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PTEN induced putative kinase 1 (PINK1) alleviates angiotensin II-induced cardiac injury by ameliorating mitochondrial dysfunction
International Journal of Cardiology 266 (2018) 198–205
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
PTEN induced putative kinase 1 (PINK1) alleviates angiotensin II-induced cardiac injury by ameliorating mitochondrial dysfunction Wenjun Xiong a,b, Jinghai Hua a,b, Zuheng Liu a,b, Wanqiang Cai a,b, Yujia Bai a,b, Qiong Zhan a,b, Wenyan Lai a,b, Qingchun Zeng a,b, Hao Ren b,c,⁎, Dingli Xu a,b,⁎⁎ a b c
State Key Laboratory of Organ Failure Research, Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou, China Key Laboratory for Organ Failure Research, Ministry of Education of the People's Republic of China, Guangzhou, China Department of Rheumatology, Nanfang Hospital, Southern Medical University, Guangzhou, China
a r t i c l e
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Article history: Received 13 December 2017 Received in revised form 17 February 2018 Accepted 12 March 2018
Keywords: PINK1 Parkin Mitophagy Angiotensin II MMP
a b s t r a c t Background: Mitochondrial quality control is crucial to the development of angiotensin II (AngII)-induced cardiac hypertrophy. PTEN induced putative kinase 1 (PINK1) is rapidly degraded in normal mitochondria but accumulates in damaged mitochondria, triggering autophagy to protect cells. PINK1 mediates mitophagy in general, but whether PINK1 mediates AngII-induced mitophagy and the effects of PINK1 on AngII-induced injury are unknown. This study was designed to investigate the function of PINK1 in an AngII stimulation model and its regulation of AngII-induced mitophagy. Methods: We studied the function of PINK1 in mitochondrial homeostasis in AngII-stimulated cardiomyocytes via RNA interference-mediated knockdown and adenovirus-mediated overexpression of the PINK1 protein. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, adenosine triphosphate (ATP) content, cell apoptosis rates and cardiomyocyte hypertrophy were measured. The expression of LC3B, Beclin1 and p62 was measured. Mitochondrial morphology was examined via electron microscopy. Mitophagy was detected by confocal microscopy based on the co-localization of lysosomes and mitochondria. Additionally, endogenous PINK1, phosphorylated PINK1, mito-PINK1, total Parkin, cyto-Parkin, mito-Parkin and phosphorylated Parkin protein levels were measured. Results: Cardiomyocytes untreated by AngII had very low levels of total and phosphorylated PINK1. However, in the AngII stimulation model, the MMP was decreased, and the levels of total and phosphorylated PINK1 were increased. After PINK1 was knocked down, Parkin translocation to the mitochondria was inhibited. Moreover, levels of phosphorylated Parkin were reduced, and autophagy markers were downregulated. MMP and ATP contents were further reduced, ROS production and the apoptotic rate were further increased, and myocardial hypertrophy was further aggravated compared with those in the AngII group. However, PINK1 overexpression promoted Parkin translocation and phosphorylation, autophagy markers were upregulated, and myocardial injury was reduced. In addition, the effects of PINK1 overexpression were reversed by autophagy inhibitors. Conclusion: Decreased MMP induced by AngII maintains the stability of PINK1, causing PINK1 autophosphorylation. PINK1 activation promotes Parkin translocation and phosphorylation and increases autophagy to clear damaged mitochondria. Thus, PINK1/Parkin-mediated mitophagy has a compensatory, protective role in AngIIinduced cytotoxicity. © 2018 Published by Elsevier B.V.
Abbreviations: PINK1, PTEN-induced putative kinase 1; AngII, angiotensin II; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; LC3B, microtubuleassociated protein 1 light chain 3 beta; ATP, adenosine triphosphate. ⁎ Correspondence to: H. Ren, Department of Rheumatology, Nanfang Hospital, Southern Medical University, 1838 Northern Guangzhou Ave, Guangzhou, Guangdong 510515, China. ⁎⁎ Correspondence to: D. Xu, Department of Cardiology, Nanfang Hospital, Southern Medical University, 1838 Northern Guangzhou Ave, Guangzhou, Guangdong 510515, China. E-mail addresses: [email protected] (H. Ren), dinglixu@fimmu.com (D. Xu).
https://doi.org/10.1016/j.ijcard.2018.03.054 0167-5273/© 2018 Published by Elsevier B.V.
1. Introduction Myocardial hypertrophy is an important adaptive response that maintains and increases cardiac output during the early period of heart failure [1–3]. Persistent stress can burden myocardial hypertrophy, ultimately resulting in ventricular remodeling, congestive heart failure or sudden death due to arrhythmia [4–6]. Activation of the renin-angiotensin system is a key factor that triggers myocardial hypertrophy. Moreover, mitochondrial function is crucial to the development
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of cardiac hypertrophy [7]. Dysfunctional mitochondria decrease mitochondrial membrane potential (MMP), produce high levels of reactive oxygen species (ROS) and are more sensitive to Ca2+-induced mitochondrial permeability transition pores. Furthermore, damaged mitochondria trigger a chain reaction, eventually resulting in lower ATP levels. Increased knowledge of mitochondrial function will help the treatment of cardiac hypertrophy-related events [8,9]. In recent years, autophagy has been recognized as a critical factor in maintaining cardiac function during myocardial hypertrophy. Autophagy is a regulated mechanism leading to the disassembly of dysfunctional proteins or organelles [10–13]. Under pathological conditions, autophagy is activated as an adaptive response to stress to promote survival. However, the role of autophagy in cardiac hypertrophy remains controversial [14,15]. Indeed, autophagic activity may perform different functions depending on the stage and severity of the disease. Activation of autophagy in the early stages of the disease may be a protective mechanism for the clearance and elimination of misfolded polyubiquitinated protein aggregates [11]. In addition, it is necessary to remove dysfunctional mitochondria to prevent the catastrophic loss of ATP in the early stages of the disease. Meanwhile, autophagy provides energy to compensate for the increased ATP requirements caused by stress overload. Cardiac autophagy may play a key role in removing damaged mitochondria, maintaining cell homeostasis and preventing apoptosis [16]. However, the point at which autophagic activity becomes autophagic death has not been defined. Thus, understanding the mechanisms of autophagic regulation may facilitate the discovery of a novel strategy to combat myocardial hypertrophy. Accumulating evidence from studies investigating PTEN-induced putative kinase 1 (PINK1) and Parkin has revealed that these two proteins participate in mitochondrial quality control during cardiac injury [17]. PINK1-activated Parkin translocates to mitochondria with low membrane potential to initiate the autophagic degradation of damaged mitochondria [18]. PINK1 mediates mitophagy in general, but it is unknown whether PINK1 mediates AngII-induced mitophagy, and the effects of PINK1 on angiotensin II (AngII)-induced injury are uncharacterized. We hypothesized that PINK1 regulates mitophagy to alleviate AngIIinduced cardiomyocyte injury by promoting Parkin translocation and phosphorylation. We knocked down and overexpressed PINK1 in cardiomyocytes to study the effects of PINK1 on cardiac function, and we found that in an AngII stimulation model, PINK1 promoted Parkin translocation and phosphorylation, thereby regulating autophagy signaling and clearing damaged mitochondria. PINK1-mediated mitophagy eliminated the damaged mitochondria-induced chain reaction, mitigating outcomes such as myocardial apoptosis and cardiac hypertrophy.
2. Materials and methods 2.1. Chemicals and reagents Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Trypsin and collagenase were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA). AngII, chloroquine diphosphate salt (CQ) and manganese chloride (Mncl2) were purchased from Sigma-Aldrich Co. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was purchased from Beyotime (Jiangsu, China). The following polyclonal primary antibodies were used in this study: anti-PINK1, anti-Parkin, Anti-Parkin (phospho S65), anti-p62, anti-Beclin1, anti-COX IV, anti-β-actin (Abcam, Cambridge, UK), anti-GAPDH (Santa Cruz Biotechnology Inc., CA, USA) and anti-LC3B (Sigma-Aldrich Co.).
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2.3. Detection of mitochondrial morphology Mitophagy was determined based on the co-localization of mitochondria and lysosomes. Cardiomyocytes were co-incubated with MitoTracker Green (100 nM) and LysoTracker Red (50 nM, Molecular Probes, Eugene, OR, USA) for 40 min. Confocal images were acquired with a Zeiss LSM 880 (Zeiss, Germany). The number of MitoTracker- and LysoTracker-positive foci was determined by using ImageJ software. Mitochondrial morphology was observed by transmission electron microscopy (TEM). Small fragments of cardiomyocytes (1 mm3 in size) were fixed for 2 h with 2.5% glutaraldehyde in 0.1 M PBS, pH 7.4. After rinsing 6× with PBS for 30 min, the cells were incubated with 1% osmium tetroxide in 0.1 M cacodylate phosphate buffer for 2 h. The cells were subsequently rinsed 3× with PBS for 10 min. The samples were then dehydrated in a graded ethanol series (50%, 70%, 90%, and 100%), rinsed with propylene oxide, permeabilized with a 1:1 mixture of propylene oxide and Epon 812, and then baked in a 38 °C oven for 3 h. The above steps were repeated using a 1:2 mixture of propylene oxide and Epon 812. Next, ultrathin sections (50–70 nm) of the samples were cut with an ultramicrotome (UC7, Leica, Germany), collected on 200-mesh copper grids and incubated with 5% uranyl acetate in ethanol (10 min) and lead citrate (15 min) for contrast enhancement. Finally, mitochondria were examined with an electron microscope (JEM-1400, Japan) operated at 80 kV. 2.4. Measurement of ROS levels Intracellular ROS production was quantified with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE, Invitrogen Molecular Probes, USA). Cardiomyocytes were incubated with culture medium containing 10 μM DCFH-DA or 10 μM DHE for 30 min at 37 °C and washed three times with PBS. Then, ROS production was detected using a fluorescence microscope (Olympus, Japan). ImageJ was used for analysis. 2.5. Measurement of MMP MMP was measured using a JC-1 kit (Sigma-Aldrich Co.). After stimulation, cells were incubated with JC-1 staining solution at 37 °C for 20 min and then washed twice with JC-1 staining buffer. MMP was assayed with a fluorescence microscope. ImageJ was used for analyses. 2.6. Apoptosis assays Apoptosis in cardiomyocytes was analyzed using an Annexin V Apoptosis Detection Kit (Roche, Shanghai, China). Cells were incubated for 15 min at 4 °C in the dark with Annexin V. After the addition of propidium iodide (PI) into the medium for 5 min, the proportion of Annexin V-positive and PI-positive cells was analyzed with a flow cytometer (Beckman Coulter, USA). 2.7. Measurement of cardiomyocyte hypertrophy The mRNA levels of hypertrophy-related genes were detected. Total RNA was purified from cardiomyocytes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA samples were reverse transcribed to generate cDNAs using a first-strand cDNA synthesis kit (GeneCopoeia, Rockville, MD, USA). qRT-PCR was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The expression of the following genes was measured: ANP, forward primer 5′-ACCTGCTAGACCACCTGGAG-3′, reverse primer 5′-CCTTGG CTGTTATCTTCGGTACCGG-3′; β-MHC, forward primer 5′-TCTGGACAGCTCCCCATTCT-3′, reverse primer 5′-CAAGGCTAACCTGGAGAAGATG-3′; GAPDH, forward primer 5′-ATCA AGAAGGTGGTGAAGCA-3′, reverse primer 5′-AAGGTGGAAGAATGGGAGTTG-3′. We utilized 10-μl reactions with the following RT-PCR conditions: 95 °C for 5 min; 40 cycles of 95 °C for 10 s, 55–60 °C for 20 s, and 72 °C for 32 s. The results were analyzed with the 2−△△Ct method and normalized to GAPDH gene expression. Cardiomyocyte surface area was observed by immunofluorescence. Cardiomyocytes were fixed and permeabilized. The cells were blocked and then incubated overnight with anti-β-actin. Then, the cells were incubated with secondary antibody to label cytoskeletal actin. Finally, the cells were stained with DAPI at room temperature. Confocal images were acquired using a Zeiss LSM 880. 2.8. Measurement of ATP levels ATP content in cardiomyocytes was measured by an ATP assay kit (Beyotime). Briefly, the cardiomyocytes were lysed by cellular ATP-releasing reagent. ATP detection solution was mixed with luciferase solution. Bioluminescence was measured with a luminometer. ATP content was estimated according to a standard curve. The results were normalized to cellular protein concentration.
2.2. Cell culture and adenoviral transduction 2.9. Immunofluorescence Neonatal rat cardiomyocytes were prepared from the hearts of Sprague-Dawley rats using enzymatic dissociation and cultured as previously described [19]. Recombinant adenoviruses for PINK1 overexpression (AD-PINK1) and knockdown (si-PINK1) were designed and synthesized by GeneChem Co. (Shanghai, China). The viruses were added to cells according to the manufacturer's instructions. Cells were stimulated with 1 μM AngII for 24 h after adenoviral transduction for 72–96 h, and then subsequent experiments and analyses were performed.
Cardiomyocytes were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.2% Triton X-100 for 10 min. The cells were blocked for 30 min and then incubated overnight with primary antibodies at 4 °C. Then, the cells were incubated with goat anti-rabbit secondary antibody (Santa Cruz Biotechnology Inc.) for 60 min at room temperature. Additionally, the cells were incubated with goat anti-mouse antibody to label cytoskeletal actin. Finally, the cells were stained with DAPI at room temperature. Confocal images were
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acquired using a Zeiss LSM 880. Quantification of fluorescent pixels was carried out by ImageJ software.
2.10. Immunoblot analysis For total protein extraction, the cardiomyocytes were lysed in lysis buffer containing protease inhibitor cocktail (KeyGEN BioTECH Inc., Jiangsu, China) for 30 min. After centrifuging at 12,000g for 15 min at 4 °C, cell suspensions were collected as whole-cell proteins. Cytoplasmic proteins and mitochondrial proteins were extracted from cells using a Cell Mitochondria Isolation Kit (Beyotime, Jiangsu, China) according to the manufacturer's instructions. Briefly, cardiomyocytes were lysed in mitochondrial fractionation reagent with PMSF for 15 min. The cell suspensions were transferred to a homogenizer and homogenized 10 to 30 times. After centrifuging at 1000g for 10 min at 4 °C, the cell suspensions were transferred to another centrifuge tube. The cell lysates continued to be centrifuged at 11,000g for 10 min at 4 °C. The supernatants were composed of cytoplasm, and the sediment contained the mitochondria. After further lysis, cytoplasmic proteins and mitochondrial proteins were collected for subsequent experiments. Approximately 30 μg of protein per sample was separated on SDS-polyacrylamide gels. To detect phosphorylated PINK1, Phos-tag™ SDS-PAGE containing 50 μmol/l Phos-tag™ acrylamide (Wako Pure Chemical Industries, Ltd, Japan) and 100 μmol/l MnCl2 was performed. After
electrophoresis, Phos-tag gels were washed with transfer buffer containing 1 mmol/l EDTA and then replaced with transfer buffer without EDTA. The gels were electrotransferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked in 2.5% non-fat milk, incubated overnight with the indicated primary antibodies and then incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). The density of the expressed protein bands was quantified by ImageJ software. 2.11. Statistical analysis The data are expressed as the mean ± SD. Statistical analyses were performed using either one-way analysis of variance or Student's t-tests (GraphPad Prism 6.0 software, San Diego, CA, USA). P-values b 0.05 were considered statistically significant.
3. Results 3.1. Knockdown of PINK1 inhibited cardiomyocyte mitophagy To study the effects of PINK1-targeting siRNA (si-PINK1) on mitophagy in the AngII model, we detected changes in mitochondrial
Fig. 1. PINK1 knockdown inhibited mitophagy and aggravated injury in cardiomyocytes stimulated with AngII. Cardiomyocytes were transfected with adenoviruses encoding si-PINK1 and si-Control and then stimulated with AngII (1 μM) for 24 h. (A) TEM showing mitochondrial morphology in cardiomyocytes. Enlarged image in the yellow frame shows the details of mitochondrial structures. (B) Fluorescence images showing lysosomal-mitochondrial interactions. Mitochondria are shown in green, lysosomes are shown in red, and Hoechst staining in the nuclei is shown in blue. (C) Immunoblotting showing the expression of Beclin1, p62 and LC3B in cardiomyocytes. Quantification relative to β-actin levels. (D) Fluorescence images using DHE fluorescent dye showing ROS production in cardiomyocytes. (E) Fluorescence images using DCFH-DA fluorescent dye showing ROS production in cardiomyocytes. (F) Fluorescence images of cardiomyocytes stained with JC-1 tracker. (G) Cells were stained with PI and Annexin V-FITC and then subjected to flow cytometric analysis. (H-I) Relative fold changes in the mRNA levels of β-MHC and ANP were determined using qRT-PCR. (J) Fluorescence images showing the surface area of cardiomyocytes. Cytoskeletal actin is shown in red, and DAPI staining in the nuclei is shown in blue. (K) Intracellular ATP levels were determined by using an ATP assay kit with a luminometer. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. si-Control + Control; #P b 0.05 vs. si-Control + AngII.
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morphology and autophagy-related protein levels. The TEM results showed that knockdown of PINK1 inhibited AngII-induced mitophagy and exacerbated AngII-induced mitochondrial fracture (Fig. 1A). Fluorescence images of the co-localization of lysosomes and mitochondria showed that knockdown of PINK1 decreased lysosomal-mitochondrial interactions (Fig. 1B). Similarly, knockdown of PINK1 negated the AngII-induced increase in the expression of autophagy-related proteins, with the exception of p62 (Fig. 1C). 3.2. Knockdown of PINK1 increased oxidative stress and cell apoptosis To evaluate the effects of si-PINK1 on AngII-induced injury, we analyzed intracellular ROS production, apoptosis and hypertrophy in cardiomyocytes. Knockdown of PINK1 significantly increased AngIIinduced ROS production as shown by DHE and DCFH staining (Fig. 1D, E). Additionally, JC-1 staining showed that the knockdown of PINK1 aggravated the AngII-induced reduction in MMP (Fig. 1F). Knockdown of
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PINK1 significantly increased AngII-induced cardiomyocyte apoptosis (Fig. 1G) and decreased ATP content (Fig. 1K). Additionally, downregulation of PINK1 enhanced AngII-increased mRNA levels of ANP and β-MHC (Fig. 1H, I) and the surface area of cardiomyocytes (Fig. 1J). 3.3. Overexpression of PINK1 enhanced cardiomyocyte mitophagy We tested the effects of PINK1 overexpression on mitophagy in the AngII model. The TEM results revealed that PINK1 overexpression increased the formation of autophagosomes (Fig. 2A). Fluorescence images of the co-localization of lysosomes and mitochondria showed that the overexpression of PINK1 further increased lysosomal-mitochondrial interactions (Fig. 2B). Additionally, western blot analysis showed that PINK1 overexpression promoted an AngII-induced increase in the expression of Beclin1 and LC3B II and reduced the expression of p62 (Fig. 2C). Therefore, PINK1 was involved in mitophagy induction in response to AngII stimulation.
Fig. 2. PINK1 overexpression upregulated mitophagy and alleviated injury in cardiomyocytes stimulated with AngII. Cardiomyocytes were transfected with adenoviruses Ad-PINK1 and Ad-Control and then stimulated with AngII (1 μM) for 24 h. (A) TEM showing mitochondrial morphology in cardiomyocytes. Enlarged image in the yellow frame shows the details of mitochondrial structures. (B) Fluorescence images showing lysosomal-mitochondrial interactions. Mitochondria are shown in green, lysosomes are shown in red, and Hoechst staining in the nuclei is shown in blue. (C) Immunoblotting showing the expression of Beclin1, p62 and LC3B in cardiomyocytes. Quantification relative to β-actin levels. (D) Fluorescence images using DHE fluorescent dye showing ROS production in cardiomyocytes. (E) Fluorescence images using DCFH-DA fluorescent dye showing ROS production in cardiomyocytes. (F) Fluorescence images of cardiomyocytes stained with JC-1 tracker. (G) Cells were stained with PI and Annexin V-FITC and then subjected to flow cytometric analysis. (H–I) Relative fold changes in the mRNA levels of β-MHC and ANP were determined using qRT-PCR. (J) Fluorescence images showing the surface area of cardiomyocytes. Cytoskeletal actin is shown in red, and DAPI staining in the nuclei is shown in blue. (K) Intracellular ATP levels were determined by using an ATP assay kit with a luminometer. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. Ad-Control + Control; #P b 0.05 vs. Ad-Control + AngII.
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3.4. Overexpression of PINK1 prevented AngII-induced oxidative stress and apoptosis Next, we investigated the effects of PINK1 overexpression during AngII-induced cardiac injury. AngII-induced ROS production was inhibited following the overexpression of PINK1 as shown by DHE and DCFH staining (Fig. 2D and E). Furthermore, PINK1 overexpression promoted the removal of damaged mitochondria due to MMP reduction (Fig. 2F). Upregulation of PINK1 significantly decreased AngII-induced cardiomyocyte apoptosis (Fig. 2G) and increased ATP levels (Fig. 2K). Additionally, PINK1 overexpression reduced AngII-increased mRNA levels of ANP and β-MHC (Fig. 2H, I) and the surface area of cardiomyocytes (Fig. 2J). Therefore, PINK1 exerted protective effects against AngII-induced cardiac injury. 3.5. Autophagy inhibition reversed the effects of PINK1 To determine whether the regulation of mitophagy by PINK1 plays a critical role in AngII-induced cardiac injury, we treated cardiomyocytes with CQ, an inhibitor of autophagy. The CQ-treated group had higher ROS levels (Fig. 3A and B) and apoptosis rates (Fig. 3C) during AngIIinduced cardiac injury than did the PINK1 overexpression group, indicating that the effects of PINK1 overexpression were reversed by the autophagy inhibitor. Therefore, PINK1 alleviated AngII-induced cardiac injury by regulating mitophagy. 3.6. The PINK1/Parkin pathway regulated mitophagy in AngII-induced cardiac injury To further elucidate the functions of PINK1 and the PINK1/Parkin pathways, we assess the expression of PINK1 and Parkin proteins and
the phosphorylation levels of PINK1 and its substrate, Parkin. With increased AngII stimulation time, the expression of PINK1 peaked at 24 h, followed by stable expression until 60 h and decreased expression at 72 h (Fig. 4A). PINK1 fluorescence intensity was increased (Fig. 4D) with decreasing MMP (Fig. 4C) when AngII was stimulated for 24 h. Additionally, AngII-induced MMP reduction promoted PINK1 mitochondrial translocation (Fig. 4I, J). Endogenous PINK1 expression and phosphorylation were increased under AngII and CCCP stimulation (Fig. 4B). Use of RNA interference-mediated knockdown and adenovirus-mediated overexpression of the PINK1 protein induced corresponding knockdown and overexpression of PINK1 (Fig. 4E, F). PINK1 knockdown inhibited Parkin mitochondrial translocation (Fig. 4G). Concurrently, elevated levels of mitochondrial Parkin (mito-Parkin) were observed in the AngII model when PINK1 was upregulated, indicating Parkin translocates from the cytoplasm to the mitochondria (Fig. 4H). Additionally, phosphorylation of Parkin was induced by PINK1 overexpression and inhibited by PINK1 knockdown (Fig. 4I, J). 4. Discussion Neurohumoral stimulation through the renin-angiotensin system plays critical roles in cardiac physiological and pathological processes [20–22]. As an important neurohumoral factor, AngII promotes myocardial hypertrophy. Using an in vitro AngII-induced cardiomyocyte hypertrophy model, we found that AngII stimulation upregulated the basal and phosphorylation levels of endogenous PINK1. We further revealed that PINK1 knockdown increased AngII-induced ROS production and apoptosis, thereby interfering with mitochondrial energy production. Additionally, the overexpression of PINK1 promoted the maintenance of cardiomyocyte function by mediating mitophagy, a process that removes dysfunctional mitochondria. These effects were associated
Fig. 3. Effects of PINK1 overexpression were reversed by an autophagy inhibitor. Cardiomyocytes were transfected with the adenoviruses Ad-PINK1 and Ad-Control and then co-treated with AngII (1 μM) for 24 h and CQ (10 μM). (A) Fluorescence images using DHE fluorescent dye showing ROS production in cardiomyocytes. (B) Fluorescence images using DCFH-DA fluorescent dye showing ROS production in cardiomyocytes. (C) Cells were stained with PI and Annexin V-FITC and then subjected to flow cytometric analysis. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. Ad-Control + Control; #P b 0.05 vs. Ad-Control + AngII; ▲P b 0.05 vs. Ad-PINK1 + AngII.
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Fig. 4. PINK1/Parkin pathway in cardiomyocytes stimulated with AngII. (A) Immunoblotting showing the expression of PINK1 for the indicated times. (B) Immunoblotting of endogenous PINK1. The asterisks show phosphorylated PINK1. Cardiomyocytes were treated with CCCP (10 μM, 1 h) or AngII (1 μM, 24 h) and subjected to SDS–PAGE on a 50-μM Phos-tag gel. (C) Fluorescence images of cardiomyocytes stained with JC-1 tracker. (D) Immunofluorescence images demonstrating the expression of PINK1 in cardiomyocytes stimulated with AngII. PINK1 staining is shown in green, representing the expression of PINK1, and DAPI staining in the nuclei is shown in blue. Cardiomyocytes were transfected with the adenoviruses si-PINK1 and si-Control and then stimulated with AngII. (E,G,I) Immunoblotting showing the expression of PINK1, total Parkin, cyto-Parkin, mito-Parkin, P-Parkin and mito-PINK1. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. si-Control + Control; #P b 0.05 vs. si-Control + AngII. Cardiomyocytes were transfected with the adenoviruses Ad-PINK1 and Ad-Control and then stimulated with AngII. (F,H,J) Immunoblotting showing the expression of PINK1, total Parkin, cyto-Parkin, mito-Parkin, P-Parkin and mito-PINK1 in cardiomyocytes. *P b 0.05 vs. Ad-Control + Control; #P b 0.05 vs. Ad-Control + AngII. (J) Schematic model of the mechanism underlying the regulatory role of PINK1 in AngII-induced cardiac injury.
with activation of the PINK1/Parkin signaling pathway. Thus, PINK1 is important for the regulation of cardiac remodeling induced by AngII. The protein kinase C (PKC) signaling pathway is crucial in the AngIIinduced cardiac hypertrophy [23]. Furthermore, cardiac hypertrophy involves a PKC-mediated increase in mitochondrial ROS production [24]. When AngII activates the hypertrophic signaling pathway, large amounts of ROS are produced. Additionally, ROS acts as a positive
feedback signal to further stimulate the hypertrophy signaling pathway, and ROS also activates the apoptotic signaling pathway. Constant activation of this pathway eventually causes damage to cardiomyocytes. ROS is primarily derived from mitochondrial dysfunction. Thus, preserving mitochondrial function integrity might be an effective strategy for the treatment of hypertrophic cardiomyocytes. Mitophagy has attracted substantial interest in recent years due to its critical role in the clearance
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of damaged mitochondria [25]. This mechanism of mitochondrial quality control is mediated by two factors: PINK1 and its downstream substrate, the E3 ubiquitin ligase Parkin [26,27]. Overall mitochondrial fitness is protected via the regulation of mitophagy [28]. Therefore, we upregulated the expression of PINK1 and observed that it exerted a partial beneficial effect by increasing autophagic activity and reducing intracellular ROS levels in AngII-induced cardiomyocytes relative to control cells. The downregulation of PINK1 induced the opposite results. Based on these findings, we confirmed that mitophagy plays a critical role during AngII-induced cell death by facilitating the degradation of damaged mitochondria. Localized to the mitochondria, PINK1 regulates mitochondrial mass via the selective degradation of dysfunctional mitochondria [28–31]. The Youle group reported that newly synthesized PINK1 is introduced into the healthy mitochondrial inner membrane and undergoes mitochondrial protease-dependent cleavage followed by proteasome degradation [32–36]. However, normal MMP is required for input, and the dissipation of MMP prevents PINK1 from reaching the intima; thus, PINK1 remains confined to the mitochondrial outer membrane. Previous studies have explained how the loss of MMP causes PINK1 to accumulate on damaged mitochondria [37–39]. Similarly, in our study, we observed that unphosphorylated and phosphorylated levels of PINK1 in cardiomyocytes treated without AngII were very low. However, in the AngII stimulation model, the MMP was decreased, and PINK1 basal and phosphorylation levels were increased. In addition, we observed that AngII-induced MMP reduction promoted PINK1 mitochondrial translocation. Conversely, opposing results have been observed in many studies; for example, PINK1 mRNA and protein levels were significantly lower in a diabetes mellitus group. Interestingly, the results of some studies are similar to ours, where the expression of PINK1 was markedly increased. We analyzed changes in PINK1 expression primarily to evaluate temporal expression patterns in cell models and under pathological conditions and observed varying levels of PINK1 at different physiological stages, such as early compensatory increase and late decrease. Upregulation of PINK1 in cells with normal MMP did not cause an increase in autophagy markers. These results indirectly suggest that mitochondria with reduced membrane potential maintain PINK1 localization on the mitochondrial membrane, causing PINK1 autophosphorylation and ultimately autophagy. Okatsu et al. demonstrated by mass spectrometry and mutation analysis that PINK1 autophosphorylation occurs at Ser228 and Ser402, and importantly, alanine mutations at these sites abolish PINK1 autophosphorylation and inhibit Parkin recruitment to depolarized mitochondria [40]. These results suggest that the reduction in membrane potential is a trigger to prevent PINK1 degradation by mitochondrial proteases and facilitate subsequent phosphorylation modifications. Therefore, we exogenously increased PINK1 levels. With AngII stimulation, we observed an increase in the exogenous expression of PINK1. Furthermore, transfer of Parkin from the cytoplasm to the mitochondria increased autophagy, decreased ROS activity, and significantly improved apoptosis and hypertrophy. Thus, we concluded that endogenous PINK1 and its phosphorylation activity were necessary for AngII-induced mitophagy, which is protective. However, endogenous PINK1 and its phosphorylation activity were not sufficient to initiate additional and more effective autophagy to antagonize the decline in MMP under AngII stimulation. Studies have shown that post-translational modification of Parkin is a key regulator of mitochondrial autophagy [41]. PINK1 promotes the recruitment of cytosolic Parkin to the mitochondrial outer membrane of depolarized mitochondria, causes the ubiquitination of mitochondrial proteins and transduces autophagy signals. This process is associated with Parkin phosphorylation at the S65 residue in the ubiquitin-like domain via PINK1-regulated phosphorylation. Our study examined the translocation and phosphorylation of Parkin. By exogenously changing PINK1 expression levels, we found that Parkin translocated from the cytoplasm to the mitochondria with PINK1 upregulation in the AngII treatment model. Additionally, levels of phosphorylated Parkin increased in
cardiomyocytes after the induction of mitophagy. Similarly, knockdown of PINK1 expression inhibited the translocation and phosphorylation of Parkin. Thus, PINK1/Parkin pathway regulates mitochondrial mass via the selective degradation of dysfunctional mitochondria. In summary, decreased MMP induced by AngII maintains the stability of PINK1, causing PINK1 autophosphorylation. Additionally, PINK1 activation promotes Parkin translocation and phosphorylation, thereby regulating autophagy signaling, increasing autophagy, clearing damaged mitochondria, reducing mitochondrial oxidative stress, and preventing apoptosis and hypertrophy. Furthermore, these effects were reversed by treatment with an autophagy inhibitor. Therefore, to a certain extent, PINK1/Parkin-mediated mitophagy plays a compensatory protective role in AngII-induced cytotoxicity, and modulation of this mechanism may be a valid approach for the treatment of myocardial hypertrophy.
Declaration of conflicts of interest The authors report that there are no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
PTEN induced putative kinase 1 (PINK1) alleviates angiotensin II-induced cardiac injury by ameliorating mitochondrial dysfunction Wenjun Xiong a,b, Jinghai Hua a,b, Zuheng Liu a,b, Wanqiang Cai a,b, Yujia Bai a,b, Qiong Zhan a,b, Wenyan Lai a,b, Qingchun Zeng a,b, Hao Ren b,c,⁎, Dingli Xu a,b,⁎⁎ a b c
State Key Laboratory of Organ Failure Research, Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou, China Key Laboratory for Organ Failure Research, Ministry of Education of the People's Republic of China, Guangzhou, China Department of Rheumatology, Nanfang Hospital, Southern Medical University, Guangzhou, China
a r t i c l e
i n f o
Article history: Received 13 December 2017 Received in revised form 17 February 2018 Accepted 12 March 2018
Keywords: PINK1 Parkin Mitophagy Angiotensin II MMP
a b s t r a c t Background: Mitochondrial quality control is crucial to the development of angiotensin II (AngII)-induced cardiac hypertrophy. PTEN induced putative kinase 1 (PINK1) is rapidly degraded in normal mitochondria but accumulates in damaged mitochondria, triggering autophagy to protect cells. PINK1 mediates mitophagy in general, but whether PINK1 mediates AngII-induced mitophagy and the effects of PINK1 on AngII-induced injury are unknown. This study was designed to investigate the function of PINK1 in an AngII stimulation model and its regulation of AngII-induced mitophagy. Methods: We studied the function of PINK1 in mitochondrial homeostasis in AngII-stimulated cardiomyocytes via RNA interference-mediated knockdown and adenovirus-mediated overexpression of the PINK1 protein. Mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, adenosine triphosphate (ATP) content, cell apoptosis rates and cardiomyocyte hypertrophy were measured. The expression of LC3B, Beclin1 and p62 was measured. Mitochondrial morphology was examined via electron microscopy. Mitophagy was detected by confocal microscopy based on the co-localization of lysosomes and mitochondria. Additionally, endogenous PINK1, phosphorylated PINK1, mito-PINK1, total Parkin, cyto-Parkin, mito-Parkin and phosphorylated Parkin protein levels were measured. Results: Cardiomyocytes untreated by AngII had very low levels of total and phosphorylated PINK1. However, in the AngII stimulation model, the MMP was decreased, and the levels of total and phosphorylated PINK1 were increased. After PINK1 was knocked down, Parkin translocation to the mitochondria was inhibited. Moreover, levels of phosphorylated Parkin were reduced, and autophagy markers were downregulated. MMP and ATP contents were further reduced, ROS production and the apoptotic rate were further increased, and myocardial hypertrophy was further aggravated compared with those in the AngII group. However, PINK1 overexpression promoted Parkin translocation and phosphorylation, autophagy markers were upregulated, and myocardial injury was reduced. In addition, the effects of PINK1 overexpression were reversed by autophagy inhibitors. Conclusion: Decreased MMP induced by AngII maintains the stability of PINK1, causing PINK1 autophosphorylation. PINK1 activation promotes Parkin translocation and phosphorylation and increases autophagy to clear damaged mitochondria. Thus, PINK1/Parkin-mediated mitophagy has a compensatory, protective role in AngIIinduced cytotoxicity. © 2018 Published by Elsevier B.V.
Abbreviations: PINK1, PTEN-induced putative kinase 1; AngII, angiotensin II; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; LC3B, microtubuleassociated protein 1 light chain 3 beta; ATP, adenosine triphosphate. ⁎ Correspondence to: H. Ren, Department of Rheumatology, Nanfang Hospital, Southern Medical University, 1838 Northern Guangzhou Ave, Guangzhou, Guangdong 510515, China. ⁎⁎ Correspondence to: D. Xu, Department of Cardiology, Nanfang Hospital, Southern Medical University, 1838 Northern Guangzhou Ave, Guangzhou, Guangdong 510515, China. E-mail addresses: [email protected] (H. Ren), dinglixu@fimmu.com (D. Xu).
https://doi.org/10.1016/j.ijcard.2018.03.054 0167-5273/© 2018 Published by Elsevier B.V.
1. Introduction Myocardial hypertrophy is an important adaptive response that maintains and increases cardiac output during the early period of heart failure [1–3]. Persistent stress can burden myocardial hypertrophy, ultimately resulting in ventricular remodeling, congestive heart failure or sudden death due to arrhythmia [4–6]. Activation of the renin-angiotensin system is a key factor that triggers myocardial hypertrophy. Moreover, mitochondrial function is crucial to the development
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of cardiac hypertrophy [7]. Dysfunctional mitochondria decrease mitochondrial membrane potential (MMP), produce high levels of reactive oxygen species (ROS) and are more sensitive to Ca2+-induced mitochondrial permeability transition pores. Furthermore, damaged mitochondria trigger a chain reaction, eventually resulting in lower ATP levels. Increased knowledge of mitochondrial function will help the treatment of cardiac hypertrophy-related events [8,9]. In recent years, autophagy has been recognized as a critical factor in maintaining cardiac function during myocardial hypertrophy. Autophagy is a regulated mechanism leading to the disassembly of dysfunctional proteins or organelles [10–13]. Under pathological conditions, autophagy is activated as an adaptive response to stress to promote survival. However, the role of autophagy in cardiac hypertrophy remains controversial [14,15]. Indeed, autophagic activity may perform different functions depending on the stage and severity of the disease. Activation of autophagy in the early stages of the disease may be a protective mechanism for the clearance and elimination of misfolded polyubiquitinated protein aggregates [11]. In addition, it is necessary to remove dysfunctional mitochondria to prevent the catastrophic loss of ATP in the early stages of the disease. Meanwhile, autophagy provides energy to compensate for the increased ATP requirements caused by stress overload. Cardiac autophagy may play a key role in removing damaged mitochondria, maintaining cell homeostasis and preventing apoptosis [16]. However, the point at which autophagic activity becomes autophagic death has not been defined. Thus, understanding the mechanisms of autophagic regulation may facilitate the discovery of a novel strategy to combat myocardial hypertrophy. Accumulating evidence from studies investigating PTEN-induced putative kinase 1 (PINK1) and Parkin has revealed that these two proteins participate in mitochondrial quality control during cardiac injury [17]. PINK1-activated Parkin translocates to mitochondria with low membrane potential to initiate the autophagic degradation of damaged mitochondria [18]. PINK1 mediates mitophagy in general, but it is unknown whether PINK1 mediates AngII-induced mitophagy, and the effects of PINK1 on angiotensin II (AngII)-induced injury are uncharacterized. We hypothesized that PINK1 regulates mitophagy to alleviate AngIIinduced cardiomyocyte injury by promoting Parkin translocation and phosphorylation. We knocked down and overexpressed PINK1 in cardiomyocytes to study the effects of PINK1 on cardiac function, and we found that in an AngII stimulation model, PINK1 promoted Parkin translocation and phosphorylation, thereby regulating autophagy signaling and clearing damaged mitochondria. PINK1-mediated mitophagy eliminated the damaged mitochondria-induced chain reaction, mitigating outcomes such as myocardial apoptosis and cardiac hypertrophy.
2. Materials and methods 2.1. Chemicals and reagents Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT, USA). Trypsin and collagenase were purchased from Sigma-Aldrich Co. (Saint Louis, MO, USA). AngII, chloroquine diphosphate salt (CQ) and manganese chloride (Mncl2) were purchased from Sigma-Aldrich Co. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was purchased from Beyotime (Jiangsu, China). The following polyclonal primary antibodies were used in this study: anti-PINK1, anti-Parkin, Anti-Parkin (phospho S65), anti-p62, anti-Beclin1, anti-COX IV, anti-β-actin (Abcam, Cambridge, UK), anti-GAPDH (Santa Cruz Biotechnology Inc., CA, USA) and anti-LC3B (Sigma-Aldrich Co.).
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2.3. Detection of mitochondrial morphology Mitophagy was determined based on the co-localization of mitochondria and lysosomes. Cardiomyocytes were co-incubated with MitoTracker Green (100 nM) and LysoTracker Red (50 nM, Molecular Probes, Eugene, OR, USA) for 40 min. Confocal images were acquired with a Zeiss LSM 880 (Zeiss, Germany). The number of MitoTracker- and LysoTracker-positive foci was determined by using ImageJ software. Mitochondrial morphology was observed by transmission electron microscopy (TEM). Small fragments of cardiomyocytes (1 mm3 in size) were fixed for 2 h with 2.5% glutaraldehyde in 0.1 M PBS, pH 7.4. After rinsing 6× with PBS for 30 min, the cells were incubated with 1% osmium tetroxide in 0.1 M cacodylate phosphate buffer for 2 h. The cells were subsequently rinsed 3× with PBS for 10 min. The samples were then dehydrated in a graded ethanol series (50%, 70%, 90%, and 100%), rinsed with propylene oxide, permeabilized with a 1:1 mixture of propylene oxide and Epon 812, and then baked in a 38 °C oven for 3 h. The above steps were repeated using a 1:2 mixture of propylene oxide and Epon 812. Next, ultrathin sections (50–70 nm) of the samples were cut with an ultramicrotome (UC7, Leica, Germany), collected on 200-mesh copper grids and incubated with 5% uranyl acetate in ethanol (10 min) and lead citrate (15 min) for contrast enhancement. Finally, mitochondria were examined with an electron microscope (JEM-1400, Japan) operated at 80 kV. 2.4. Measurement of ROS levels Intracellular ROS production was quantified with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE, Invitrogen Molecular Probes, USA). Cardiomyocytes were incubated with culture medium containing 10 μM DCFH-DA or 10 μM DHE for 30 min at 37 °C and washed three times with PBS. Then, ROS production was detected using a fluorescence microscope (Olympus, Japan). ImageJ was used for analysis. 2.5. Measurement of MMP MMP was measured using a JC-1 kit (Sigma-Aldrich Co.). After stimulation, cells were incubated with JC-1 staining solution at 37 °C for 20 min and then washed twice with JC-1 staining buffer. MMP was assayed with a fluorescence microscope. ImageJ was used for analyses. 2.6. Apoptosis assays Apoptosis in cardiomyocytes was analyzed using an Annexin V Apoptosis Detection Kit (Roche, Shanghai, China). Cells were incubated for 15 min at 4 °C in the dark with Annexin V. After the addition of propidium iodide (PI) into the medium for 5 min, the proportion of Annexin V-positive and PI-positive cells was analyzed with a flow cytometer (Beckman Coulter, USA). 2.7. Measurement of cardiomyocyte hypertrophy The mRNA levels of hypertrophy-related genes were detected. Total RNA was purified from cardiomyocytes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA samples were reverse transcribed to generate cDNAs using a first-strand cDNA synthesis kit (GeneCopoeia, Rockville, MD, USA). qRT-PCR was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The expression of the following genes was measured: ANP, forward primer 5′-ACCTGCTAGACCACCTGGAG-3′, reverse primer 5′-CCTTGG CTGTTATCTTCGGTACCGG-3′; β-MHC, forward primer 5′-TCTGGACAGCTCCCCATTCT-3′, reverse primer 5′-CAAGGCTAACCTGGAGAAGATG-3′; GAPDH, forward primer 5′-ATCA AGAAGGTGGTGAAGCA-3′, reverse primer 5′-AAGGTGGAAGAATGGGAGTTG-3′. We utilized 10-μl reactions with the following RT-PCR conditions: 95 °C for 5 min; 40 cycles of 95 °C for 10 s, 55–60 °C for 20 s, and 72 °C for 32 s. The results were analyzed with the 2−△△Ct method and normalized to GAPDH gene expression. Cardiomyocyte surface area was observed by immunofluorescence. Cardiomyocytes were fixed and permeabilized. The cells were blocked and then incubated overnight with anti-β-actin. Then, the cells were incubated with secondary antibody to label cytoskeletal actin. Finally, the cells were stained with DAPI at room temperature. Confocal images were acquired using a Zeiss LSM 880. 2.8. Measurement of ATP levels ATP content in cardiomyocytes was measured by an ATP assay kit (Beyotime). Briefly, the cardiomyocytes were lysed by cellular ATP-releasing reagent. ATP detection solution was mixed with luciferase solution. Bioluminescence was measured with a luminometer. ATP content was estimated according to a standard curve. The results were normalized to cellular protein concentration.
2.2. Cell culture and adenoviral transduction 2.9. Immunofluorescence Neonatal rat cardiomyocytes were prepared from the hearts of Sprague-Dawley rats using enzymatic dissociation and cultured as previously described [19]. Recombinant adenoviruses for PINK1 overexpression (AD-PINK1) and knockdown (si-PINK1) were designed and synthesized by GeneChem Co. (Shanghai, China). The viruses were added to cells according to the manufacturer's instructions. Cells were stimulated with 1 μM AngII for 24 h after adenoviral transduction for 72–96 h, and then subsequent experiments and analyses were performed.
Cardiomyocytes were fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.2% Triton X-100 for 10 min. The cells were blocked for 30 min and then incubated overnight with primary antibodies at 4 °C. Then, the cells were incubated with goat anti-rabbit secondary antibody (Santa Cruz Biotechnology Inc.) for 60 min at room temperature. Additionally, the cells were incubated with goat anti-mouse antibody to label cytoskeletal actin. Finally, the cells were stained with DAPI at room temperature. Confocal images were
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acquired using a Zeiss LSM 880. Quantification of fluorescent pixels was carried out by ImageJ software.
2.10. Immunoblot analysis For total protein extraction, the cardiomyocytes were lysed in lysis buffer containing protease inhibitor cocktail (KeyGEN BioTECH Inc., Jiangsu, China) for 30 min. After centrifuging at 12,000g for 15 min at 4 °C, cell suspensions were collected as whole-cell proteins. Cytoplasmic proteins and mitochondrial proteins were extracted from cells using a Cell Mitochondria Isolation Kit (Beyotime, Jiangsu, China) according to the manufacturer's instructions. Briefly, cardiomyocytes were lysed in mitochondrial fractionation reagent with PMSF for 15 min. The cell suspensions were transferred to a homogenizer and homogenized 10 to 30 times. After centrifuging at 1000g for 10 min at 4 °C, the cell suspensions were transferred to another centrifuge tube. The cell lysates continued to be centrifuged at 11,000g for 10 min at 4 °C. The supernatants were composed of cytoplasm, and the sediment contained the mitochondria. After further lysis, cytoplasmic proteins and mitochondrial proteins were collected for subsequent experiments. Approximately 30 μg of protein per sample was separated on SDS-polyacrylamide gels. To detect phosphorylated PINK1, Phos-tag™ SDS-PAGE containing 50 μmol/l Phos-tag™ acrylamide (Wako Pure Chemical Industries, Ltd, Japan) and 100 μmol/l MnCl2 was performed. After
electrophoresis, Phos-tag gels were washed with transfer buffer containing 1 mmol/l EDTA and then replaced with transfer buffer without EDTA. The gels were electrotransferred to nitrocellulose membranes (Millipore, USA). The membranes were blocked in 2.5% non-fat milk, incubated overnight with the indicated primary antibodies and then incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). The density of the expressed protein bands was quantified by ImageJ software. 2.11. Statistical analysis The data are expressed as the mean ± SD. Statistical analyses were performed using either one-way analysis of variance or Student's t-tests (GraphPad Prism 6.0 software, San Diego, CA, USA). P-values b 0.05 were considered statistically significant.
3. Results 3.1. Knockdown of PINK1 inhibited cardiomyocyte mitophagy To study the effects of PINK1-targeting siRNA (si-PINK1) on mitophagy in the AngII model, we detected changes in mitochondrial
Fig. 1. PINK1 knockdown inhibited mitophagy and aggravated injury in cardiomyocytes stimulated with AngII. Cardiomyocytes were transfected with adenoviruses encoding si-PINK1 and si-Control and then stimulated with AngII (1 μM) for 24 h. (A) TEM showing mitochondrial morphology in cardiomyocytes. Enlarged image in the yellow frame shows the details of mitochondrial structures. (B) Fluorescence images showing lysosomal-mitochondrial interactions. Mitochondria are shown in green, lysosomes are shown in red, and Hoechst staining in the nuclei is shown in blue. (C) Immunoblotting showing the expression of Beclin1, p62 and LC3B in cardiomyocytes. Quantification relative to β-actin levels. (D) Fluorescence images using DHE fluorescent dye showing ROS production in cardiomyocytes. (E) Fluorescence images using DCFH-DA fluorescent dye showing ROS production in cardiomyocytes. (F) Fluorescence images of cardiomyocytes stained with JC-1 tracker. (G) Cells were stained with PI and Annexin V-FITC and then subjected to flow cytometric analysis. (H-I) Relative fold changes in the mRNA levels of β-MHC and ANP were determined using qRT-PCR. (J) Fluorescence images showing the surface area of cardiomyocytes. Cytoskeletal actin is shown in red, and DAPI staining in the nuclei is shown in blue. (K) Intracellular ATP levels were determined by using an ATP assay kit with a luminometer. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. si-Control + Control; #P b 0.05 vs. si-Control + AngII.
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morphology and autophagy-related protein levels. The TEM results showed that knockdown of PINK1 inhibited AngII-induced mitophagy and exacerbated AngII-induced mitochondrial fracture (Fig. 1A). Fluorescence images of the co-localization of lysosomes and mitochondria showed that knockdown of PINK1 decreased lysosomal-mitochondrial interactions (Fig. 1B). Similarly, knockdown of PINK1 negated the AngII-induced increase in the expression of autophagy-related proteins, with the exception of p62 (Fig. 1C). 3.2. Knockdown of PINK1 increased oxidative stress and cell apoptosis To evaluate the effects of si-PINK1 on AngII-induced injury, we analyzed intracellular ROS production, apoptosis and hypertrophy in cardiomyocytes. Knockdown of PINK1 significantly increased AngIIinduced ROS production as shown by DHE and DCFH staining (Fig. 1D, E). Additionally, JC-1 staining showed that the knockdown of PINK1 aggravated the AngII-induced reduction in MMP (Fig. 1F). Knockdown of
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PINK1 significantly increased AngII-induced cardiomyocyte apoptosis (Fig. 1G) and decreased ATP content (Fig. 1K). Additionally, downregulation of PINK1 enhanced AngII-increased mRNA levels of ANP and β-MHC (Fig. 1H, I) and the surface area of cardiomyocytes (Fig. 1J). 3.3. Overexpression of PINK1 enhanced cardiomyocyte mitophagy We tested the effects of PINK1 overexpression on mitophagy in the AngII model. The TEM results revealed that PINK1 overexpression increased the formation of autophagosomes (Fig. 2A). Fluorescence images of the co-localization of lysosomes and mitochondria showed that the overexpression of PINK1 further increased lysosomal-mitochondrial interactions (Fig. 2B). Additionally, western blot analysis showed that PINK1 overexpression promoted an AngII-induced increase in the expression of Beclin1 and LC3B II and reduced the expression of p62 (Fig. 2C). Therefore, PINK1 was involved in mitophagy induction in response to AngII stimulation.
Fig. 2. PINK1 overexpression upregulated mitophagy and alleviated injury in cardiomyocytes stimulated with AngII. Cardiomyocytes were transfected with adenoviruses Ad-PINK1 and Ad-Control and then stimulated with AngII (1 μM) for 24 h. (A) TEM showing mitochondrial morphology in cardiomyocytes. Enlarged image in the yellow frame shows the details of mitochondrial structures. (B) Fluorescence images showing lysosomal-mitochondrial interactions. Mitochondria are shown in green, lysosomes are shown in red, and Hoechst staining in the nuclei is shown in blue. (C) Immunoblotting showing the expression of Beclin1, p62 and LC3B in cardiomyocytes. Quantification relative to β-actin levels. (D) Fluorescence images using DHE fluorescent dye showing ROS production in cardiomyocytes. (E) Fluorescence images using DCFH-DA fluorescent dye showing ROS production in cardiomyocytes. (F) Fluorescence images of cardiomyocytes stained with JC-1 tracker. (G) Cells were stained with PI and Annexin V-FITC and then subjected to flow cytometric analysis. (H–I) Relative fold changes in the mRNA levels of β-MHC and ANP were determined using qRT-PCR. (J) Fluorescence images showing the surface area of cardiomyocytes. Cytoskeletal actin is shown in red, and DAPI staining in the nuclei is shown in blue. (K) Intracellular ATP levels were determined by using an ATP assay kit with a luminometer. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. Ad-Control + Control; #P b 0.05 vs. Ad-Control + AngII.
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3.4. Overexpression of PINK1 prevented AngII-induced oxidative stress and apoptosis Next, we investigated the effects of PINK1 overexpression during AngII-induced cardiac injury. AngII-induced ROS production was inhibited following the overexpression of PINK1 as shown by DHE and DCFH staining (Fig. 2D and E). Furthermore, PINK1 overexpression promoted the removal of damaged mitochondria due to MMP reduction (Fig. 2F). Upregulation of PINK1 significantly decreased AngII-induced cardiomyocyte apoptosis (Fig. 2G) and increased ATP levels (Fig. 2K). Additionally, PINK1 overexpression reduced AngII-increased mRNA levels of ANP and β-MHC (Fig. 2H, I) and the surface area of cardiomyocytes (Fig. 2J). Therefore, PINK1 exerted protective effects against AngII-induced cardiac injury. 3.5. Autophagy inhibition reversed the effects of PINK1 To determine whether the regulation of mitophagy by PINK1 plays a critical role in AngII-induced cardiac injury, we treated cardiomyocytes with CQ, an inhibitor of autophagy. The CQ-treated group had higher ROS levels (Fig. 3A and B) and apoptosis rates (Fig. 3C) during AngIIinduced cardiac injury than did the PINK1 overexpression group, indicating that the effects of PINK1 overexpression were reversed by the autophagy inhibitor. Therefore, PINK1 alleviated AngII-induced cardiac injury by regulating mitophagy. 3.6. The PINK1/Parkin pathway regulated mitophagy in AngII-induced cardiac injury To further elucidate the functions of PINK1 and the PINK1/Parkin pathways, we assess the expression of PINK1 and Parkin proteins and
the phosphorylation levels of PINK1 and its substrate, Parkin. With increased AngII stimulation time, the expression of PINK1 peaked at 24 h, followed by stable expression until 60 h and decreased expression at 72 h (Fig. 4A). PINK1 fluorescence intensity was increased (Fig. 4D) with decreasing MMP (Fig. 4C) when AngII was stimulated for 24 h. Additionally, AngII-induced MMP reduction promoted PINK1 mitochondrial translocation (Fig. 4I, J). Endogenous PINK1 expression and phosphorylation were increased under AngII and CCCP stimulation (Fig. 4B). Use of RNA interference-mediated knockdown and adenovirus-mediated overexpression of the PINK1 protein induced corresponding knockdown and overexpression of PINK1 (Fig. 4E, F). PINK1 knockdown inhibited Parkin mitochondrial translocation (Fig. 4G). Concurrently, elevated levels of mitochondrial Parkin (mito-Parkin) were observed in the AngII model when PINK1 was upregulated, indicating Parkin translocates from the cytoplasm to the mitochondria (Fig. 4H). Additionally, phosphorylation of Parkin was induced by PINK1 overexpression and inhibited by PINK1 knockdown (Fig. 4I, J). 4. Discussion Neurohumoral stimulation through the renin-angiotensin system plays critical roles in cardiac physiological and pathological processes [20–22]. As an important neurohumoral factor, AngII promotes myocardial hypertrophy. Using an in vitro AngII-induced cardiomyocyte hypertrophy model, we found that AngII stimulation upregulated the basal and phosphorylation levels of endogenous PINK1. We further revealed that PINK1 knockdown increased AngII-induced ROS production and apoptosis, thereby interfering with mitochondrial energy production. Additionally, the overexpression of PINK1 promoted the maintenance of cardiomyocyte function by mediating mitophagy, a process that removes dysfunctional mitochondria. These effects were associated
Fig. 3. Effects of PINK1 overexpression were reversed by an autophagy inhibitor. Cardiomyocytes were transfected with the adenoviruses Ad-PINK1 and Ad-Control and then co-treated with AngII (1 μM) for 24 h and CQ (10 μM). (A) Fluorescence images using DHE fluorescent dye showing ROS production in cardiomyocytes. (B) Fluorescence images using DCFH-DA fluorescent dye showing ROS production in cardiomyocytes. (C) Cells were stained with PI and Annexin V-FITC and then subjected to flow cytometric analysis. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. Ad-Control + Control; #P b 0.05 vs. Ad-Control + AngII; ▲P b 0.05 vs. Ad-PINK1 + AngII.
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Fig. 4. PINK1/Parkin pathway in cardiomyocytes stimulated with AngII. (A) Immunoblotting showing the expression of PINK1 for the indicated times. (B) Immunoblotting of endogenous PINK1. The asterisks show phosphorylated PINK1. Cardiomyocytes were treated with CCCP (10 μM, 1 h) or AngII (1 μM, 24 h) and subjected to SDS–PAGE on a 50-μM Phos-tag gel. (C) Fluorescence images of cardiomyocytes stained with JC-1 tracker. (D) Immunofluorescence images demonstrating the expression of PINK1 in cardiomyocytes stimulated with AngII. PINK1 staining is shown in green, representing the expression of PINK1, and DAPI staining in the nuclei is shown in blue. Cardiomyocytes were transfected with the adenoviruses si-PINK1 and si-Control and then stimulated with AngII. (E,G,I) Immunoblotting showing the expression of PINK1, total Parkin, cyto-Parkin, mito-Parkin, P-Parkin and mito-PINK1. Data are presented as the mean ± SD. (n = 3). *P b 0.05 vs. si-Control + Control; #P b 0.05 vs. si-Control + AngII. Cardiomyocytes were transfected with the adenoviruses Ad-PINK1 and Ad-Control and then stimulated with AngII. (F,H,J) Immunoblotting showing the expression of PINK1, total Parkin, cyto-Parkin, mito-Parkin, P-Parkin and mito-PINK1 in cardiomyocytes. *P b 0.05 vs. Ad-Control + Control; #P b 0.05 vs. Ad-Control + AngII. (J) Schematic model of the mechanism underlying the regulatory role of PINK1 in AngII-induced cardiac injury.
with activation of the PINK1/Parkin signaling pathway. Thus, PINK1 is important for the regulation of cardiac remodeling induced by AngII. The protein kinase C (PKC) signaling pathway is crucial in the AngIIinduced cardiac hypertrophy [23]. Furthermore, cardiac hypertrophy involves a PKC-mediated increase in mitochondrial ROS production [24]. When AngII activates the hypertrophic signaling pathway, large amounts of ROS are produced. Additionally, ROS acts as a positive
feedback signal to further stimulate the hypertrophy signaling pathway, and ROS also activates the apoptotic signaling pathway. Constant activation of this pathway eventually causes damage to cardiomyocytes. ROS is primarily derived from mitochondrial dysfunction. Thus, preserving mitochondrial function integrity might be an effective strategy for the treatment of hypertrophic cardiomyocytes. Mitophagy has attracted substantial interest in recent years due to its critical role in the clearance
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of damaged mitochondria [25]. This mechanism of mitochondrial quality control is mediated by two factors: PINK1 and its downstream substrate, the E3 ubiquitin ligase Parkin [26,27]. Overall mitochondrial fitness is protected via the regulation of mitophagy [28]. Therefore, we upregulated the expression of PINK1 and observed that it exerted a partial beneficial effect by increasing autophagic activity and reducing intracellular ROS levels in AngII-induced cardiomyocytes relative to control cells. The downregulation of PINK1 induced the opposite results. Based on these findings, we confirmed that mitophagy plays a critical role during AngII-induced cell death by facilitating the degradation of damaged mitochondria. Localized to the mitochondria, PINK1 regulates mitochondrial mass via the selective degradation of dysfunctional mitochondria [28–31]. The Youle group reported that newly synthesized PINK1 is introduced into the healthy mitochondrial inner membrane and undergoes mitochondrial protease-dependent cleavage followed by proteasome degradation [32–36]. However, normal MMP is required for input, and the dissipation of MMP prevents PINK1 from reaching the intima; thus, PINK1 remains confined to the mitochondrial outer membrane. Previous studies have explained how the loss of MMP causes PINK1 to accumulate on damaged mitochondria [37–39]. Similarly, in our study, we observed that unphosphorylated and phosphorylated levels of PINK1 in cardiomyocytes treated without AngII were very low. However, in the AngII stimulation model, the MMP was decreased, and PINK1 basal and phosphorylation levels were increased. In addition, we observed that AngII-induced MMP reduction promoted PINK1 mitochondrial translocation. Conversely, opposing results have been observed in many studies; for example, PINK1 mRNA and protein levels were significantly lower in a diabetes mellitus group. Interestingly, the results of some studies are similar to ours, where the expression of PINK1 was markedly increased. We analyzed changes in PINK1 expression primarily to evaluate temporal expression patterns in cell models and under pathological conditions and observed varying levels of PINK1 at different physiological stages, such as early compensatory increase and late decrease. Upregulation of PINK1 in cells with normal MMP did not cause an increase in autophagy markers. These results indirectly suggest that mitochondria with reduced membrane potential maintain PINK1 localization on the mitochondrial membrane, causing PINK1 autophosphorylation and ultimately autophagy. Okatsu et al. demonstrated by mass spectrometry and mutation analysis that PINK1 autophosphorylation occurs at Ser228 and Ser402, and importantly, alanine mutations at these sites abolish PINK1 autophosphorylation and inhibit Parkin recruitment to depolarized mitochondria [40]. These results suggest that the reduction in membrane potential is a trigger to prevent PINK1 degradation by mitochondrial proteases and facilitate subsequent phosphorylation modifications. Therefore, we exogenously increased PINK1 levels. With AngII stimulation, we observed an increase in the exogenous expression of PINK1. Furthermore, transfer of Parkin from the cytoplasm to the mitochondria increased autophagy, decreased ROS activity, and significantly improved apoptosis and hypertrophy. Thus, we concluded that endogenous PINK1 and its phosphorylation activity were necessary for AngII-induced mitophagy, which is protective. However, endogenous PINK1 and its phosphorylation activity were not sufficient to initiate additional and more effective autophagy to antagonize the decline in MMP under AngII stimulation. Studies have shown that post-translational modification of Parkin is a key regulator of mitochondrial autophagy [41]. PINK1 promotes the recruitment of cytosolic Parkin to the mitochondrial outer membrane of depolarized mitochondria, causes the ubiquitination of mitochondrial proteins and transduces autophagy signals. This process is associated with Parkin phosphorylation at the S65 residue in the ubiquitin-like domain via PINK1-regulated phosphorylation. Our study examined the translocation and phosphorylation of Parkin. By exogenously changing PINK1 expression levels, we found that Parkin translocated from the cytoplasm to the mitochondria with PINK1 upregulation in the AngII treatment model. Additionally, levels of phosphorylated Parkin increased in
cardiomyocytes after the induction of mitophagy. Similarly, knockdown of PINK1 expression inhibited the translocation and phosphorylation of Parkin. Thus, PINK1/Parkin pathway regulates mitochondrial mass via the selective degradation of dysfunctional mitochondria. In summary, decreased MMP induced by AngII maintains the stability of PINK1, causing PINK1 autophosphorylation. Additionally, PINK1 activation promotes Parkin translocation and phosphorylation, thereby regulating autophagy signaling, increasing autophagy, clearing damaged mitochondria, reducing mitochondrial oxidative stress, and preventing apoptosis and hypertrophy. Furthermore, these effects were reversed by treatment with an autophagy inhibitor. Therefore, to a certain extent, PINK1/Parkin-mediated mitophagy plays a compensatory protective role in AngII-induced cytotoxicity, and modulation of this mechanism may be a valid approach for the treatment of myocardial hypertrophy.
Declaration of conflicts of interest The authors report that there are no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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