glutaraldehyde and 4% paraformaldehyde in 0.1M phosphatebuffer for 2 h at 4°C. After washing in phosphate buffer, the cellswere post-fixed with 1% osmium tetroxide in 0.1M phosphatebuffer. The samples were dehydrated and infiltrated withpropylene oxide, embedded in epoxy resin. Ultrathin sectionswere placed on copper grids and double-stained with uranylacetate and lead citrate. The ultrastructure of myocardium cell wasobserved with a transmission electron microscope (H-7650;Hitachi, Tokyo, Japan) at 80 kV accelerating voltage.

Immunofluorescence analysis

H9c2 cells were cultured on glass-bottom dish. After indicatedtreatments, the cells were fixed with 4% formaldehyde for 30minat room temperature and then were blocked in 5% normal goatserum/0.3% Triton X-100 in PBS for 1 h at room temperature.Subsequently, the cells were incubated with antibodies againstParkin (1:100 dilution) overnight at 4°C. The sections were rinsedwith PBS and sequentially incubated respectively with TRITC-conjugated anti-rabbit secondary antibody (1:200, Zhongshan

Goldenbridge Biotechnology, Beijing, China) in a humidifiedcontainer for 1 h at 37°C. Finally, MitoTracker Green (1:5000dilution, Beyotime, Nanjing, China) was used to visualizemitochondria. DAPI counterstaining was performed to stain thenuclei. Images were viewed by a Leica TCS-SP2 confocal scanningmicroscope (Leica, Heidelberg, Germany). Mander’s overlapcoefficient was used to quantify the degree of colocalization byImage Pro-Plus software (Zinchuk et al., 2007). Five randomly-selected fields from one coverslip were included to get an average,and experiments were repeated independent at least 3 times.

Statistical analysis

Data are presented as means� SEM. Statistical analysis wasprocessedwith one-way ANOVA followed by Tukey post-hoc testor Student’s t test. P< 0.05 was considered as statisticallysignificant. The number of experimental samples used in eachgroup is presented in the figure legends.

ResultsH/R caused defective mitophagy

Cardiomyocytes are sensitive to ischemia/reperfusion damageand undergo apoptosis due to insufficient oxygen supply. Wefirst determined the time course of changes in cell injury afterreoxygenation. H9c2 cells were subjected to 8 h of hypoxia

Fig. 4. M2 receptor siRNA attenuated ACh-mediated mitophagy. (A–E) Expressions of autophagy/mitophagy markers including LC3, p62,PINK1, and Parkin after M2 receptor siRNA transfection. (F and G) Co-localization of Parkin and MitoTracker Green-labeled mitochondria.Fluorescent images were captured by confocal microscopy and columns represent the Manders overlap coefficient of Parkin andmitochondria. At least 78 cells from three independent experiments for each groupwere included. Scale bar: 50mm. �P< 0.05 and ��P< 0.01versus control group. #P< 0.05 versus H/R group.

JOURNAL OF CELLULAR PHYSIOLOGY

A C h I N D U C E D P I N K 1 / P A R K I N - D E P E N D E N T M I T O P H A G Y 1175

Page 6: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

followed by 4, 8, or 16 h of reoxygenation. As shown inFigure 1A–C, reoxygenation induced a moderate decrease incell viability concurrent with an increase in the expression ofcleaved-caspase 3.

Autophagy is a lysosome-dependent cellular catabolicprocess accompanied with LC3-II conversion from LC3-I andp62 degradation, which removes impaired cellular componentsin a timely manner (Mizushima et al., 2010). Thereby, wemonitored the time course of autophagy as indicated by theratio of LC3-II/L3-I and p62 expression. Compared withcontrol cells, H/R sustained defective autophagy, as judged bydecreased levels of LC3-II/LC3-I and accumulation of p62(Fig. 1D–F). Of note, mitophagy as a selective form ofautophagy induces autophagic clearance of dysfunctionalmitochondria through specialized molecules to sense damagedmitochondria (Jin and Youle, 2012; Hattori et al., 2014). Wenext sought to determine the dynamic changes of PINK1/Parkin-mediated mitophagy at the different time ofreoxygenation. As Figure 1D, G, and H illustrated, the proteinexpression of PINK1 and Parkin in cytosol lysates fell to thelowest at 4 h of reoxygenation and then increased slightly,which is counter to the expression inmitochondrial extraction.These results suggested that H/R reduced PINK1/Parkin-mediated mitophagy due to insufficient mitochondrial

translocation of PINK1 and Parkin. H/R in H9c2 cells causeddefective autophagy and mitophagy, and ultimately led to celldeath. According to these results, hypoxia 8 h followed byreoxygenation 4 h was chosen in the subsequent experiment.

ACh enhanced cytoprotective mitophagy

ACh (10�8�10�5M) administered at the beginning ofreoxygenation restored cell survival and decreasedexpression of caspase 3 in a concentration-dependentmanner (Fig. 2A–C). Likewise, western blot analysisshowed that ACh treatment especially at10�7�10�5M markedly increased the ratio of LC3-II/LC3-Iaccompanied with the loss of p62 (Fig. 2D–F), indicatingthat ACh improved H/R-induced impaired autophagy,which is in accordance with previous observations (Zhaoet al., 2013). We further examined whetherPINK1/Parkin translocation to mitochondria wasreverted by ACh. As shown in Figure 2D, G, and H, AChadministration increased the expression of PINK1 andParkin in mitochondrial fraction of H9c2 cells subjected toreoxygenation, and its effects appeared concentrationdependent. Taken together, these data indicated that AChtreatment enhanced cytoprotective mitophagy.

Fig. 5. ACh-induced mitophagy protected mitochondrial structure and function against H/R injury. (A) Ultrastructural examination of H9c2cells. Upper panel represents H9c2 cells subjected to H/R, H/RþACh, and H/RþAChþ 3-MA, respectively. Lower panel represents parallelmitochondria morphology (N, nucleus; M, mitochondria). Black bar: 1mm. (B) ATP content. (C and D) JC-1 staining. The ratio of Red/Greenfluorescence reflects change in the mitochondrial membrane potential. Scale bar: 50mm. ��P< 0.01 versus control group. #P< 0.05 and##P< 0.01 versus H/R group. &P< 0.05 and &&P< 0.01 versus H/RþACh group. (n¼ 6).

JOURNAL OF CELLULAR PHYSIOLOGY

1176 S U N E T A L .

Page 7: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

Considering its effectivity and hypotoxicity, ACh at aconcentration of 10�6M was applied for the remainingexperiments.

ACh activated mitophagy through M2 receptor

It is well known that ACh modulates heart function viamuscarinic receptors. In the heart, most functionalresponses are associated with M2 receptor (Harvey, 2012).To determine the role of M2 receptor in ACh-inducedmitophagy, the specific M2 receptor antagonist METH wasused and itself had no obvious effect on cell viability eitherunder control or H/R condition (Supplementary Fig. S1A).As shown in Figure 3A–C, METH treatment partiallysuppressed the activation of autophagy by ACh in H9c2cells, showing as reduction of LC3-II/LC3-I andaccumulation of p62. Similarly, after M2 receptor inhibitionwith METH, H9c2 cells exhibited a marked decrease of bothPINK1 and Parkin mitochondrial translocation by westernblot analysis (Fig. 3A, D, and E). To verify the observation,Parkin translocation was measured by immunofluorescenceand the overlap between Parkin and MitoTracker Green-labeled mitochondria was quantified. ACh treatmentincreased the overlap coefficient between Parkin andmitochondria following H/R; while METH blocked ACh-induced mitochondrial translocation of Parkin (Fig. 3F andG). In addition to the pharmacologic inhibitor, M2 receptorsiRNA was utilized to confirm this finding. In addition, M2receptor siRNA did not affect cell viability (SupplementaryFig. S2). As expected, ACh failed to improve autophagy/

mitophagy in H9c2 cells transfected with M2 receptorsiRNA (Fig. 4). These results strongly suggested that theeffects of ACh on mitophagy were mediated through M2receptor.

ACh-mediated mitophagy alleviated mitochondrialdysfunction following H/R

Depletion of autophagy has been found to severely blunt theloss of damaged mitochondria (Huang et al., 2011). Since AChalso induces cardiac autophagy, we hypothesize that ACh mayconfer mitoprotection through induction of mitophagy.Thus, 3-MA (an autophagy inhibitor) was co-administeredwith ACh at the onset of reoxygenation and itself had noobvious effect on cell viability either under control or H/Rcondition (Supplementary Fig. S1B). Transmission ElectronMicroscopy is a reliable method to monitor dysfunctionalmitochondria. As shown in Figure 5A, H/R exhibited celledema, disruption of cardiomyocyte integration. Moreover,mitochondria in H/R groupwere swollen and their cristae werebarely distinguishable. On the other hand, cells treated withACh restored the fine structure of cristae. Furthermore,apparent nuclear apoptosis was noted in 3-MA group.

Mitochondria are critical organelles to ATP productionfor cellular metabolism and ATP content incardiomyocytes was detected using an ATP bioluminescentassay. Our results showed that H/R injury significantly ledto the depletion of cellular ATP reserves and AChtreatment fully restored the energy production in H9c2cells, apparently favorable to the cell survival. However,

Fig. 6. ACh alleviated damaged mitochondria-based cell injury. (A and B) Measurement of ROS by DCFH-DA after 4 h of reoxygenation.Scale bar: 50mm. (C and D) Western blot analysis for cytochrome c release. (E) Activity of caspase 3. ��P< 0.01 versus control group.##P< 0.01 versus H/R group. &P< 0.05 and &&P< 0.01 versus H/RþACh group. (n¼ 6).

JOURNAL OF CELLULAR PHYSIOLOGY

A C h I N D U C E D P I N K 1 / P A R K I N - D E P E N D E N T M I T O P H A G Y 1177

Page 8: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

3-MA dramatically abrogated the beneficial effects of AChon ATP generation (Fig. 5B). Mitochondrial function wasalso evaluated by examining the alteration in membranepotentials. In general, normal H9c2 cells hadpredominantly high membrane potential and were stainedwith JC-1 aggregates emitted red fluorescence. H/R cellsexhibited severely depolarized mitochondrial membranepotential with higher levels of JC-1 monomers emittedgreen fluorescence. Such a fluorescence shift suggested ageneral drop of mitochondrial membrane potential inH9c2 cells exposed to H/R. ACh relieved membranepotential decline, which appeared as the significantincrease of the Red/Green ratio compared to that in H/Rgroup. Likewise, this effect was blocked by 3-MA(Fig. 5C and D).

Substantial ROS production is a marker of mitochondrialdysfunction. The DCFH-DA was used to monitor the levelsof ROS in cardiomyocytes. As shown in Figure 6A and B, ROSelevated remarkably followed by H/R and treatment withACh significantly reduced ROS generation compared withuntreated cells. 3-MA prevented ACh-induced inhibition ofROS production. In addition, damaged mitochondria lead torupture of outer mitochondrial membrane and release ofpro-apoptotic proteins including cytochrome c into

cytoplasm where they promote caspases activation (Whelanand Zuckerbraun, 2013; Kamat et al., 2014). Our findingsshowed that ACh attenuated cytochrome c release andcaspase 3 activity. However, 3-MA treated cells abolished theprotection of ACh (Fig. 6C–E). Overall, the data indicatedthat mitochondria clearance is an autophagy-dependentprocess and mitophagy is one of the mechanisms forACh-mediated mitoprotection.

ACh-mediated mitoprotection was dependent onPINK1/Parkin

PINK1 and parkin are linked in a common pathway whichcontrol mitochondrial function and autophagy. Todetermine whether PINK1/Parkin was required for ACh-mediated cardioprotection, we used siRNA todownregulate PINK1 and Parkin. In preliminary experiment,transient transfection of PINK1 and Parkin decreasedprotein levels compared with negative control siRNA(Supplementary Fig. S3A and B). In addition, PINK1 andParkin siRNA did not affect cell viability (SupplementaryFig. S3C and D). Knockdown of PINK1 and Parkin werevulnerable to apoptosis than those treated with controlsiRNA, appeared as increase of caspase 3 activity in PINK1

Fig. 7. PINK1/Parkin-mediated mitophagy is involvedin ACh-elicited mitoprotection. (A) Activity of caspase 3 in H9c2 cells treated withPINK1/Parkin siRNA. (B) ATP content. (C-D) Measurement of ROS by DCFH-DA. Scale bar: 50mm.�P< 0.05 and ��P< 0.01 versus NC siRNAgroup. (n¼ 6).

JOURNAL OF CELLULAR PHYSIOLOGY

1178 S U N E T A L .

Page 9: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

and Parkin siRNA group (Fig. 7A). Furthermore, followingPINK1 and Parkin siRNA knockdown, cells exhibited asignificantly decrease in ATP content (Fig. 7B) and greaterlevel of ROS production (Fig. 7C and D) compared to that inthe negative control siRNA group when both treated withH/R and ACh. These data highlighted the importance ofPINK1/Parkin in the ACh-induced mitoprotection.

Discussion

In this study, PINK1/Parkin-dependent mitophagy was impaired inmyocardial I/R injury. To our best knowledge, we identified for thefirst time that ACh promoted translocation of PINK1 and Parkinfrom cytosol to mitochondria and increased co-localization ofParkin with mitochondria. Our data also revealed the loss of M2receptor in conjunction with reduced mitophagy, indicating M2receptor mediated ACh-elicited mitophagy. Meanwhile, inhibitionof mitophagy increased cardiac toxicity and destructedmitochondrial function, further demonstrating that mitophagy isrequired for the benefit of ACh in cardiomyocytoes. This workprovided a novel cardioprotective mechanism of ACh against I/Rinjury possibly by M2 receptor and PINK1/Parkin-dependentmitophagy (Fig. 8).

Mitochondria perform a dual role in the life and death ofcardiomyocytes (Bourke et al., 2013). Healthy mitochondriaproduce energy in the form of ATP that drives virtually allbiological processes, but damaged organelles becomecytotoxic factories producing pathological ROS. In order toprevent cell death and maintain mitochondrial function,damaged mitochondria should be degraded before apoptosisor necrosis is triggered. Autophagy is the only mechanism bywhich entire organelles are engulfed and recycled (Liu et al.,2013), and mitophagy provides appropriate mitochondrialpruning and remove dysfunctional mitochondria to reducedamage (de Vries and Przedborski, 2013; Lu et al., 2013;Redmann et al., 2014). It is of interest to defining the role ofmitophagy in cardiovascular diseases. Removal of damaged orunwanted mitochondria will leave more robust mitochondriato resist I/R injury. Our findings showed that H/R stimuliresulted in defective autophagy/mitophagy concurrent withcell injury, appeared as decrease in cell viability and increasein the expression of cleaved-caspase 3. These resultsindicated that impaired mitophagy occurred incardiomyocytes subjected to H/R, which was in line withprevious data in the hepatocytes (Kim et al., 2008). Thus, it isconceivable that promoting mitophagy appropriately could

Fig. 8. ACh induced cytoprotective mitophagy through M2 receptor and PINK1/Parkin signal pathway against hypoxia/reoxygenation injuryin H9c2 cells. (1) ACh promoted cytoprotective mitophagy through enhanced mitochondrial translocation of PINK1/Parkin. (2) M2 receptorantagonist METH and siRNA abolished the effects of ACh on mitophagy. (3) Activation of mitophagy improved mitochondrial structure andfunction (maintaining ATP content and membrane potential), meanwhile ACh alleviated damaged mitochondria-depengent cell injury (ROSoverproduction, release of pro-apoptosis factors). (4) Autophagy inhibitor 3-MA and PINK1/Parkin siRNA abrogated the ACh - inducedmitoprotection.

JOURNAL OF CELLULAR PHYSIOLOGY

A C h I N D U C E D P I N K 1 / P A R K I N - D E P E N D E N T M I T O P H A G Y 1179

Page 10: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

provide benefits in cardiomyocytes and represent a strategictarget for I/R injury.

There is emerging evidence suggesting that improved vagaltone and ACh treatment have significant protective effects onmitochondria, including lower oxidative stress, increased ATPproduction, and attenuated mitochondrial calcium overload(Jiang et al., 2014; Miao et al., 2014; He et al., 2015).Furthermore, ACh has been found to be beneficial in autophagyactivation (Zhao et al., 2013). ACh may therefore present as apotential regulator for mitophagy activation. As expected, arecovery of mitophagy after ACh administration duringreoxygenation was observed and ACh-elicited mitophagy wasprevented by M2 receptor antagonist and siRNA. Thus, it ispossible that ACh activated mitophagy through M2 receptor.

It should be noted that impaired mitochondrial eliminationconstitutes a major injury mechanism in diverse diseases (deVries and Przedborski, 2013; Lu et al., 2013; Redmann et al.,2014). Decreased mitophagy can cause inflammation and thedeath of cell population, which will result in degenerativediseases (de Vries and Przedborski, 2013). Similarly, inhibitionof mitophagy leads to cell death and exacerbates myocardialdysfunction (Kubli et al., 2013; Siddall et al., 2013). Although theprocess for mitophagy is exploited much in brain research, therelationship between mitophagy and cardiovascular diseases islittle known (Bourke et al., 2013). The present studydemonstrated that ACh reserved mitochondrial structure andfunction, including inhibition of mitochondrial swelling,increased ATP production, and recovered membranepotential. Additionally, ACh relieved the damagedmitochondrial-based cell injury, such as oxidative stress andapoptosis-promoting. These mitochondrial protection of AChwere reversed by the autophagy inhibitor 3-MA, suggestingthat mitophagy was an autophagy-related process andfacilitated the benefits of ACh-mediated mitoprotection.However, the effects of ACh on mitophagy in vivo inexperimental animals required further investigation.

Currently, PINK1/Parkin-directed mitophagy provided amechanistic link between mitochondrial damage andautophagic clearance (Geisler et al., 2010). We furtherexplored the signaling pathway mediated by ACh incardiomyocytoes. PINK1 and Parkin participate in mitophagyregulation in mammals (Palikaras and Tavernarakis, 2012). Acombination of mitochondrial dysfunction and insufficientmitophagy (such as PINK1 and Parkin mutant) contributes tomultiple pathologies (Green et al., 2011). According to ourstudy, PINK1 and Parkin were mainly localized in the cytosolfollowing H/R, andmore disruptedmitochondria were found inH/R group. Kubli et al. have found that Parkin deficiencyresulted in myocardial infarction (Kubli et al., 2013). Consistentwith this, we observed that depletion of PINK1 and Parkin withsiRNA resulted in an enhanced susceptibility to myocardialdamage, since damaged mitochondria will still hydrolyze ATPand increase ROS production. These findings confirmed thatPINK1/Parkin mediated-mitophagy activation plays a criticalrole in adapting to stress.

Generally, our findings offered compelling evidence thatACh initiated a mitophagy process against I/R injury. AChmediated cytoprotective mitophagy through enhancingPINK1/Parkin translocation to mitochondria via a M2receptor-dependent manner. These results may provide novelpotential targets for mitochondrial quality control-relatedtherapeutic avenue in ischemic heart disease.

Acknowledgments

This work is supported by National Natural ScienceFoundation of China (No.81473203), Major International(Regional) Joint Research Project of National Natural ScienceFoundation of China (No. 81120108002), Specialized Research

Fund for the Doctoral Program of Higher Education (No.20130201130008). We are grateful to Dr. WendyW. Zang forEnglish editing.

Literature Cited

AshrafiG, Schwarz TL. 2013. The pathways of mitophagy for quality control and clearance ofmitochondria. Cell Death Differ 20:31–42.

Bourke LT, Knight RA, Latchman DS, Stephanou A, McCormick J. 2013. Signal transducerand activator of transcription-1 localizes to the mitochondria and modulates mitophagy.JAKSTAT 2:e25666.

De Ferrari GM, Schwartz PJ. 2011. Vagus nerve stimulation: From pre-clinical to clinicalapplication: Challenges and future directions. Heart Fail Rev 16:195–203.

de Vries RL, Przedborski S. 2013. Mitophagy and Parkinson’s disease: Be eaten to stayhealthy. Mol Cell Neurosci 55:37–43.

Geisler S, Holmstr€om KM, Skujat D, Fiesel FC, Rothfuss OC, Kahle PJ, Springer W.2010. PINK1/Parkin-mediated mitophagy is dependent on VDAC1. Nat Cell Biol12:119–131.

Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Blaha MJ, Dai S, Ford ES, Fox CS,Franco S, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Huffman MD, JuddSE, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Mackey RH, Magid DJ,Marcus GM, Marelli A, Matchar DB, McGuire DK, Mohler ER, Moy CS, Mussolino ME,Neumar RW,Nichol G, PandeyDK, Paynter NP, ReevesMJ, Sorlie PD, Stein J, Towfighi A,Turan TN, Virani SS, Wong ND, Woo D, Turner MB. 2014. Executive summary: Heartdisease and stroke statistics-2014 update: A report from the American heart association.Circulation 129:399–410.

Green DR, Galluzzi L, Kroemer G. 2011. Mitochondria and the autophagy-inflammation-celldeath axis in organismal aging. Science 333:1109–1112.

Hammerling BC, Gustafsson AB. 2014. Mitochondrial quality control in the myocardium:Cooperation between protein degradation and mitophagy. J Mol Cell Cardiol75:122–130.

Harvey RD. 2012. Muscarinic receptor agonists and antagonists: Effects on cardiovascularfunction. Handb Exp Pharmacol 208:299–316.

Hattori N, Saiki S, Imai Y. 2014. Regulation by mitophagy. Int J Biochem Cell Biol53C:147–150.

Hausenloy DJ, Yellon DM. 2013. Myocardial ischemia-reperfusion injury: A neglectedtherapeutic target. J Clin Invest 123:92–100.

He X, Bi XY, Lu XZ, Zhao M, Yu XJ, Sun L, Xu M, Wier WG, Zang WJ. 2015. Reduction ofmitochondria endoplasmic reticulum interactions by acetylcholine protects humanumbilical vein endothelial cells from hypoxia/reoxygenation injury. Arterioscler ThrombVasc Biol 35:1623–1634.

Hiraki T, Baker W, Greenberg JH. 2012. Effect of vagus nerve stimulation during transientfocal cerebral ischemia on chronic outcome in rats. J Neurosci Res 90:887–894.

Huang C, Andres AM, Ratliff EP, Hernandez G, Lee P, Gottlieb RA. 2011. Preconditioninginvolves selective mitophagy mediated by Parkin and p62/SQSTM1. PLoS ONE 6:e20975.

Jiang HK, Miao Y,Wang YH, Zhao M, Feng ZH, Yu XJ, Liu JK, ZangWJ. 2014. Aerobic intervaltraining protects against myocardial infarction-induced oxidative injury by enhancingantioxidase system andmitochondrial biosynthesis. Clin ExpPharmacol Physiol 41:192–201.

Jin SM, Youle RJ. 2012. PINK1- and Parkin-mediatedmitophagy at a glance. J Cell Sci 125(Pt 4)795–799.

Kamat PK, Kalani A, Kyles P, Tyagi SC, Tyagi N. 2014. Autophagy of mitochondria: Apromising therapeutic target for neurodegenerative disease. Cell Biochem Biophys70:707–719.

Kim JS, Nitta T, Mohuczy D, O’Malley KA, Moldawer LL, Dunn Jr WA, Behrns KE. 2008.Impaired autophagy: Amechanism ofmitochondrial dysfunction in anoxic rat hepatocytes.Hepatology 47:1725–1736.

Kong SS, Liu JJ, Hwang TC, Yu XJ, Zhao M, Zhao M, Yuan BX, Lu Y, Kang YM,Wang B, ZangWJ. 2012. Optimizing the parameters of vagus nerve stimulation by uniform design in ratswith acute myocardial infarction. PLoS ONE 7:e42799.

Kroemer G, Marino G, Levine B. 2010. Autophagy and the integrated stress response. MolCell 40:280–293.

Kubli DA, Zhang X, Lee Y, Hanna RA, Quinsay MN, Nguyen CK, Jimenez R, Petrosyan S,Murphy AN, Gustafsson AB. 2013. Parkin protein deficiency exacerbates cardiac injuryand reduces survival following myocardial infarction. J Biol Chem 288:915–926.

Liang JM, Xu HY, Zhang XJ, Li X, Zhang HB, Ge PF. 2013. Role of mitochondrial function inthe protective effects of ischaemic postconditioning on ischaemia/reperfusion cerebraldamage. J Int Med Res 41:618–627.

Liu K, Sun Y, Gu Z, Shi N, Zhang T, Sun X. 2013. Mitophagy in ischaemia/reperfusion inducedcerebral Injury. Neurochem Res 38:1295–1300.

Lopez-Armada MJ, Riveiro-Naveira RR, Vaamonde-Garcia C, Valcarcel-Ares MN. 2013.Mitochondrial dysfunction and the inflammatory response. Mitochondrion 13:106–118.

Lu H, Li G, Liu L, Feng L, Wang X, Jin H. 2013. Regulation and function of mitophagy indevelopment and cancer. Autophagy 9:1720–1736.

Lu J, Zang WJ, Yu XJ, Chen LN, Zhang CH, Jia B. 2005. Effects of ischaemia-mimetic factorson isolated rat ventricular myocytes. Exp Physiol 90:497–505.

Manfrini O, Pizzi C, Viecca M, Bugiardini R. 2008. Abnormalities of cardiac autonomicnervous activity correlate with expansive coronary artery remodeling. Atherosclerosis197:183–189.

Mastitskaya S, Marina N, Gourine A, Gilbey MP, Spyer KM, Teschemacher AG, Kasparov S,Trapp S, Ackland GL, Gourine AV. 2012. Cardioprotection evoked by remote ischaemicpreconditioning is critically dependent on the activity of vagal pre-ganglionic neurones.Cardiovasc Res 95:487–494.

Miao Y, Bi XY, Zhao M, Jiang HK, Liu JJ, Li DL, Yu XJ, Yang YH, Huang N, Zang WJ. 2014.Acetylcholine inhibits tumor necrosis factor alpha activated endoplasmic reticulumapoptotic pathway via EGFR-PI3K signaling in cardiomyocytes. J Cell Physiol230:767–774.

Mizushima N, Yoshimori T, Levine B. 2010. Methods in mammalian autophagy research. Cell140:313–326.

Ong SB, Gustafsson AB. 2012. New roles for mitochondria in cell death in the reperfusedmyocardium. Cardiovasc Res 94:190–196.

Palikaras K, Tavernarakis N. 2012. Mitophagy in neurodegeneration and aging. Front Genet3:297.

Przyklenk K, Dong Y, Undyala VV, Whittaker P. 2012. Autophagy as a therapeutic target forischemia-reperfusion injury? Concepts, controversies and challenges. Cardiovasc Res94:197–205.

JOURNAL OF CELLULAR PHYSIOLOGY

1180 S U N E T A L .

Page 11: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

Redmann M, Dodson M, Boyer-Guittaut M, Darley-Usmar V, Zhang J. 2014. Mitophagymechanisms and role in human diseases. Int J Biochem Cell Biol 53:127–133.

Schwartz PJ, De Ferrari GM. 2011. Sympathetic-parasympathetic interaction inhealth and disease: Abnormalities and relevance in heart failure. Heart Fail Rev16:101–107.

Shimizu S, Honda S, Arakawa S, Yamaguchi H. 2014. Alternative macroautophagy andmitophagy. Int J Biochem Cell Biol 50:64–66.

Siddall HK, Yellon DM, Ong SB, Mukherjee UA, Burke N, Hall AR, Angelova PR,Ludtmann MH, Deas E, Davidson SM, Mocanu MM, Hausenloy DJ. 2013. Loss ofPINK1 increases the heart’s vulnerability to ischemia-reperfusion Injury. PLoS ONE8:e62400.

Sun L, Zang WJ, Wang H, Zhao M, Yu XJ, He X, Miao Y, Zhou J. 2014. Acetylcholinepromotes ROS detoxification against hypoxia/reoxygenation-induced oxidative stressthrough FoxO3a/PGC-1alpha dependent superoxide dismutase. Cell Physiol Biochem34:1614–1625.

Sun L, Zhao M, Yu XJ, Wang H, He X, Liu JK, Zang WJ. 2013. Cardioprotection byacetylcholine: A novel mechanism via mitochondrial biogenesis and function involving thePGC-1alpha pathway. J Cell Physiol 228:1238–1248.

Walters AM, Porter GJ, Brookes PS. 2012. Mitochondria as a drug target in ischemic heartdisease and cardiomyopathy. Circ Res 111:1222–1236.

Whelan SP, Zuckerbraun BS. 2013. Mitochondrial signaling: Forwards, backwards, and inbetween. Oxid Med Cell Longev 2013:351613.

Yu SW, Andrabi SA, Wang H, Kim NS, Poirier GG, Dawson TM, Dawson VL. 2006.Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death.Proc Natl Acad Sci USA 103:18314–18319.

Zhang J. 2013. Autophagy and mitophagy in cellular damage control. Redox Biol1:19–23.

Zhao M, Sun L, Yu XJ, Miao Y, Liu JJ, Wang H, Ren J, ZangWJ. 2013. Acetylcholine mediatesAMPK-dependent autophagic cytoprotection in H9c2 cells during hypoxia/reoxygenationinjury. Cell Physiol Biochem 32:601–613.

Zinchuk V, Zinchuk O, Okada T. 2007. Quantitative colocalization analysis of multicolorconfocal immu-nofluorescence microscopy images: Pushing pixels to explore biologicalphenomena. Acta Histochem Cytochem 40:101–111.

Supporting Information

Additional supporting information may be found in the onlineversion of this article at the publisher’s web-site.

JOURNAL OF CELLULAR PHYSIOLOGY

A C h I N D U C E D P I N K 1 / P A R K I N - D E P E N D E N T M I T O P H A G Y 1181

Page 12: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by ...download.xuebalib.com/3ec6v4GRgCJ6.pdfAcetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through

本文献由“学霸图书馆-文献云下载”收集自网络,仅供学习交流使用。

学霸图书馆(www.xuebalib.com)是一个“整合众多图书馆数据库资源,

提供一站式文献检索和下载服务”的24 小时在线不限IP

图书馆。

图书馆致力于便利、促进学习与科研,提供最强文献下载服务。

图书馆导航:

图书馆首页 文献云下载 图书馆入口 外文数据库大全 疑难文献辅助工具