- Email: [email protected]
Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3
Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3 Mingming Zhang M.D., Zhijing Zhao M.D., pH.D., Min Shen M.D., pH.D., Yingmei Zhang M.D., pH.D., Jianhong Duan M.D., Yanjie Guo M.D., Dongwei Zhang M.D., Jianqiang Hu M.D., Jie Lin M.D., Wanrong Man M.D., Lichao Hou M.D. Ph.D., Haichang Wang M.D., pH.D., Dongdong Sun M.D., pH.D. PII: DOI: Reference:
S0925-4439(16)30221-6 doi: 10.1016/j.bbadis.2016.09.003 BBADIS 64549
To appear in:
BBA - Molecular Basis of Disease
Received date: Revised date: Accepted date:
13 July 2016 29 August 2016 3 September 2016
Please cite this article as: Mingming Zhang, Zhijing Zhao, Min Shen, Yingmei Zhang, Jianhong Duan, Yanjie Guo, Dongwei Zhang, Jianqiang Hu, Jie Lin, Wanrong Man, Lichao Hou, Haichang Wang, Dongdong Sun, Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3, BBA - Molecular Basis of Disease (2016), doi: 10.1016/j.bbadis.2016.09.003
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ACCEPTED MANUSCRIPT Title: Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3
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Authors:
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Mingming Zhang1,2*, M.D., Zhijing Zhao2*, M.D., Ph.D., Min Shen2*, M.D., Ph.D., Yingmei Zhang3, M.D., Ph.D., Jianhong Duan4, M.D., Yanjie Guo2, M.D., Dongwei Zhang1, M.D., Jianqiang Hu1,2, M.D., Jie Lin1,2, M.D., Wanrong Man1,2, M.D., Lichao Hou5# M.D., Ph.D.,
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Haichang Wang1,2#, M.D., Ph.D., Dongdong Sun1,2#, M.D., Ph.D. 1
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Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, China;
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Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an, China;
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Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital,
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Fudan University, Shanghai, China;
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University, Xi’an, China;
Department of Anesthesia, Xijing Hospital, Fourth Military Medical University, Xi’an, China;
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5
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State Key Laboratory of Military Stomatology, School of Stomatology, Fourth Military Medical
*Contributed equally to this work.
Correspondence: Dongdong Sun, 1 Xinsi Road, Department of Cardiology, Tangdu Hospital,
Fourth Military Medical University, Xi’an, Shaanxi, 710038, China. Fax: 86 29 84775183; Tel: 86 29 84775183; E-mail: [email protected] (DS). Haichang Wang, 1 Xinsi Road, Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, 710038, China. Fax: 86 29 84773469; Tel: 86 29 84773469; E-mail: [email protected] (HW). Lichao Hou, 127 West Changle Road, Department of Anesthesia, Xijing Hospital, Fourth Military
ACCEPTED MANUSCRIPT Medical University, Xi’an, Shaanxi, 710032, China. Fax: 86 29 84775337; Tel: 86 29 84775337; E-mail: [email protected]
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Number of figures: 8
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Word count: 6517
ACCEPTED MANUSCRIPT Abstract Myocardial infarction (MI), which is characterized by chamber dilation and left ventricular (LV)
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dysfunction, represents a major cause of morbidity and mortality worldwide. Polydatin (PD), a
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monocrystalline and polyphenolic drug isolated from a traditional Chinese herb (Polygonum cuspidatum), alleviates mitochondrial dysfunction. We investigated the effects and underlying mechanisms of PD in post-MI cardiac dysfunction. We constructed an MI model by left anterior
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descending (LAD) coronary artery ligation using wild-type (WT) and Sirt3 knockout (Sirt3−/−)
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mice. Cardiac function, cardiomyocytes autophagy levels, apoptosis and mitochondria biogenesis in mice that underwent cardiac MI injury were compared between groups. PD significantly
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improved cardiac function, increased autophagy levels and decreased cardiomyocytes apoptosis
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after MI. Furthermore, PD improved mitochondrial biogenesis, which is evidenced by increased
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ATP content, citrate synthase (CS) activity and complexes I/II/III/IV/V activities in the cardiomyocytes subjected to MI injury. Interestingly, Sirt3 knockout abolished the protective
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effects of PD administration. PD inhibited apoptosis in cultured neonatal mouse ventricular myocytes subjected to hypoxia for 6 h to simulate MI injury. PD increased GFP-LC3 puncta, and reduced the accumulation of protein aggresomes and p62 in cardiomyocytes after hypoxia. Interestingly, the knock-down of Sirt3 nullified the PD-induced beneficial effects. Thus, the protective effects of PD are associated with the up-regulation of autophagy and improvement of mitochondrial biogenesis through Sirt3 activity. Keywords: Polydatin, PD; Myocardial infarction, MI; Autophagy; Sirt3; Mitochondria
ACCEPTED MANUSCRIPT Introduction Myocardial infarction (MI) induced by coronary artery occlusion is the major cause of death
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worldwide [1, 2]. Approximately 3 - 4 million individuals suffer from myocardial infarction (MI)
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each year [3]. Percutaneous coronary intervention (PCI) and intensive pharmacotherapy restore coronary perfusion and reduce adverse ventricular remodeling [4]. However, the development of cardiac dysfunction after MI remains a major challenge [5, 6].
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Polydatin (PD) is a monocrystalline and polyphenolic drug extracted from the traditional
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Chinese herb Polygonum cuspidatum. Previous studies have shown that PD may be beneficial in the treatment of cardiac ischemia/reperfusion (I/R) injury and alleviate acute kidney injury by
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inhibiting mitochondrial dysfunction [7, 8]. Furthermore, PD ameliorates injury to the small
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intestine via Sirt3 activation-mediated mitochondrial protection and alleviates multiorgan
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dysfunction [9, 10]. However, the effects and underlying mechanism of PD against MI have not been fully elucidated.
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Autophagy has an essential role in maintaining intracellular homeostasis via removing damaged or excess organelles and protein aggregates [11, 12]. Basal levels of autophagy are important for maintaining cellular homeostasis and protecting cells against excess or dysfunctional organelles [12-14]. Defects in autophagy cause cardiac dysfunction [12, 15, 16]. Conversely, enhancing autophagy promotes cell survival in response to cardiac ischemic injury and heart failure [12, 14, 17]. Mitochondria are cellular organelles mainly responsible for cellular respiration and energy production. Mitochondrial dysfunction is expected to cause diminished energy production and increased cell death, en route to the onset and progression of MI [18, 19]. Impaired mitochondrial
ACCEPTED MANUSCRIPT biogenesis has been implicated in the etiology of MI [18, 20, 21]. Understanding the mechanisms underlying mitochondrial dysfunction is pertinent to finding novel targets and therapeutic
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strategies to restore mitochondrial integrity and optimize treatment for MI.
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Sirtuins are highly conserved NAD+-dependent protein deacetylases, seven homologs of which have been identified in mammals (sirt1-7) [22-24]. Among the sirtuin family, Sirtuin-3 (Sirt3) is localized to mitochondria and regulates some mitochondrial metabolic pathways. Previous studies
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have suggested that Sirt3 protects cardiomyocytes from stress-mediated cell death, preserves
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contractile function in response to a chronic increase in workload and interrupts the development of cardiac hypertrophy [24, 25]. Sirt3 is also involved in ATP synthesis and maintenance in many
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tissues, including the heart, liver, and kidney [26]. Furthermore, sirt3 attenuates oxidative stress
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and improves mitochondrial respiration in cardiomyocytes [27]. Consistently, Sirt3 deficiency
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impairs mitochondrial and contractile function in the heart [24]. Interestingly, sirt3 also regulates autophagy processes [28]. Sirt3 has been recently recognized as an important regulator of AMPK
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activation [29-31]. We, therefore, attempted to elucidate whether Sirt3 was involved in mediating the protective effects of PD. Here, we sought to determine whether PD is capable of blocking MI injury by activating autophagy. We also investigated whether the protective effect of PD on autophagy was mediated via the Sirt3/AMPK pathway.
ACCEPTED MANUSCRIPT Methods Animals and treatment
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All animal protocols were approved by the Fourth Military Medical University Ethic
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Committee on Animal Care and all experiments were performed in adherence with the National
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Institutes of Health Guidelines on the Use of Laboratory Animals. Sirt3 knockout (Sirt3−/−) mice were established at K&D gene technology (WuHan, China) (C57BL/6 background). Western blot
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analysis and real-time PCR (RT-PCR) were used to screen Sirt3−/− mice. Eight- to 12- week-old Sirt3−/− mice were randomly allocated to the following groups, with n=20 each: (1) Wild-type +
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sham group (Sham); (2) Sirt3−/− + sham group (Sirt3−/−); (3) Myocardial infarction group (MI); (4) Sirt3−/− + Myocardial infarction group (Sirt3−/− + MI); (5) Myocardial infarction + PD group (MI +
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PD); (6)Sirt3−/− + Myocardial infarction + PD group (Sirt3−/− + MI + PD). Before constructing the MI model, PD (7.5 mg/kg) was dissolved in normal saline and was
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injected intraperitoneally for 10 days. A concentration of 10µM of PD was applied in the experiments in vitro. For autophagy inhibition, mice were injected intraperitoneally with
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autophagy inhibitor 3-methyladenine (3-MA) (10 mg/kg per day) for ten consecutive days. Myocardial infarction surgery As previously described [32] , the MI animal model was constructed by left anterior descending (LAD) coronary artery ligation. A left thoracic incision was used to open the chest. A 6–0 silk suture slipknot was placed at the proximal one-third of the left anterior descending artery. Sham-operated control animals underwent a similar surgery without ligation of the artery. Echocardiography Twenty-four hours after MI, mice were sedated with 2 % isoflurane inhalation and studied on an echocardiography system (Sequoia Acuson, 15-MHz linear transducer;Siemens, Erlangen,
ACCEPTED MANUSCRIPT Germany). Left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS)
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were calculated by using computer algorithms. The left ventricular pressure (LVP) was measured
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via a Millar Mikro-tip catheter transducer that was inserted into the left ventricular cavity through the left carotid artery. The first derivative of the left ventricular pressure (±LV dp/dt max) was obtained by computer algorithms and an interactive videographics program (Po-Ne-Mah
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Transmission electron microscopy (TEM)
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Physiology Platform P3 Plus, Gould Instrument Systems, Valley View, Ohio).
Mitochondrial ultrastructure and autophagosomes were examined using TEM. Cardiac
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specimens from the peri-infarct region were prepared as previously described [33, 34]. Heart
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tissue was removed from the animal and quickly rinsed in PBS. The tissue was placed in a petri
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dish with 0.5% glutaraldehyde and 0.2% tannic acid in PBS, diced into 2 mm cubes, and then transferred to modified Karnovsky's fixative (4% formaldehyde and 2.5% glutaraldehyde
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containing 8 mM CaCl2 in 0.1 M sodium cacodylate buffer (pH 7.4)). Samples were washed with PBS and post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h to produce osmium black. Samples were then dehydrated using a graded series of ethanol and embedded in Epon/SPURR resin (EM Science) that was polymerized at 65°C overnight. Sections of heart tissues were prepared with a diamond knife on a Reichert-Jung Ultracut-E ultramicrotome and stained with UrAc (20 min) followed by 0.2% lead citrate (2.5 min). Images were photographed with a Jeol JEM-1200EX electron microscope. Determination of cardiomyocyte apoptosis Cleaved Caspase-3, Bax and Bcl-2 were detected by Western blot evaluation. Flow cytometric
ACCEPTED MANUSCRIPT analysis of cellular apoptosis was performed as previously described [33]. In brief, cardiomyocytes were harvested and stained with annexin V (Invitrogen) and propidium iodide (PI).
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Data acquisition and analysis were performed using a flow cytometer (FACSort- B0008; Becton-
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Dickinson, Franklin Lakes, NJ, USA) and Cell Quest Pro software, respectively. All of these assays were performed in a blinded manner.
Primary neonatal mouse ventricular cardiomyocyte culture
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Primary cultures of ventricular cardiomyocytes were prepared from 1-day-old non-transgenic
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mice. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone, SV30087.02) and 1% penicillinstreptomycin and maintained at 37 °C in 5 % CO2 as previously
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described [35]. The neonatal mice (1 day old) were disinfected with 75 % ethanol and then killed
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by decapitation. The chest was opened and the heart was rapidly removed and placed in cold PBS
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solution. The myocardium specimen was cut into small pieces and washed, followed by digestion with collagenase type 2. After that, the cell suspension was centrifuged (800 g for 5 min). The
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supernatant was then removed, and the cell pellet was resuspended in medium supplemented with 10% fetal bovine serum. These steps were repeated until the tissue fragments had disappeared. The dissociated cells were replated in a culture flask at 37 °C for 1 h to enrich the culture with cardiomyocytes. The non-adherent cardiomyocytes were collected and were then plated onto gelatin-coated plates. The preparation was carried out at 37 °C, in the presence of 95% O2 and 5% CO2. Transduction of adenoviruses The adenoviruses harboring GFP-LC3 (GFP-LC3) were purchased from GeneChem Technology Ltd (Shanghai, China). The adenoviruses harboring Sirt3 shRNA (Ad-sh-Sirt3)
ACCEPTED MANUSCRIPT (MOI:100, 1×109 TU / ml) were purchased from Hanbio Technology Ltd (Shanghai, China) and were transduced 24 h after transduction of GFP-LC3. After 36 h, the cardiomyocytes were
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incubated in hypoxic conditions for 6 h.
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Detection of GFP-LC3, aggresomes and p62
Fluorescence microscopic detection of GFP-LC3, aggresomes and p62 was conducted according to the manufacturer’s instructions as previously described [35].
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Construction of short hairpin RNA (shRNA) adenoviral expression vectors
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The generation of GFP-LC3 adenoviruses has been described [35]. Cardiac mitochondria isolation
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Mitochondria were isolated from the hearts of the mice as previously described [33].
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Still-beating hearts were removed from mice anesthetized with 1% pentobarbital sodium solution.
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We used ice-cold medium A (120 mM NaCl, 2 mM MgCl2, 20 mM HEPES, 1 mM EGTA, and 5 g/l bovine serum albumin; pH 7.4) to rinse the hearts to remove any residual blood. Cardiac tissue
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was minced in ice-cold medium A and homogenized. The homogenate was centrifuged (600×g, 10 min, 4 °C). The supernatant was subsequently centrifuged (17,000×g, 10 min, 4 °C). The pellet containing the mitochondria was re-suspended in medium A and then centrifuged (7,000×g, 10 min, 4 °C). After the last centrifugation, the pellet was re-suspended in medium B (2 mM HEPES, 300 mM sucrose, 0.1 mM EGTA; pH 7.4) and re-centrifuged (3,500×g, 10 min, 4 °C). The resulting pellet containing the heart mitochondria was suspended in a small volume of medium B. All work was performed on wet ice at 0 °C. Citrate synthase (CS), chain complex activities and ATP content Citrate synthase (CS) and electron transport chain complex activities (Complex I, II, III, IV, and
ACCEPTED MANUSCRIPT V) were detected using a citrate synthase activity assay kit (Sigma, USA). An ATP bioluminescent assay kit (Beyotime, China) was used to measure the ATP content of the myocardium according to
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the standard protocol.
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Calcium retention capacity (mCRC)
To test the sensitivity of the mitochondrial permeability transition pore (mPTP) opening to calcium, the mitochondrial calcium retention capacity (mCRC) was determined as the capacity of
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mitochondria to uptake calcium before permeability transition.
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ROS production and manganese superoxide dismutase (MnSOD) activity The production of ROS was measured in frozen tissue by electron paramagnetic resonance
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(EPR) spectroscopy as previously described [33]. The ROS levels were expressed in arbitrary
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units per milligram of wet tissue. MnSOD was assayed as Vives-Bauza previously described [33]
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and is expressed in units/mg. Western blot evaluation
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Protein concentration quantitation was determined by Bradford assay (Bio-Rad Laboratories, Hercules, Calif), and protein was then separated by SDS-PAGE; the following antibodies were used: Sirt3 (1:1000, Cell Signaling, Danvers, MA, USA), p62, Beclin1 (1:1000, Abcam, Cambridge, MA, UK), Cleaved caspase-3, Bax, Bcl-2 (1:1000, Sigma, St Louis, MO, USA), LC3A/B, Atg5, Adenine mononucleotide protein kinase (AMPK), p-AMPK (Thr172), Anti-ULK1, anti-p-ULK1 (Ser757), anti-FoxO3a, anti-p-FoxO3a (Ser253) (1:1000, Cell Signaling, Danvers, MA, USA), β-actin, GAPDH (1:500, Santa Cruz, CA, USA), and secondary antibodies (Anti-mouse/rabbit IgG) conjugated with horseradish peroxidase (1:5000, Cell Signaling, Danvers, MA, USA). The blots were visualized with a chemiluminescence system (Amersham Bioscience,
ACCEPTED MANUSCRIPT Buchinghamshire, UK). The signals were quantified by Image Pro Plus software (Media Cybernetics).
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Statistical analysis
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Continuous variables were expressed as the mean ± SEM. Comparison between groups were subjected to ANOVA followed by Bonferroni correction for post hoc t-test. Two-sided tests have been used throughout, and P values < 0.05 were considered statistically significant. SPSS software
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package version 14.0 (SPSS, Chicago, IL) was used for data analysis.
ACCEPTED MANUSCRIPT Results Treatment with PD improves cardiac function in mice after MI
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Twenty-four hours of permanent coronary ligation caused severe cardiac dysfunction as
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evidenced by decreased LVEF, LVFS and ± LV dp / dt max with increased LVEDV and LVESV
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(Figure 1A-1G). PD significantly increased LVEF, LVFS and ± LV dp / dt max, suggesting that treatment with PD significantly improved LV systolic function after MI (Figure 1A-1E). In
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PD-treated mice, the increases of LVEDV and LVESV were significantly attenuated compared with the MI mice (Figure 1F, 1G), suggesting that PD significantly attenuated LV dysfunction
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after MI. We next examined whether PD improved cardiac function through activating Sirt3. After
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24 hours of coronary ligation, both LVEF and LVFS were significantly decreased in the WT and
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the Sirt3-/- hearts subjected to MI injury (Figure 1A-1C). Specifically, PD slightly but insignificantly improved LVEF, LVFS and ± LV dp/dt max in the Sirt3-/- mice subjected to MI
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injury (Figure 1A-1E). Meanwhile, Sirt3 knockout abolished the effects of PD administration on LVEDV and LVESV (Figure 1F-1G).
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PD attenuated alterations in mitochondrial ultrastructure and increased autophagosomes after MI
Mitochondrial ultrastructure was examined using TEM. In the sham-operated hearts, the mitochondria were intact and had tightly packed cristae, which formed longitudinal rows between myofibrils. In the Sirt3-/- hearts, some mitochondria were swollen and had disoriented cristae, breakage, and mitochondrial disarrangement (Figure 2), whereas, in the MI mice, most of the mitochondria were markedly damaged with abnormal cristae or matrix areas. Most mitochondria displayed sharply defined cristae in PD-treated mice. A small number of swollen mitochondria were observed, and no obvious vacuole was noted in the PD-treated group (Figure 2). The
ACCEPTED MANUSCRIPT PD-treated mice exhibited moderate mitochondrial damage (Figure 2). In Sirt3-/- hearts after MI, the mitochondria injury was increased, as evidenced by the lack of cristae and matrix in some
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mitochondria, resulting in what appeared to be vacuoles. PD slightly but insignificantly revised
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the mitochondria ultrastructure disorder in Sirt3-/- hearts after MI (Figure 2). These data demonstrate that mitochondrial function was impaired following MI. PD revised the mitochondria ultrastructure disorder in MI hearts but not in Sirt3-/- hearts after MI (Figure 2).
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Autophagosomes, evaluated using transmission electron microscopy (TEM), were
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significantly increased in the MI group compared with the WT group (Figure 2). PD significantly further increased the number of autophagosomes after MI compared to the MI group (Figure 2).
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Sirt3 knockout inhibited autophagy, which was evidenced by decreased autophagosomes (Figure
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2). PD insignificantly further increased autophagosomes in the Sirt3-/- mice after MI injury (Figure
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2).
PD improved mitochondrial function after MI
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Mitochondrial respiration function was examined (Figure 3A-3C). Our data revealed that mitochondria isolated from the MI mice had significantly lower ATP content, CS activity and complexes I/II/III/IV/V activities compared to the mitochondria from the hearts of the sham-operated group (Figure 3A-3C). ATP content, CS activity and complexes I/II/III/IV/V activities of the cardiomyocyte mitochondria were significantly increased in the PD-treated group after MI (Figure 3A-3C). Together, these data suggest that mitochondrial function was impaired in MI mice. Interestingly, PD alleviated these aberrant changes after MI injury. However, PD slightly but insignificantly alleviated mitochondrial damage and had no effects on ATP content, CS activity and complexes I/II/III/IV/V activities in the Sirt3-/- mice subjected to MI injury ((Figure 3A-3C).
ACCEPTED MANUSCRIPT PD administration increased mCRC (Figure 3D), indicating that sensitivity to calcium-induced mPTP opening was decreased. ROS levels (Figure 3E) assessed by EPR spectroscopy and
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mitochondrial MnSOD activity (Figure 3F) were decreased in the PD treated group compared to
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the MI group. However, PD slightly but insignificantly increased mCRC and decreased ROS levels and mitochondrial MnSOD activity in the Sirt3-/- mice subjected to MI injury ((Figure 3D-3F).
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PD prevented post-infarction induced apoptosis
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Consistent with these observations, treatment with PD significantly reduced cardiomyocyte apoptosis induced by MI, as determined by Western blot (Figure4A-4D). PD significantly
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decreased levels of pro-apoptotic proteins Bax and Cleaved Caspase-3 and increased the level of
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anti-apoptotic protein Bcl-2 compared with the MI group (Figure 4A-4D). These data indicate that
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more viable cardiomyocytes were presented in the peri-infarct area in mice treated with PD. However, PD reduced cardiomyocyte apoptosis in the post-MI cardiomyocytes without affecting
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the Sirt3-/- mice after MI injury (Figure 4A-4D). Coincidentally, PD administration had no effect on the Cleaved Caspase-3, Bax and Bcl-2 protein levels in the Sirt3-/- mice after MI injury (Figure 4A-4D).
The Sirt3/ AMPK pathway was directly involved in PD-mediated cardioprotection against MI We hypothesized that Sirt3, a member of the sirtuin family, may also regulate AMPK, ULK1 and FoxO3a phosphorylation. Sirt3 knockout inhibited the phosphorylation of AMPK and ULK1, as well as upregulated the phosphorylation of FoxO3a, which was evidenced by decreased ratios of p-AMPK/AMPK and p-ULK1/ULK1and an increased ratio of p- FoxO3a / FoxO3a (Figure 5).
ACCEPTED MANUSCRIPT In addition, the phosphorylation of AMPK and ULK1 were partially blocked in MI mice (Figure 5). Western blot analysis revealed the increase in Sirt3 and the ratios of p-AMPK/AMPK and
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p-ULK1/ULK1 and the decrease in the ratio of p- FoxO3a / FoxO3a in the PD-treated mouse
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hearts compared with the non-treated hearts (Figure 5). PD insignificantly increased the ratio of p-AMPK/AMPK and p-ULK1/ULK1 and decreased the ratios of p- FoxO3a / FoxO3a in the Sirt3-/- mice after MI injury (Figure 5).
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These data suggest that Sirt3/AMPK signaling is involved in MI-induced cardiomyocyte injury
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and that PD reduces cardiomyocyte susceptibility to MI injury, in part through upregulation of Sirt3/AMPK signaling.
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PD activates cardiomyocyte autophagy regulatory signaling
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Sirt3 is a known key factor for the regulation of mitochondrial biogenesis and energy
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metabolism. Sirt3 was markedly decreased in the peri-infarct zone in mice with MI (Figure 6), whereas PD significantly increased Sirt3 expression in MI mice (Figure 6). In consistence,
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Western blot analysis revealed an increase in the ratio of LC3-II/LC3-Ι and a reduction of p62 in the PD-treated mouse hearts compared with the non-treated hearts (Figure 6). Sirt3 knockout inhibited autophagy, which was evidenced by the decreased ratio of LC3-II/LC3-Ι and increased p62 expression (Figure 6). PD insignificantly further increased autophagy levels in the Sirt3-/mice after MI injury (Figure 6). Assessment of cardiac function revealed that 3-MA significantly depressed LVEF, as well as LVFS, and increased LVESV and LVEDV in mice after MI. Moreover, the protective effects of PD were reversed by 3-MA administration in mice after MI (Figure 6). PD prevented hypoxia-induced apoptosis
ACCEPTED MANUSCRIPT Consistent with these observations, treatment with PD significantly reduced cardiomyocyte apoptosis induced by hypoxia, as determined by flow cytometry analysis (Figure 7). PD
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significantly decreased apoptotic index compared with the hypoxia group (Figure 7). However,
Sirt3 knockdown after hypoxia injury (Figure 7).
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PD reduced cardiomyocytes apoptosis in the cardiomyocyte after hypoxia without affecting the
PD stimulates autophagic flux in the cardiomyocytes subjected to hypoxia injury
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More GFP-LC3 puncta and increased accumulation of aggresomes and P62 were observed in
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the hypoxia group compared with the control group (Figure 8). Interestingly, PD pretreatment significantly increased the number of GFP-LC3 puncta and attenuated the accumulation of
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aggresomes and p62 in the cardiomyocytes subjected to hypoxia injury (Figure 8).
The
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stimulated autophagic flux exerted by PD administration was abolished by Sirt3 knockdown
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(Figure 8).
ACCEPTED MANUSCRIPT Discussion MI remains a major health care problem worldwide. Cardiac dysfunction subsequent to MI is
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associated with a substantially higher fatality rate [36, 37]. Understanding the mechanisms
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regulating cardiomyocyte death and survival after MI is fundamental to reducing the risk of
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developing cardiac dysfunction [38, 39]. In the present study, we provide direct evidence that pretreatment with PD alleviated cardiac dysfunction after MI.
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Autophagy degrades damaged protein aggregates and organelles, which maintains organelle function and protein quality [13]. Up-regulation of autophagy in the conditions of myocardial
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ischemic injury and heart failure is generally compensatory. Autophagy enhancement is critical to
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cell survival by removing damaged organelles and protein aggregates [7, 40]. Cardiomyocytes
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damage is often accompanied by decreased LC-3 expression and the accumulation of p62 which indicates insufficient autophagy. Interestingly, PD pretreatment significantly promoted
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cardiomyocyte autophagy after MI, as evidenced by increased LC3 expression and less accumulation of p62. Furthermore, PD increased autophagosome formation in cardiomyocytes
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subjected to MI injury. In vitro experiments also demonstrated that PD pretreatment significantly promoted autophagy in neonatal mice cardiomyocytes subjected to hypoxia injury, as evidenced by more GFP-LC3 puncta and less accumulation of aggresomes and p62. Most notably, the protective effects of PD in mice after MI were reversed by the administration of 3-MA, an inhibitor of autophagy. These observations provide evidence to support the notion that PD exerts its protective effects by inducing autophagy in mice subjected to MI injury. Previous research indicates that Sirt3 deficiency impairs mitochondrial function in the heart and sirt3 also controls autophagy levels [24, 28]. We, therefore, constructed an MI injury model in the WT and sirt3-/- mice with or without PD pretreatment to elucidate the molecular mechanism
ACCEPTED MANUSCRIPT underlying the protective effects of PD. We used TEM and Western blot analysis to detect cardiomyocyte autophagy levels among different groups. Autophagy was found to be enhanced
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after PD treatment in the WT mice that underwent MI injury. However, in the Sirt3 -/- mice, PD
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failed to up-regulate cardiomyocyte autophagy. These results indicate that PD-induced autophagy might play a pivotal role in its protection effects, and PD may exert its protective effects against MI through activating the Sirt3/AMPK pathway. In line with these observations, PD improved
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cardiac function, decreased cardiomyocyte apoptosis, increased autophagy levels and alleviated
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mitochondrial injury solely in the WT mice subjected to MI injury without affecting the Sirt3 -/mice after MI. Moreover, the protective effects of PD on neonatal mice cardiomyocyte autophagy
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after hypoxia were abolished by sirt3 knockdown.
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Mitochondria are by far the most abundant organelle in the heart. Around ninety percent of
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adenosine triphosphate (ATP) is produced in the mitochondria to supply the energy requirements for heart contraction [23, 41]. Mitochondria play a key role in mediating cellular homeostasis [19,
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20, 42-45]. As a result, maintenance of mitochondrial biogenesis and function are essential for cardiomyocyte survival. In the present study, PD increased ATP content, CS activity and complexes I/II/III/IV/V activities and alleviated mitochondrial ultrastructure injury in the cardiomyocytes subjected to MI injury. Interestingly, the protective effects of PD on mitochondrial biogenesis were abolished by sirt3 knockout. These results indicate that PD may exert its protective effects on mitochondrial biogenesis against MI through Sirt3 activation. Substantial loss of cardiac myocytes after MI can lead to contractile dysfunction and heart failure. Our study revealed the anti-apoptotic effects of PD through the activation of Sirt3, as
ACCEPTED MANUSCRIPT proven via decreased Bax and cleaved Caspase-3. Sirt3 knockout abolished the anti-apoptotic effects of PD after MI. Thus, PD may also exert anti-apoptotic effects through Sirt3 activation.
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In conclusion, the present study provides evidence that PD enhances cardiac function after MI.
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The protective effects of PD are associated with the upregulation of autophagy, down-regulation of apoptosis and improvement of mitochondrial biogenesis through sirt3 activation. PD may represent a potential drug to treat or prevent cardiac dysfunction caused by MI.
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Limitations
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Other members of RISK pathway may participate in the link between PD and its cardiac protection effects. To further demonstrate the efficacy of PD administration on cardiomyocyte
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mitochondrial biogenesis and function, the AMPK-related pathway should be measured in future
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studies. Further studies should be performed to investigate whether PD regulates mitophagy by
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different mechanisms in mice subjected to MI injury. Acknowledgements
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This work was supported by the National Natural Science Foundation of China (No. 81570318, No. 81570361, No. 81300149), and the Shaanxi Social Development and Scientific Problem Tackling Program (2015SF097, 2015KW-036). Conflict of Interest None declared.
ACCEPTED MANUSCRIPT References [1] F. Deng, Y. Xia, M. Fu, Y. Hu, F. Jia, Y. Rahardjo, Y. Duan, L. He, J. Chang, Influence of heart
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failure on the prognosis of patients with acute myocardial infarction in southwestern China, Exp
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ACCEPTED MANUSCRIPT Figure legends Figure 1 PD improves cardiac dysfunction in mice after MI
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A: Representative echocardiographic images at 24 h after MI; B: LV ejection fraction (LVEF); C:
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LV fraction shortening (LVFS); D: LV end-diastolic volume (LVEDV); E: LV end-systolic volume (LVESV); F: +LV dp/dt; G: -LV dp/dt. Mean ± SEM (n=20), *P<0.05 vs Sham group; #P<0.05 vs ＆
Sirt3-/- group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI; P<0.05 vs MI+PD.
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Figure 2 PD attenuated alterations in mitochondrial ultrastructure and increased
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autophagosomes after MI
Representative images of the ultrastructural morphology of mitochondria (yellow arrow) and the
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typical autophagosomes (red arrow) in mice hearts. (Magnification: upper panel ×8200; medial
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panel ×20500; lower panel ×43000).
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Figure 3 PD improved mitochondrial function after MI A-B: ATP content and citrate synthase (CS) activity; C: Enzymatic activities of complexes I-V in
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cardiomyocyte mitochondria; D: The mitochondrial calcium retention capacity (mCRC); E: ROS levels; F: Manganese superoxide dismutase (MnSOD). Mean ± SEM (n=20), *P<0.05 vs Sham ＆
group; #P<0.05 vs Sirt3-/- group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI; P<0.05 vs MI+PD. Figure 4 PD inhibits apoptosis in cardiomyocytes after MI A-D: Protein expression with representative gel blots of Cleaved Caspase-3, Bcl-2 and Bax. Mean ± SEM (n=20), *P<0.05 vs Sham group; #P<0.05 vs Sirt3-/- group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI;
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P<0.05 vs MI+PD.
Figure 5 Sirt3 and phosphorylation of AMPK and ULK1 in the myocardium from mice treated with or without MI.
ACCEPTED MANUSCRIPT A: Representative gel blots; B: Sirt3; C: p-AMPK to AMPK ratio; D: p-ULK1 to ULK1 ratio; E: p- FoxO3a to FoxO3a ratio. Mean ± SEM (n=20), *P<0.05 vs Sham group; #P<0.05 vs Sirt3-/-
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group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI; P<0.05 vs MI+PD.
myocardium from mice treated with or without MI.
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Figure 6 Western blot analysis of the autophagy markers p62, LC3 and Atg5 in the
A: Representative gel blots of p62, LC3, Atg5 and GAPDH (loading control) using specific
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antibodies; B: Beclin1; C: Atg5; D: p62; E: LC3II-to-GAPDH ratio; All proteins were normalized
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to the loading control GAPDH; Mean ± SEM (n=20), *P<0.05 vs Sham group; #P<0.05 vs Sirt3-/group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI;
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P<0.05 vs MI+PD. F: LV ejection fraction
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(LVEF); G: LV fraction shortening (LVFS); H: LV end-diastolic volume (LVEDV); I: LV
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end-systolic volume (LVESV); Mean ± SEM (n=20), *P<0.05 vs MI group; #P<0.05 vs MI+3MA
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group; §P < 0.05 vs MI+PD group.
Figure 7 PD inhibits apoptosis in cardiomyocytes after hypoxia
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A: Apoptosis of the primary cardiomyocytes determined by Cy3-annexinV/PI double staining and flow cytometry. Region B2: late apoptotic cells (Cy3/PI, where Cy3 is cyanine-3 and PI is propidium iodide); Region B3: vital cells; Region B4: early apoptotic cells. B: Quantitative analysis of apoptotic index. Mean ± SEM, *P<0.05 vs Con group; #P<0.05 vs Ad-sh-Sirt3 group; §
P < 0.05 vs H; $P<0.05 vs Ad-sh-Sirt3+H;
＆
P<0.05 vs H+PD.
Figure 8 PD augments cardiomyocyte autophagy flux subjected to hypoxia through Sirt3 activation A-B: Protein expression with representative gel blots of Sirt3; C: Representative images of GFP depicting GFP-LC3 puncta in cardiomyocytes shown in panel; D: Quantitative analysis of the
ACCEPTED MANUSCRIPT number of GFP-LC3 puncta; E: Representative images showing immunofluorescence staining for p62 (orange), DAPI (blue) and ProteoStat aggresome detection reagent (red) in cultured
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cardiomyocytes. F: The number of cardiomyocytes with perinuclear colocalization of p62 and
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aggresomes (p62/aggresomes), indicated by yellow color in the merged images was counted (n = 50 in each group). Mean ± SEM, *P<0.05 vs Con group; #P<0.05 vs Ad-sh-Sirt3 group; §P < 0.05
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vs H; $P<0.05 vs Ad-sh-Sirt3+H; &P<0.05 vs H+PD.
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PD enhances cardiac function after MI in mice. PD up-regulates autophagy, inhibits apoptosis and improves mitochondrial biogenesis. In Sirt3-/- mice subjected to MI injury, the protective effects of PD are abolished.
S0925-4439(16)30221-6 doi: 10.1016/j.bbadis.2016.09.003 BBADIS 64549
To appear in:
BBA - Molecular Basis of Disease
Received date: Revised date: Accepted date:
13 July 2016 29 August 2016 3 September 2016
Please cite this article as: Mingming Zhang, Zhijing Zhao, Min Shen, Yingmei Zhang, Jianhong Duan, Yanjie Guo, Dongwei Zhang, Jianqiang Hu, Jie Lin, Wanrong Man, Lichao Hou, Haichang Wang, Dongdong Sun, Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3, BBA - Molecular Basis of Disease (2016), doi: 10.1016/j.bbadis.2016.09.003
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ACCEPTED MANUSCRIPT Title: Polydatin protects cardiomyocytes against myocardial infarction injury by activating Sirt3
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Authors:
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Mingming Zhang1,2*, M.D., Zhijing Zhao2*, M.D., Ph.D., Min Shen2*, M.D., Ph.D., Yingmei Zhang3, M.D., Ph.D., Jianhong Duan4, M.D., Yanjie Guo2, M.D., Dongwei Zhang1, M.D., Jianqiang Hu1,2, M.D., Jie Lin1,2, M.D., Wanrong Man1,2, M.D., Lichao Hou5# M.D., Ph.D.,
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Haichang Wang1,2#, M.D., Ph.D., Dongdong Sun1,2#, M.D., Ph.D. 1
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Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, China;
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Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an, China;
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Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital,
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Fudan University, Shanghai, China;
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University, Xi’an, China;
Department of Anesthesia, Xijing Hospital, Fourth Military Medical University, Xi’an, China;
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State Key Laboratory of Military Stomatology, School of Stomatology, Fourth Military Medical
*Contributed equally to this work.
Correspondence: Dongdong Sun, 1 Xinsi Road, Department of Cardiology, Tangdu Hospital,
Fourth Military Medical University, Xi’an, Shaanxi, 710038, China. Fax: 86 29 84775183; Tel: 86 29 84775183; E-mail: [email protected] (DS). Haichang Wang, 1 Xinsi Road, Department of Cardiology, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, 710038, China. Fax: 86 29 84773469; Tel: 86 29 84773469; E-mail: [email protected] (HW). Lichao Hou, 127 West Changle Road, Department of Anesthesia, Xijing Hospital, Fourth Military
ACCEPTED MANUSCRIPT Medical University, Xi’an, Shaanxi, 710032, China. Fax: 86 29 84775337; Tel: 86 29 84775337; E-mail: [email protected]
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Number of figures: 8
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ACCEPTED MANUSCRIPT Abstract Myocardial infarction (MI), which is characterized by chamber dilation and left ventricular (LV)
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dysfunction, represents a major cause of morbidity and mortality worldwide. Polydatin (PD), a
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monocrystalline and polyphenolic drug isolated from a traditional Chinese herb (Polygonum cuspidatum), alleviates mitochondrial dysfunction. We investigated the effects and underlying mechanisms of PD in post-MI cardiac dysfunction. We constructed an MI model by left anterior
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descending (LAD) coronary artery ligation using wild-type (WT) and Sirt3 knockout (Sirt3−/−)
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mice. Cardiac function, cardiomyocytes autophagy levels, apoptosis and mitochondria biogenesis in mice that underwent cardiac MI injury were compared between groups. PD significantly
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improved cardiac function, increased autophagy levels and decreased cardiomyocytes apoptosis
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after MI. Furthermore, PD improved mitochondrial biogenesis, which is evidenced by increased
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ATP content, citrate synthase (CS) activity and complexes I/II/III/IV/V activities in the cardiomyocytes subjected to MI injury. Interestingly, Sirt3 knockout abolished the protective
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effects of PD administration. PD inhibited apoptosis in cultured neonatal mouse ventricular myocytes subjected to hypoxia for 6 h to simulate MI injury. PD increased GFP-LC3 puncta, and reduced the accumulation of protein aggresomes and p62 in cardiomyocytes after hypoxia. Interestingly, the knock-down of Sirt3 nullified the PD-induced beneficial effects. Thus, the protective effects of PD are associated with the up-regulation of autophagy and improvement of mitochondrial biogenesis through Sirt3 activity. Keywords: Polydatin, PD; Myocardial infarction, MI; Autophagy; Sirt3; Mitochondria
ACCEPTED MANUSCRIPT Introduction Myocardial infarction (MI) induced by coronary artery occlusion is the major cause of death
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worldwide [1, 2]. Approximately 3 - 4 million individuals suffer from myocardial infarction (MI)
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each year [3]. Percutaneous coronary intervention (PCI) and intensive pharmacotherapy restore coronary perfusion and reduce adverse ventricular remodeling [4]. However, the development of cardiac dysfunction after MI remains a major challenge [5, 6].
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Polydatin (PD) is a monocrystalline and polyphenolic drug extracted from the traditional
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Chinese herb Polygonum cuspidatum. Previous studies have shown that PD may be beneficial in the treatment of cardiac ischemia/reperfusion (I/R) injury and alleviate acute kidney injury by
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inhibiting mitochondrial dysfunction [7, 8]. Furthermore, PD ameliorates injury to the small
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intestine via Sirt3 activation-mediated mitochondrial protection and alleviates multiorgan
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dysfunction [9, 10]. However, the effects and underlying mechanism of PD against MI have not been fully elucidated.
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Autophagy has an essential role in maintaining intracellular homeostasis via removing damaged or excess organelles and protein aggregates [11, 12]. Basal levels of autophagy are important for maintaining cellular homeostasis and protecting cells against excess or dysfunctional organelles [12-14]. Defects in autophagy cause cardiac dysfunction [12, 15, 16]. Conversely, enhancing autophagy promotes cell survival in response to cardiac ischemic injury and heart failure [12, 14, 17]. Mitochondria are cellular organelles mainly responsible for cellular respiration and energy production. Mitochondrial dysfunction is expected to cause diminished energy production and increased cell death, en route to the onset and progression of MI [18, 19]. Impaired mitochondrial
ACCEPTED MANUSCRIPT biogenesis has been implicated in the etiology of MI [18, 20, 21]. Understanding the mechanisms underlying mitochondrial dysfunction is pertinent to finding novel targets and therapeutic
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strategies to restore mitochondrial integrity and optimize treatment for MI.
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Sirtuins are highly conserved NAD+-dependent protein deacetylases, seven homologs of which have been identified in mammals (sirt1-7) [22-24]. Among the sirtuin family, Sirtuin-3 (Sirt3) is localized to mitochondria and regulates some mitochondrial metabolic pathways. Previous studies
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have suggested that Sirt3 protects cardiomyocytes from stress-mediated cell death, preserves
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contractile function in response to a chronic increase in workload and interrupts the development of cardiac hypertrophy [24, 25]. Sirt3 is also involved in ATP synthesis and maintenance in many
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tissues, including the heart, liver, and kidney [26]. Furthermore, sirt3 attenuates oxidative stress
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and improves mitochondrial respiration in cardiomyocytes [27]. Consistently, Sirt3 deficiency
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impairs mitochondrial and contractile function in the heart [24]. Interestingly, sirt3 also regulates autophagy processes [28]. Sirt3 has been recently recognized as an important regulator of AMPK
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activation [29-31]. We, therefore, attempted to elucidate whether Sirt3 was involved in mediating the protective effects of PD. Here, we sought to determine whether PD is capable of blocking MI injury by activating autophagy. We also investigated whether the protective effect of PD on autophagy was mediated via the Sirt3/AMPK pathway.
ACCEPTED MANUSCRIPT Methods Animals and treatment
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All animal protocols were approved by the Fourth Military Medical University Ethic
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Committee on Animal Care and all experiments were performed in adherence with the National
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Institutes of Health Guidelines on the Use of Laboratory Animals. Sirt3 knockout (Sirt3−/−) mice were established at K&D gene technology (WuHan, China) (C57BL/6 background). Western blot
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analysis and real-time PCR (RT-PCR) were used to screen Sirt3−/− mice. Eight- to 12- week-old Sirt3−/− mice were randomly allocated to the following groups, with n=20 each: (1) Wild-type +
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sham group (Sham); (2) Sirt3−/− + sham group (Sirt3−/−); (3) Myocardial infarction group (MI); (4) Sirt3−/− + Myocardial infarction group (Sirt3−/− + MI); (5) Myocardial infarction + PD group (MI +
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PD); (6)Sirt3−/− + Myocardial infarction + PD group (Sirt3−/− + MI + PD). Before constructing the MI model, PD (7.5 mg/kg) was dissolved in normal saline and was
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injected intraperitoneally for 10 days. A concentration of 10µM of PD was applied in the experiments in vitro. For autophagy inhibition, mice were injected intraperitoneally with
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autophagy inhibitor 3-methyladenine (3-MA) (10 mg/kg per day) for ten consecutive days. Myocardial infarction surgery As previously described [32] , the MI animal model was constructed by left anterior descending (LAD) coronary artery ligation. A left thoracic incision was used to open the chest. A 6–0 silk suture slipknot was placed at the proximal one-third of the left anterior descending artery. Sham-operated control animals underwent a similar surgery without ligation of the artery. Echocardiography Twenty-four hours after MI, mice were sedated with 2 % isoflurane inhalation and studied on an echocardiography system (Sequoia Acuson, 15-MHz linear transducer;Siemens, Erlangen,
ACCEPTED MANUSCRIPT Germany). Left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS)
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were calculated by using computer algorithms. The left ventricular pressure (LVP) was measured
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via a Millar Mikro-tip catheter transducer that was inserted into the left ventricular cavity through the left carotid artery. The first derivative of the left ventricular pressure (±LV dp/dt max) was obtained by computer algorithms and an interactive videographics program (Po-Ne-Mah
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Transmission electron microscopy (TEM)
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Physiology Platform P3 Plus, Gould Instrument Systems, Valley View, Ohio).
Mitochondrial ultrastructure and autophagosomes were examined using TEM. Cardiac
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specimens from the peri-infarct region were prepared as previously described [33, 34]. Heart
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tissue was removed from the animal and quickly rinsed in PBS. The tissue was placed in a petri
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dish with 0.5% glutaraldehyde and 0.2% tannic acid in PBS, diced into 2 mm cubes, and then transferred to modified Karnovsky's fixative (4% formaldehyde and 2.5% glutaraldehyde
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containing 8 mM CaCl2 in 0.1 M sodium cacodylate buffer (pH 7.4)). Samples were washed with PBS and post-fixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h to produce osmium black. Samples were then dehydrated using a graded series of ethanol and embedded in Epon/SPURR resin (EM Science) that was polymerized at 65°C overnight. Sections of heart tissues were prepared with a diamond knife on a Reichert-Jung Ultracut-E ultramicrotome and stained with UrAc (20 min) followed by 0.2% lead citrate (2.5 min). Images were photographed with a Jeol JEM-1200EX electron microscope. Determination of cardiomyocyte apoptosis Cleaved Caspase-3, Bax and Bcl-2 were detected by Western blot evaluation. Flow cytometric
ACCEPTED MANUSCRIPT analysis of cellular apoptosis was performed as previously described [33]. In brief, cardiomyocytes were harvested and stained with annexin V (Invitrogen) and propidium iodide (PI).
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Data acquisition and analysis were performed using a flow cytometer (FACSort- B0008; Becton-
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Dickinson, Franklin Lakes, NJ, USA) and Cell Quest Pro software, respectively. All of these assays were performed in a blinded manner.
Primary neonatal mouse ventricular cardiomyocyte culture
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Primary cultures of ventricular cardiomyocytes were prepared from 1-day-old non-transgenic
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mice. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone, SV30087.02) and 1% penicillinstreptomycin and maintained at 37 °C in 5 % CO2 as previously
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described [35]. The neonatal mice (1 day old) were disinfected with 75 % ethanol and then killed
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by decapitation. The chest was opened and the heart was rapidly removed and placed in cold PBS
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solution. The myocardium specimen was cut into small pieces and washed, followed by digestion with collagenase type 2. After that, the cell suspension was centrifuged (800 g for 5 min). The
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supernatant was then removed, and the cell pellet was resuspended in medium supplemented with 10% fetal bovine serum. These steps were repeated until the tissue fragments had disappeared. The dissociated cells were replated in a culture flask at 37 °C for 1 h to enrich the culture with cardiomyocytes. The non-adherent cardiomyocytes were collected and were then plated onto gelatin-coated plates. The preparation was carried out at 37 °C, in the presence of 95% O2 and 5% CO2. Transduction of adenoviruses The adenoviruses harboring GFP-LC3 (GFP-LC3) were purchased from GeneChem Technology Ltd (Shanghai, China). The adenoviruses harboring Sirt3 shRNA (Ad-sh-Sirt3)
ACCEPTED MANUSCRIPT (MOI:100, 1×109 TU / ml) were purchased from Hanbio Technology Ltd (Shanghai, China) and were transduced 24 h after transduction of GFP-LC3. After 36 h, the cardiomyocytes were
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incubated in hypoxic conditions for 6 h.
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Detection of GFP-LC3, aggresomes and p62
Fluorescence microscopic detection of GFP-LC3, aggresomes and p62 was conducted according to the manufacturer’s instructions as previously described [35].
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Construction of short hairpin RNA (shRNA) adenoviral expression vectors
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The generation of GFP-LC3 adenoviruses has been described [35]. Cardiac mitochondria isolation
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Mitochondria were isolated from the hearts of the mice as previously described [33].
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Still-beating hearts were removed from mice anesthetized with 1% pentobarbital sodium solution.
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We used ice-cold medium A (120 mM NaCl, 2 mM MgCl2, 20 mM HEPES, 1 mM EGTA, and 5 g/l bovine serum albumin; pH 7.4) to rinse the hearts to remove any residual blood. Cardiac tissue
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was minced in ice-cold medium A and homogenized. The homogenate was centrifuged (600×g, 10 min, 4 °C). The supernatant was subsequently centrifuged (17,000×g, 10 min, 4 °C). The pellet containing the mitochondria was re-suspended in medium A and then centrifuged (7,000×g, 10 min, 4 °C). After the last centrifugation, the pellet was re-suspended in medium B (2 mM HEPES, 300 mM sucrose, 0.1 mM EGTA; pH 7.4) and re-centrifuged (3,500×g, 10 min, 4 °C). The resulting pellet containing the heart mitochondria was suspended in a small volume of medium B. All work was performed on wet ice at 0 °C. Citrate synthase (CS), chain complex activities and ATP content Citrate synthase (CS) and electron transport chain complex activities (Complex I, II, III, IV, and
ACCEPTED MANUSCRIPT V) were detected using a citrate synthase activity assay kit (Sigma, USA). An ATP bioluminescent assay kit (Beyotime, China) was used to measure the ATP content of the myocardium according to
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the standard protocol.
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Calcium retention capacity (mCRC)
To test the sensitivity of the mitochondrial permeability transition pore (mPTP) opening to calcium, the mitochondrial calcium retention capacity (mCRC) was determined as the capacity of
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mitochondria to uptake calcium before permeability transition.
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ROS production and manganese superoxide dismutase (MnSOD) activity The production of ROS was measured in frozen tissue by electron paramagnetic resonance
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(EPR) spectroscopy as previously described [33]. The ROS levels were expressed in arbitrary
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units per milligram of wet tissue. MnSOD was assayed as Vives-Bauza previously described [33]
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and is expressed in units/mg. Western blot evaluation
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Protein concentration quantitation was determined by Bradford assay (Bio-Rad Laboratories, Hercules, Calif), and protein was then separated by SDS-PAGE; the following antibodies were used: Sirt3 (1:1000, Cell Signaling, Danvers, MA, USA), p62, Beclin1 (1:1000, Abcam, Cambridge, MA, UK), Cleaved caspase-3, Bax, Bcl-2 (1:1000, Sigma, St Louis, MO, USA), LC3A/B, Atg5, Adenine mononucleotide protein kinase (AMPK), p-AMPK (Thr172), Anti-ULK1, anti-p-ULK1 (Ser757), anti-FoxO3a, anti-p-FoxO3a (Ser253) (1:1000, Cell Signaling, Danvers, MA, USA), β-actin, GAPDH (1:500, Santa Cruz, CA, USA), and secondary antibodies (Anti-mouse/rabbit IgG) conjugated with horseradish peroxidase (1:5000, Cell Signaling, Danvers, MA, USA). The blots were visualized with a chemiluminescence system (Amersham Bioscience,
ACCEPTED MANUSCRIPT Buchinghamshire, UK). The signals were quantified by Image Pro Plus software (Media Cybernetics).
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Statistical analysis
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Continuous variables were expressed as the mean ± SEM. Comparison between groups were subjected to ANOVA followed by Bonferroni correction for post hoc t-test. Two-sided tests have been used throughout, and P values < 0.05 were considered statistically significant. SPSS software
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package version 14.0 (SPSS, Chicago, IL) was used for data analysis.
ACCEPTED MANUSCRIPT Results Treatment with PD improves cardiac function in mice after MI
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Twenty-four hours of permanent coronary ligation caused severe cardiac dysfunction as
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evidenced by decreased LVEF, LVFS and ± LV dp / dt max with increased LVEDV and LVESV
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(Figure 1A-1G). PD significantly increased LVEF, LVFS and ± LV dp / dt max, suggesting that treatment with PD significantly improved LV systolic function after MI (Figure 1A-1E). In
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PD-treated mice, the increases of LVEDV and LVESV were significantly attenuated compared with the MI mice (Figure 1F, 1G), suggesting that PD significantly attenuated LV dysfunction
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after MI. We next examined whether PD improved cardiac function through activating Sirt3. After
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24 hours of coronary ligation, both LVEF and LVFS were significantly decreased in the WT and
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the Sirt3-/- hearts subjected to MI injury (Figure 1A-1C). Specifically, PD slightly but insignificantly improved LVEF, LVFS and ± LV dp/dt max in the Sirt3-/- mice subjected to MI
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injury (Figure 1A-1E). Meanwhile, Sirt3 knockout abolished the effects of PD administration on LVEDV and LVESV (Figure 1F-1G).
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PD attenuated alterations in mitochondrial ultrastructure and increased autophagosomes after MI
Mitochondrial ultrastructure was examined using TEM. In the sham-operated hearts, the mitochondria were intact and had tightly packed cristae, which formed longitudinal rows between myofibrils. In the Sirt3-/- hearts, some mitochondria were swollen and had disoriented cristae, breakage, and mitochondrial disarrangement (Figure 2), whereas, in the MI mice, most of the mitochondria were markedly damaged with abnormal cristae or matrix areas. Most mitochondria displayed sharply defined cristae in PD-treated mice. A small number of swollen mitochondria were observed, and no obvious vacuole was noted in the PD-treated group (Figure 2). The
ACCEPTED MANUSCRIPT PD-treated mice exhibited moderate mitochondrial damage (Figure 2). In Sirt3-/- hearts after MI, the mitochondria injury was increased, as evidenced by the lack of cristae and matrix in some
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mitochondria, resulting in what appeared to be vacuoles. PD slightly but insignificantly revised
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the mitochondria ultrastructure disorder in Sirt3-/- hearts after MI (Figure 2). These data demonstrate that mitochondrial function was impaired following MI. PD revised the mitochondria ultrastructure disorder in MI hearts but not in Sirt3-/- hearts after MI (Figure 2).
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Autophagosomes, evaluated using transmission electron microscopy (TEM), were
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significantly increased in the MI group compared with the WT group (Figure 2). PD significantly further increased the number of autophagosomes after MI compared to the MI group (Figure 2).
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Sirt3 knockout inhibited autophagy, which was evidenced by decreased autophagosomes (Figure
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2). PD insignificantly further increased autophagosomes in the Sirt3-/- mice after MI injury (Figure
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2).
PD improved mitochondrial function after MI
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Mitochondrial respiration function was examined (Figure 3A-3C). Our data revealed that mitochondria isolated from the MI mice had significantly lower ATP content, CS activity and complexes I/II/III/IV/V activities compared to the mitochondria from the hearts of the sham-operated group (Figure 3A-3C). ATP content, CS activity and complexes I/II/III/IV/V activities of the cardiomyocyte mitochondria were significantly increased in the PD-treated group after MI (Figure 3A-3C). Together, these data suggest that mitochondrial function was impaired in MI mice. Interestingly, PD alleviated these aberrant changes after MI injury. However, PD slightly but insignificantly alleviated mitochondrial damage and had no effects on ATP content, CS activity and complexes I/II/III/IV/V activities in the Sirt3-/- mice subjected to MI injury ((Figure 3A-3C).
ACCEPTED MANUSCRIPT PD administration increased mCRC (Figure 3D), indicating that sensitivity to calcium-induced mPTP opening was decreased. ROS levels (Figure 3E) assessed by EPR spectroscopy and
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mitochondrial MnSOD activity (Figure 3F) were decreased in the PD treated group compared to
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the MI group. However, PD slightly but insignificantly increased mCRC and decreased ROS levels and mitochondrial MnSOD activity in the Sirt3-/- mice subjected to MI injury ((Figure 3D-3F).
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PD prevented post-infarction induced apoptosis
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Consistent with these observations, treatment with PD significantly reduced cardiomyocyte apoptosis induced by MI, as determined by Western blot (Figure4A-4D). PD significantly
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decreased levels of pro-apoptotic proteins Bax and Cleaved Caspase-3 and increased the level of
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anti-apoptotic protein Bcl-2 compared with the MI group (Figure 4A-4D). These data indicate that
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more viable cardiomyocytes were presented in the peri-infarct area in mice treated with PD. However, PD reduced cardiomyocyte apoptosis in the post-MI cardiomyocytes without affecting
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the Sirt3-/- mice after MI injury (Figure 4A-4D). Coincidentally, PD administration had no effect on the Cleaved Caspase-3, Bax and Bcl-2 protein levels in the Sirt3-/- mice after MI injury (Figure 4A-4D).
The Sirt3/ AMPK pathway was directly involved in PD-mediated cardioprotection against MI We hypothesized that Sirt3, a member of the sirtuin family, may also regulate AMPK, ULK1 and FoxO3a phosphorylation. Sirt3 knockout inhibited the phosphorylation of AMPK and ULK1, as well as upregulated the phosphorylation of FoxO3a, which was evidenced by decreased ratios of p-AMPK/AMPK and p-ULK1/ULK1and an increased ratio of p- FoxO3a / FoxO3a (Figure 5).
ACCEPTED MANUSCRIPT In addition, the phosphorylation of AMPK and ULK1 were partially blocked in MI mice (Figure 5). Western blot analysis revealed the increase in Sirt3 and the ratios of p-AMPK/AMPK and
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p-ULK1/ULK1 and the decrease in the ratio of p- FoxO3a / FoxO3a in the PD-treated mouse
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hearts compared with the non-treated hearts (Figure 5). PD insignificantly increased the ratio of p-AMPK/AMPK and p-ULK1/ULK1 and decreased the ratios of p- FoxO3a / FoxO3a in the Sirt3-/- mice after MI injury (Figure 5).
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These data suggest that Sirt3/AMPK signaling is involved in MI-induced cardiomyocyte injury
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and that PD reduces cardiomyocyte susceptibility to MI injury, in part through upregulation of Sirt3/AMPK signaling.
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PD activates cardiomyocyte autophagy regulatory signaling
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Sirt3 is a known key factor for the regulation of mitochondrial biogenesis and energy
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metabolism. Sirt3 was markedly decreased in the peri-infarct zone in mice with MI (Figure 6), whereas PD significantly increased Sirt3 expression in MI mice (Figure 6). In consistence,
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Western blot analysis revealed an increase in the ratio of LC3-II/LC3-Ι and a reduction of p62 in the PD-treated mouse hearts compared with the non-treated hearts (Figure 6). Sirt3 knockout inhibited autophagy, which was evidenced by the decreased ratio of LC3-II/LC3-Ι and increased p62 expression (Figure 6). PD insignificantly further increased autophagy levels in the Sirt3-/mice after MI injury (Figure 6). Assessment of cardiac function revealed that 3-MA significantly depressed LVEF, as well as LVFS, and increased LVESV and LVEDV in mice after MI. Moreover, the protective effects of PD were reversed by 3-MA administration in mice after MI (Figure 6). PD prevented hypoxia-induced apoptosis
ACCEPTED MANUSCRIPT Consistent with these observations, treatment with PD significantly reduced cardiomyocyte apoptosis induced by hypoxia, as determined by flow cytometry analysis (Figure 7). PD
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significantly decreased apoptotic index compared with the hypoxia group (Figure 7). However,
Sirt3 knockdown after hypoxia injury (Figure 7).
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PD reduced cardiomyocytes apoptosis in the cardiomyocyte after hypoxia without affecting the
PD stimulates autophagic flux in the cardiomyocytes subjected to hypoxia injury
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More GFP-LC3 puncta and increased accumulation of aggresomes and P62 were observed in
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the hypoxia group compared with the control group (Figure 8). Interestingly, PD pretreatment significantly increased the number of GFP-LC3 puncta and attenuated the accumulation of
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aggresomes and p62 in the cardiomyocytes subjected to hypoxia injury (Figure 8).
The
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stimulated autophagic flux exerted by PD administration was abolished by Sirt3 knockdown
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(Figure 8).
ACCEPTED MANUSCRIPT Discussion MI remains a major health care problem worldwide. Cardiac dysfunction subsequent to MI is
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associated with a substantially higher fatality rate [36, 37]. Understanding the mechanisms
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regulating cardiomyocyte death and survival after MI is fundamental to reducing the risk of
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developing cardiac dysfunction [38, 39]. In the present study, we provide direct evidence that pretreatment with PD alleviated cardiac dysfunction after MI.
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Autophagy degrades damaged protein aggregates and organelles, which maintains organelle function and protein quality [13]. Up-regulation of autophagy in the conditions of myocardial
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ischemic injury and heart failure is generally compensatory. Autophagy enhancement is critical to
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cell survival by removing damaged organelles and protein aggregates [7, 40]. Cardiomyocytes
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damage is often accompanied by decreased LC-3 expression and the accumulation of p62 which indicates insufficient autophagy. Interestingly, PD pretreatment significantly promoted
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cardiomyocyte autophagy after MI, as evidenced by increased LC3 expression and less accumulation of p62. Furthermore, PD increased autophagosome formation in cardiomyocytes
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subjected to MI injury. In vitro experiments also demonstrated that PD pretreatment significantly promoted autophagy in neonatal mice cardiomyocytes subjected to hypoxia injury, as evidenced by more GFP-LC3 puncta and less accumulation of aggresomes and p62. Most notably, the protective effects of PD in mice after MI were reversed by the administration of 3-MA, an inhibitor of autophagy. These observations provide evidence to support the notion that PD exerts its protective effects by inducing autophagy in mice subjected to MI injury. Previous research indicates that Sirt3 deficiency impairs mitochondrial function in the heart and sirt3 also controls autophagy levels [24, 28]. We, therefore, constructed an MI injury model in the WT and sirt3-/- mice with or without PD pretreatment to elucidate the molecular mechanism
ACCEPTED MANUSCRIPT underlying the protective effects of PD. We used TEM and Western blot analysis to detect cardiomyocyte autophagy levels among different groups. Autophagy was found to be enhanced
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after PD treatment in the WT mice that underwent MI injury. However, in the Sirt3 -/- mice, PD
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failed to up-regulate cardiomyocyte autophagy. These results indicate that PD-induced autophagy might play a pivotal role in its protection effects, and PD may exert its protective effects against MI through activating the Sirt3/AMPK pathway. In line with these observations, PD improved
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cardiac function, decreased cardiomyocyte apoptosis, increased autophagy levels and alleviated
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mitochondrial injury solely in the WT mice subjected to MI injury without affecting the Sirt3 -/mice after MI. Moreover, the protective effects of PD on neonatal mice cardiomyocyte autophagy
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after hypoxia were abolished by sirt3 knockdown.
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Mitochondria are by far the most abundant organelle in the heart. Around ninety percent of
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adenosine triphosphate (ATP) is produced in the mitochondria to supply the energy requirements for heart contraction [23, 41]. Mitochondria play a key role in mediating cellular homeostasis [19,
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20, 42-45]. As a result, maintenance of mitochondrial biogenesis and function are essential for cardiomyocyte survival. In the present study, PD increased ATP content, CS activity and complexes I/II/III/IV/V activities and alleviated mitochondrial ultrastructure injury in the cardiomyocytes subjected to MI injury. Interestingly, the protective effects of PD on mitochondrial biogenesis were abolished by sirt3 knockout. These results indicate that PD may exert its protective effects on mitochondrial biogenesis against MI through Sirt3 activation. Substantial loss of cardiac myocytes after MI can lead to contractile dysfunction and heart failure. Our study revealed the anti-apoptotic effects of PD through the activation of Sirt3, as
ACCEPTED MANUSCRIPT proven via decreased Bax and cleaved Caspase-3. Sirt3 knockout abolished the anti-apoptotic effects of PD after MI. Thus, PD may also exert anti-apoptotic effects through Sirt3 activation.
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In conclusion, the present study provides evidence that PD enhances cardiac function after MI.
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The protective effects of PD are associated with the upregulation of autophagy, down-regulation of apoptosis and improvement of mitochondrial biogenesis through sirt3 activation. PD may represent a potential drug to treat or prevent cardiac dysfunction caused by MI.
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Limitations
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Other members of RISK pathway may participate in the link between PD and its cardiac protection effects. To further demonstrate the efficacy of PD administration on cardiomyocyte
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mitochondrial biogenesis and function, the AMPK-related pathway should be measured in future
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studies. Further studies should be performed to investigate whether PD regulates mitophagy by
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different mechanisms in mice subjected to MI injury. Acknowledgements
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This work was supported by the National Natural Science Foundation of China (No. 81570318, No. 81570361, No. 81300149), and the Shaanxi Social Development and Scientific Problem Tackling Program (2015SF097, 2015KW-036). Conflict of Interest None declared.
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ACCEPTED MANUSCRIPT Figure legends Figure 1 PD improves cardiac dysfunction in mice after MI
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A: Representative echocardiographic images at 24 h after MI; B: LV ejection fraction (LVEF); C:
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LV fraction shortening (LVFS); D: LV end-diastolic volume (LVEDV); E: LV end-systolic volume (LVESV); F: +LV dp/dt; G: -LV dp/dt. Mean ± SEM (n=20), *P<0.05 vs Sham group; #P<0.05 vs ＆
Sirt3-/- group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI; P<0.05 vs MI+PD.
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Figure 2 PD attenuated alterations in mitochondrial ultrastructure and increased
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autophagosomes after MI
Representative images of the ultrastructural morphology of mitochondria (yellow arrow) and the
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typical autophagosomes (red arrow) in mice hearts. (Magnification: upper panel ×8200; medial
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panel ×20500; lower panel ×43000).
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Figure 3 PD improved mitochondrial function after MI A-B: ATP content and citrate synthase (CS) activity; C: Enzymatic activities of complexes I-V in
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cardiomyocyte mitochondria; D: The mitochondrial calcium retention capacity (mCRC); E: ROS levels; F: Manganese superoxide dismutase (MnSOD). Mean ± SEM (n=20), *P<0.05 vs Sham ＆
group; #P<0.05 vs Sirt3-/- group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI; P<0.05 vs MI+PD. Figure 4 PD inhibits apoptosis in cardiomyocytes after MI A-D: Protein expression with representative gel blots of Cleaved Caspase-3, Bcl-2 and Bax. Mean ± SEM (n=20), *P<0.05 vs Sham group; #P<0.05 vs Sirt3-/- group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI;
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Figure 5 Sirt3 and phosphorylation of AMPK and ULK1 in the myocardium from mice treated with or without MI.
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group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI; P<0.05 vs MI+PD.
myocardium from mice treated with or without MI.
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Figure 6 Western blot analysis of the autophagy markers p62, LC3 and Atg5 in the
A: Representative gel blots of p62, LC3, Atg5 and GAPDH (loading control) using specific
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antibodies; B: Beclin1; C: Atg5; D: p62; E: LC3II-to-GAPDH ratio; All proteins were normalized
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to the loading control GAPDH; Mean ± SEM (n=20), *P<0.05 vs Sham group; #P<0.05 vs Sirt3-/group; §P < 0.05 vs MI; $P<0.05 vs Sirt3-/-+MI;
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P<0.05 vs MI+PD. F: LV ejection fraction
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(LVEF); G: LV fraction shortening (LVFS); H: LV end-diastolic volume (LVEDV); I: LV
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end-systolic volume (LVESV); Mean ± SEM (n=20), *P<0.05 vs MI group; #P<0.05 vs MI+3MA
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group; §P < 0.05 vs MI+PD group.
Figure 7 PD inhibits apoptosis in cardiomyocytes after hypoxia
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A: Apoptosis of the primary cardiomyocytes determined by Cy3-annexinV/PI double staining and flow cytometry. Region B2: late apoptotic cells (Cy3/PI, where Cy3 is cyanine-3 and PI is propidium iodide); Region B3: vital cells; Region B4: early apoptotic cells. B: Quantitative analysis of apoptotic index. Mean ± SEM, *P<0.05 vs Con group; #P<0.05 vs Ad-sh-Sirt3 group; §
P < 0.05 vs H; $P<0.05 vs Ad-sh-Sirt3+H;
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P<0.05 vs H+PD.
Figure 8 PD augments cardiomyocyte autophagy flux subjected to hypoxia through Sirt3 activation A-B: Protein expression with representative gel blots of Sirt3; C: Representative images of GFP depicting GFP-LC3 puncta in cardiomyocytes shown in panel; D: Quantitative analysis of the
ACCEPTED MANUSCRIPT number of GFP-LC3 puncta; E: Representative images showing immunofluorescence staining for p62 (orange), DAPI (blue) and ProteoStat aggresome detection reagent (red) in cultured
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cardiomyocytes. F: The number of cardiomyocytes with perinuclear colocalization of p62 and
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aggresomes (p62/aggresomes), indicated by yellow color in the merged images was counted (n = 50 in each group). Mean ± SEM, *P<0.05 vs Con group; #P<0.05 vs Ad-sh-Sirt3 group; §P < 0.05
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PD enhances cardiac function after MI in mice. PD up-regulates autophagy, inhibits apoptosis and improves mitochondrial biogenesis. In Sirt3-/- mice subjected to MI injury, the protective effects of PD are abolished.