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Inhibition of Drp1 by Mdivi-1 attenuates cerebral ischemic injury via inhibition of the mitochondria-dependent apoptotic pathway after cardiac arrest
Neuroscience 311 (2015) 67–74
INHIBITION OF DRP1 BY MDIVI-1 ATTENUATES CEREBRAL ISCHEMIC INJURY VIA INHIBITION OF THE MITOCHONDRIA-DEPENDENT APOPTOTIC PATHWAY AFTER CARDIAC ARREST Y. LI, a,by P. WANG, a,by J. WEI, c R. FAN, d Y. ZUO, e M. SHI, c H. WU, a,b M. ZHOU, a,b J. LIN, a,b M. WU, a,b X. FANG a,b AND Z. HUANG a,b*
Key words: cardiac arrest, cardiopulmonary resuscitation, Drp1, Mdivi-1, apoptosis.
a Department of Emergency Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
INTRODUCTION
b Institute of Cardiopulmonary Cerebral Resuscitation, Sun Yat-sen University, Guangzhou, China
Cardiac arrest is a leading cause of death worldwide. After the long-term efforts by the American Heart Association and related organizations to update and disseminate resuscitation guidelines, the in-hospital mortality among successfully resuscitated patients remains up to 70% (Neumar et al., 2008; Nichol et al., 2010). Moreover, nearly 60% of survival patients had moderate to severe cognitive deficits due to cerebral ischemia–reperfusion injury at three months after cardiac arrest (Roine et al., 1993). Neurological injury after cardiac arrest is a major contributor to morbidity and mortality in survivors of resuscitation (Neumar et al., 2008). Although therapeutic hypothermia was the first recommended protection demonstrated to improve neurologic outcomes in comatose survivors after cardiac arrest (Bernard et al., 2002; Hypothermia after Cardiac Arrest Study Group, 2002), it is limited by practical difficulties in implementation. Alternative approaches should be thus developed to further improve neurologic outcomes in patients after cardiac arrest. Mitochondria are important organelles in all cell types, but they are particularly important in the nervous system. Mitochondria are essential to neuronal processes such as energy production, Ca2+ regulation, maintenance of plasma membrane potential, protein folding by chaperones (Chan, 2006). There is mounting evidence that the mitochondria-dependent apoptosis is involved in the neuron damage during the cerebral ischemia–reperfusion (Mattson et al., 2001; Chen et al., 2013). Mitochondria exist in dynamic networks that continuously undergo fusion and fission, which play an important role in maintaining their function in neurons (Knott et al., 2008). Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has recently been demonstrated to be an intrinsic component of multiple mitochondria-dependent apoptosis pathways (Martinou and Youle, 2011). Drp1 usually resides in an inactive form in the cytosol and on activation translocates to the mitochondria. Inhibition of Drp1 translocation onto the mitochondria by mitochondrial division inhibitor-1 (Mdivi-1) prevents mitochondrial fission (Tanaka and Youle, 2008). Mdivi-1 is a derivative of quinazolinone
c
Department of Emergency Medicine, People’s Hospital of Baoan District, Shenzhen, China d Department of Emergency Medicine, Zhongshan People’s Hospital, Zhongshan, China e
Department of Cardiovascular Medicine, The First Affiliated Hospital, Guangxi Medical University, Nanning, China
Abstract—Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has been reported to be involved in mitochondria apoptosis pathways. However, the role of Drp1 in a rat model of cardiac arrest remains unknown. In this study, we found that activation of Drp1 in the mitochondria was increased after cardiac arrest and inhibition of Drp1 by 1.2 mg/kg of mitochondrial division inhibitor-1 (Mdivi-1) administration after the restoration of spontaneous circulation (ROSC) significantly protected against cerebral ischemic injury, shown by the increased 72-h survival rate and improved neurological function. Moreover, the increase of the vital neuron and the reduction of cytochrome c (CytC) release, apoptosis-inducing factor (AIF) translocation and caspase-3 activation in the brain indicate that this protection might result from the suppression of neuron apoptosis. Altogether, these results indicated that Drp1 is activated after cardiac arrest and the inhibition of Drp1 is protective against cerebral ischemic injury in a rat of cardiac arrest model via inhibition of the mitochondrial apoptosis pathway. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
*Correspondence to: Z. Huang, Department of Emergency Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107# West Yanjiang Road, Guangzhou 510120, China. Tel: +86-20-81332084; fax: +86-20-81332410. E-mail address: [email protected] (Z. Huang). y These authors contributed equally. Abbreviations: AIF, apoptosis-inducing factor; CA-1, cornu ammonis 1; CPR, cardiopulmonary resuscitation; CytC, cytochrome c; DMSO, dimethyl sulfoxide; Drp1, dynamin-related protein1; HE, Hematoxylin– eosin; MAP, mean aortic pressure; Mdivi-1, mitochondrial division inhibitor-1; NDS, neurologic deficit score; OPC, overall performance category; ROSC, restoration of spontaneous circulation; TUNEL, TdTmediated dUTP nick-end labeling. http://dx.doi.org/10.1016/j.neuroscience.2015.10.020 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 67
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and serves as a selective inhibitor of mitochondrial fission protein Drp1 (Cassidy-Stone et al., 2008). Recent studies have shown that Mdivi-1 could protect the heart against ischemia/reperfusion injury (Ong et al., 2010) and block apoptotic cell death and increase retinal ganglion cell survival in ischemic mouse retina (Park et al., 2011). In addition, pretreatment with Mdivi-1 could provide neuroprotection against transient ischemic brain damage in vivo (Grohm et al., 2012). Therefore, we hypothesize that inhibition of Drp1 could represent a potential therapeutic strategy in a cardiac arrest model. Herein, we showed that cardiac arrest-induced global cerebral ischemia result in mitochondrial fission protein Drp1 activation in vivo. Importantly, we found that inhibition of Drp1 by Midvi-1 could protect against cerebral ischemic injury after cardiac arrest probably via the suppression of cytochrome c (CytC) and apoptosisinducing factor (AIF)-dependent mitochondrial apoptosis pathway.
EXPERIMENTAL PROCEDURES Animals and drug preparation Male Sprague–Dawley rats (weight, 350–450 g) were purchased from the Laboratory Animal Center of Sun Yat-Sen University. Animals were housed light and temperature controlled environment. Food and water were supplied ad libitum. Animal experiments were followed the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-Sen University. All drugs used were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified. Mdivi1 was dissolved in dimethyl sulfoxide (DMSO) and used at a concentration of 0.24 and 1.2 mg/kg individually. DMSO vehicle was used as a control for cardiac arrestinduced mice. Cardiac arrest model All rats were fasted on the night prior to the experiment. After the induction of anesthesia by an intraperitoneally injection of 45 mg/kg of pentobarbital sodium, rats were intubated with a 14-gauge cannula mounted on a blunt needle with a 145° angled tip. A 23-guage catheter (PE-50) was advanced through the left femoral artery into the aorta to measure mean aortic pressure (MAP). Another 23-guage catheter (PE-50) was inserted into the left femoral vein for intravenous infusion. End-tidal PaCO2 (PETCO2) was measured with a side-stream infrared CO2 analyzer (CAPSTAR-100, CWE Inc., Ardmore, PA, USA) interposed between the tracheal cannula and the respirator. All hemodynamic data include MAP and electrocardiogram (ECG) lead II was continuously monitored via a WinDaq data-acquisition system (DataQ, Akron, OH, USA). Rectal core temperature was monitored and maintained at 36.5 ± 0.5 °C with a heat lamp. Anesthetized rats were paralyzed with 2 mg/kg vecuronium bromide and baseline measurements were accomplished. Asphyxia was induced by clamping the
endotracheal tube. After a short induction of apnea, cardiac arrest was determined by absent pulsation of aortic artery, defined as MAP <20 mmHg. Precordial compression was begun and mechanical ventilation with 100% FiO2 was performed 6 min after the onset of cardiac arrest. Precordial compression at a rate of 250/min was synchronized to conduct a compression/ ventilation ratio of 5:1. Adrenaline (20 lg/kg) was injected after 2 min of cardiopulmonary resuscitation (CPR). In unsuccessfully resuscitated animals, the 30 s of CPR and adrenaline administrations were repeated and maintained until restoration of spontaneous circulation (ROSC) or 10 min after cardiac arrest. ROSC was defined as the return of supraventricular cardiac rhythm with mean aortic pressure (MAP) over 60 mmHg for P5 min. Animals were observed for 1 h after successful resuscitation. Experimental design After ROSC, the animals were randomized into the following three groups: (1) vehicle group; (2) Mdivi-1 low-dose group (Mdivi-1 dose1 group); (3) Mdivi-1 highdose group (Mdivi-1 dose2 group). Animals in the vehicle group were given an intravenous injection of 0.1% DMSO after 1 min of ROSC. Rats in Mdivi-1 dose1 group and Mdivi-1 dose2 group were intravenously infused with Mdivi-1 at doses of 0.24 and 1.2 mg/kg, respectively. Animals underwent identical anesthetic and surgical procedures except cardiac arrest were used as the sham group. Survival study and neurological outcome evaluation The survival rate after cardiac arrest was observed until 72 h. At 24, 48 and 72 h after ROSC, the neurologic function scores were performed blindly, using an overall performance category (OPC) scoring system (OPC: 1 = normal; 2 = mild disability; 3 = moderate disability; 4 = severe disability/coma; 5 = death/brain death) and neurologic deficit score (NDS, 0–10%, normal; 100%, brain death) (Neumar et al., 1995). All survived animals were sacrificed at 72 h after surgery and the brain tissues were removed for the following examinations. HE staining and TUNEL (TdT-mediated dUTP nick-end labeling) assay Separated left hemisphere of rats fixed in 4% paraformaldehyde and the paraffin-embedded brain tissues were cut into sections of 4-lm. Hematoxylin– eosin (HE) staining (Beyotime, Hangzhou, China) and TUNEL assay (Roche, Boston, MA, USA) were performed for in situ detection of neuronal injury, according to the manufacture’s instruction. Six visual fields from each brain tissue sample, in the hippocampal cornu ammonis 1 (CA-1) region were randomly selected and the HE-stained neuronal cells were counted. The immunohistochemical score (IHS) is determined by the percentage of positive-stained cells and the intensity of staining for TUNEL staining. All analyses were performed blinded.
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Western blot analysis To obtain the mitochondrial, cytosolic and nuclear protein fractions, separated right hemisphere were homogenized in lysis buffer. Thirty micrograms of protein were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Thirty micrograms of protein were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunolabelling was performed using mouse monoclonal Drp1 antibody (diluted 1:200; Abcam, Boston, MA, USA), rabbit polyclonal cleaved caspase-3 antibody (diluted 1:1000; CST, Beverly, MA, USA), rabbit monoclonal CytC antibody (diluted 1:1,000; CST, USA), rabbit monoclonal AIF antibody (diluted 1:1,000; CST, USA), rabbit monoclonal Histone H2A.X antibody (diluted 1:1,000; CST, USA), rabbit monoclonal Cyclooxygenase (COX)-IV antibody (diluted 1:1,000; CST, USA), rabbit monoclonal b-actin antibody (diluted 1:1,000; CST, USA). Immunoreactivity was developed by chemiluminescence kit and exposed to film, and quantitative analysis of band intensity was carried out by Quantity One Software (BIO-RAD, Hercules, CA, USA).
Statistical analysis Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL). Comparison of the same parameters among groups was done using one-way analysis of variance (ANOVA), and the difference between pairs of means was tested post hoc with Fisher’s Least Significant Difference (LSD) test. A two-tailed Fisher’s exact test was employed to compare proportions of OPC values between groups (favorable vs. unfavorable outcome, OPC 1–2 vs. OPC 3–5). The difference in survival rates among the various groups was compared using log-rank test. A P-value <0.05 was considered statistically significant.
RESULTS Mdivi-1 inhibited Drp1 activation induced by cardiac arrest Western blot analysis demonstrated that Drp1 protein level increased in the mitochondria and decreased in the cytosol of brain after cardiac arrest (Fig. 1), indicating that ischemic injury induced by cardiac arrest lead to the trigger of Drp1 translocation onto mitochondria. As expected, treatment with Mdivi-1 at the dose of 1.2 mg/kg significantly attenuated cardiac arrestinduced translocation of cytosolic Drp1 (P < 0.05). However, lower dose of Mdivi-1 did not show a similar effect. Taken together, these results suggest that Mdivi-1 of 1.2 mg/kg could inhibit Drp1 activation induced by cardiac arrest.
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Inhibition of Drp1 by Mdivi-1 improved neurological outcome after ROSC To determine the effect of Drp1 inhibition by Mdivi-1 on the mortality and morbidity after cardiac arrest, total of 50 rats were subjected to asphyxial cardiac arrest. Of those, 42 rats achieved ROSC and were randomly allocated into vehicle (n = 14), Mdivi-1 dose1 (n = 14) and Mdivi-1 dose2 (n = 14). Rats were intravenously infused with either 0.1% DMSO or Mdivi-1 (0.24 or 1.2 mg/kg) after 1 min of ROSC. All survived animals showed no significant differences in body weight, basal body temperature, arterial blood gas, end-tidal PaCO2, heart rate and mean arterial pressure of rats as compared with animals in the sham group (n = 8) before inducing cardiac arrest (Table 1). At 72 h after ROSC, the survival rates of vehicle group, Mdivi-1 dose1 group and Mdivi-1 dose2 group were 21.4% (3/14), 28.6% (4/14) and 64.3% (9/14), respectively. The survival rates of rats of vehicle and Mdivi-1 dose1 group were significantly lower than those of the sham group (100%, P < 0.05). Compared with the vehicle-treated group, treatment with 1.2 mg/kg of Mdivi-1 significantly improved the survival rate after ROSC (threefold, P < 0.05), although the lower dose (0.24 mg/kg) did not show any effect (Fig. 2). OPC were not significantly different between Mdivi-1 dose2 group and vehicle group at 24 and 48 h and similar results were observed according to NDS. In the Mdivi-1 dose2 group, six of 14 animals achieved the favorable outcome (OPC 1 or 2) at 72 h after ROSC, compared to one of 14 in Mdivi-1 dose1 group and 0 of 14 in the vehicle group (Fig. 3). Treatment with 1.2 mg/kg of Mdivi-1 significantly improved the OPC score compared to the vehicle-treated group (P < 0.05). At 72 h after ROSC, survived animals treated with Mdivi-1 at the dose of 1.2 mg/kg showed better neurological function by NDS scores than those of vehicle-treated animals (P < 0.05) (Fig. 4). However, no significant differences were found between vehicle and Mdivi-1 dose1 group at the different time points (P > 0.05). Taken together, these data suggest that 1.2 mg/kg of Mdivi-1 treatment improves neurological outcomes after cardiac arrest. Inhibition of Drp1 by Mdivi-1 attenuated neuronal ischemic injury after ROSC To evaluate the effects on brain neurons after inhibition of Drp1, HE staining and TUNEL assay were performed to detect neuronal ischemic injury at 72 h after ROSC. The number of vital neurons in the hippocampal CA-1 region was significantly reduced, and the nucleus was compressed in animals of the vehicle-treated group. As compared, treatment with 1.2 mg/kg of Mdivi-1 significantly increased the number of vital neurons with normal nucleus, although the number of ischemic neurons in Mdivi-1 dose1 group was not significantly reduced (Fig. 5A, C). Apoptotic neurons were mainly found around the ischemic hippocampal CA-1 region as illustrated by TUNEL staining. Few TUNEL-positive neurons were found in the sham group, where
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Fig. 1. The protein levels of Drp1 in the brain after cardiac arrest. (A) Western blot analysis and (B) quantitative analysis of Drp1 expression at 72 h after ROSC. Cyclooxygenase (COX)-IV and b-actin were used as internal controls of mitochondrial and cytosolic subfractions, respectively. * P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. Mito, mitochondria; Cyto, cytoplasm.
Table 1. Basic characteristics
Body weight (g) HR (bpm) MAP (mmHg) PETCO2 (mmHg) Temperature (°C) PH PaCO2 (mmHg) PaO2 (mmHg) Lactate (mmol/L)
Sham (n = 8)
CPR (n = 14)
Mdivi-1 dose1 (n = 14)
Mdivi-1 dose2 (n = 14)
403.38 ± 12.97 361.88 ± 34.01 131.50 ± 18.09 39.25 ± 3.49 36.65 ± 0.26 7.40 ± 0.01 39.38 ± 3.16 95.13 ± 2.59 0.89 ± 0.08
400.50 ± 15.97 376.09 ± 31.17 131.93 ± 14.44 39.21 ± 3.64 36.54 ± 0.28 7.41 ± 0.02 39.28 ± 3.47 94.50 ± 2.24 0.90 ± 0.09
402.29 ± 14.86 374.50 ± 30.89 132.71 ± 14.25 38.57 ± 3.30 36.60 ± 0.22 7.42 ± 0.02 38.64 ± 3.13 94.07 ± 2.67 0.94 ± 0.13
400.14 ± 17.69 373.64 ± 35.03 133.57 ± 16.25 38.85 ± 2.82 36.52 ± 0.25 7.41 ± 0.01 38.93 ± 2.62 94.00 ± 2.94 0.93 ± 0.11
HR, heart rate; MAP, mean aortic pressure; PETCO2, end-tidal PaCO2.
Inhibition of Drp1 by Mdivi-1 inhibited mitochondriadependent apoptosis in the brain
Fig. 2. Inhibition of Drp1 by Mdivi-1 increased survival after cardiac arrest. *P < 0.05 vs. Sham group revealed by log-rank test; # P < 0.05 vs. Vehicle group.
numerous apoptotic neurons were evident in the vehicletreated group. As compared, less apoptotic neurons were observed (P < 0.05) in Mdivi-1 dose2 group. However, no difference in the scores was seen between vehicle and Mdivi-1 dose1 group (P > 0.05) (Fig. 5B, D).
To further find out the underlying mechanism of attenuating cerebral ischemia induced by cardiac arrest after inhibition of Drp1, we examine the molecules involved in apoptosis. As shown in Figs. 6 and 7. Caspases regulate cell apoptosis. CytC release activates caspase-3 and AIF is a caspase-independent mitochondrial death effector. Consistent with prominent increases in activated cleavage product of caspase-3, mitochondrial CytC and AIF were released into the cytosol and nucleus in the vehicle-treated group, respectively. As compared, treatment with 1.2 mg/kg of Mdivi-1 significantly inhibited release of CytC, AIF translocation and caspase-3 activation (P < 0.05). In contrast, there were no significant differences in the protein expression of cleaved caspase-3, CytC and AIF between the vehicle group and Mdivi-1 dose1 group
Fig. 3. Inhibition of Drp1 by Mdivi-1 improved OPC score after cardiac arrest. (A) 24 h, (B) 48 h, (C) 72 h. Score 1 and 2 were defined as a good outcome of neurological function, whereas score 3, 4, and 5 were defined as a poor outcome. Each dot represents one rat. *P < 0.05 vs. Sham group revealed by Fisher’s exact test; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. OPC, overall performance categories.
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Fig. 4. Inhibition of Drp1 by Mdivi-1 improved NDS of survival rats after cardiac arrest. Data are expressed as mean ± SD. #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. NDS, neurological deficit scores.
(P > 0.05). Taken together, these data suggest that Mdivi-1 (1.2 mg/kg) treatment reduced cardiac arrestinduced apoptosis by inhibiting release of CytC, translocation of AIF, caspase-3 activation.
DISCUSSION In this study, cerebral ischemia–reperfusion injury after cardiac arrest led to a prominent increase of Drp1 activation in the mitochondria. Most importantly, we provide the first direct evidence that the inhibition of
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Drp1 by Mdivi-1 improves neurological outcome and attenuated neuronal damage after cardiac arrest. The underlying mechanism maybe through the inhibition of mitochondria-dependent apoptosis pathway. These results suggested that Drp1 activation contribute to cerebral ischemia after cardiac arrest. Given the fact that Mdivi-1, the selective Drp1 inhibitor was delivered after ROSC, a clinically relevant intervention time, inhibition of Drp1 may be a promising therapeutic target for improving neurological outcome after cardiac arrest. We found cardiac arrest induced Drp1 activation in this study. The activity of Drp1 can be regulated by several post-modifications of Drp1, such as phosphorylation, sumoylation, ubiquitination and S-nitrosylation (Chang and Blackstone, 2010). Studies suggests that dephosphorylation of Drp1 at Serine 637 by calcineurin accelerates Drp1 activity (Cribbs and Strack, 2007). Cerebral ischemia–reperfusion could result in activation of the phosphatase calcineurin (Phillis et al., 2002), thus calcineurin may contribute to Drp1 activation after cardiac arrest. Drp1 accumulate on mitochondria and result in mitochondria-dependent apoptosis (Frank et al., 2001; Karbowski and Youle, 2003; Arnoult, 2007). Evidence suggests that inhibition of Drp1 by overexpression of a
Fig. 5. Inhibition of Drp1 by Mdivi-1 attenuated neuronal injury in the hippocampal CA-1 region after cardiac arrest. (A) Representative pictures of HE staining with a magnifying power of 400; (B) Representative pictures of TUNEL staining with a magnifying power of 400; (C) Proportions of ischemic neurons per total neurons; (D) IHS of TUNEL staining. Data are expressed as mean ± SD.*P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. IHS, immunohistochemical score.
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Fig. 6. Effect of inhibition of Drp1 by Mdivi-1 on protein levels of CytC and AIF after cardiac arrest. (A) Western blot analysis and (B) quantitative analysis of CytC expression at 72 h after ROSC (C) quantitative analysis of AIF expression at 72 h after ROSC. Histone, Cyclooxygenase (COX)-IV and b-actin were used as internal controls of nuclear, mitochondrial and cytosolic subfractions, respectively. Data are expressed as mean ± SD. * P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. Mito, mitochondria; Cyto, cytoplasm; Nu, nucleus; CytC, cytochrome c; AIF, apoptosis-inducing factor.
Fig. 7. Effect of inhibition of Drp1 by Mdivi-1 on protein levels of caspase-3 after cardiac arrest. (A) Western blot analysis and (B) quantitative analysis of caspase-3 expression at 72 h after ROSC. b-actin were used as internal controls of cytosolic subfractions. Data are expressed as mean ± SD. *P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group.
dominant negative mutant of Drp1 or knockdown of Drp1 could prevent the release of CytC and AIF from mitochondria and attenuate apoptosis (Brooks et al., 2011), which is further confirmed by our data. Drp1 is required for CytC release and subsequently apoptosis execution (Frank et al., 2001; Lee et al., 2004). However, opposite views support that Drp1 is not prerequisite for apoptosis activation (Breckenridge et al., 2008). Regardless, ischemicreperfusion injury after cardiac arrest in our animal model promotes the Drp-1 activity and ultimately results in apoptosis execution. CytC and AIF as pro-apoptotic proteins are all localized in the mitochondria of healthy neurons, and the regulated release of these proteins by death signals plays a vital role in the execution of the mitochondriadependent apoptotic pathway (Wang and Youle, 2009). AIF release results in caspase-independent apoptosis (Zhang et al., 2002), while CytC release activates caspase-3 and finally results in caspase-dependent apoptosis (Sheridan and Martin, 2010). In this study, we find
that inhibition of Drp1 by Mdivi-1 prevented cardiac arrest-induced CytC release, AIF translocation and caspase-3 activation. Thus, the mitochondria-dependent apoptotic pathway may be involved in the protective mechanism of Drp1 inhibition against global cerebral ischemia induced by cardiac arrest. Global cerebral ischemia mostly leads to hippocampal damage, which results in necrotic and apoptotic neuronal death usually detectable 3 days after ischemia (Sugawara et al., 2000). The hippocampus is the most sensitive region to cerebral ischemia, which is closely related to cognitive dysfunction after ischemia–reperfusion injury. In the present study, therefore, the hippocampal CA-1 region of rats surviving until 72 h was selected for pathological examination. HE staining and TUNEL assay revealed various degrees of neuronal necrosis and apoptosis in rats after cardiac arrest, and the degree of pathological damage was consistent with the general neurological function impairment. Additionally, inhibition
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of Drp1 by Mdivi-1 improved survival and neurological function in our model. These neurological benefits could be associated with the attenuation of the cardiac arrestinduced neuronal necrosis and apoptosis following Mdivi-1 administration after ROSC, adding more supportive evidences for the neuroprotective effects of inhibition of Drp1. Consistent with previous studies (Ong et al., 2010; Xie et al., 2013), we found that 1.2 mg/kg of Mdivi-1 inhibited Drp1 translocation to the mitochondria and thereby attenuated cerebral ischemic injury from cardiac arrest, although Mdivi-1 (0.24 mg/kg) had no benefits on neurological outcomes, indicating that Mdivi-1 may offer therapeutic effects in a dose-dependent manner. In our model of cardiac arrest, we found that Drp1 translocation to the mitochondria could be suppressed by Mdivi-1. Importantly, this suppression markedly improves the neurological outcome and survival after cardiac arrest. Since our interventions of Drp1 activity were based with pharmacologically use of Mdivi-1, our results still require a further confirmation by gene approach or other novel inhibitors of Drp1.
CONCLUSION In summary, we suggest that Drp1 plays an important role in cerebral ischemic injury after cardiac arrest and pharmacological inhibition of Drp1 might be a novel strategy for neuroprotection after cardiac arrest. The underlying mechanism may be via suppression of the mitochondrial apoptosis pathway. Acknowledgments—This work was supported by the National Natural Science Foundation of China (81501137) and the Science and Technology Foundation of Guangdong Province, China (2015A030310042)
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(Accepted 11 October 2015) (Available online 20 October 2015)
INHIBITION OF DRP1 BY MDIVI-1 ATTENUATES CEREBRAL ISCHEMIC INJURY VIA INHIBITION OF THE MITOCHONDRIA-DEPENDENT APOPTOTIC PATHWAY AFTER CARDIAC ARREST Y. LI, a,by P. WANG, a,by J. WEI, c R. FAN, d Y. ZUO, e M. SHI, c H. WU, a,b M. ZHOU, a,b J. LIN, a,b M. WU, a,b X. FANG a,b AND Z. HUANG a,b*
Key words: cardiac arrest, cardiopulmonary resuscitation, Drp1, Mdivi-1, apoptosis.
a Department of Emergency Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
INTRODUCTION
b Institute of Cardiopulmonary Cerebral Resuscitation, Sun Yat-sen University, Guangzhou, China
Cardiac arrest is a leading cause of death worldwide. After the long-term efforts by the American Heart Association and related organizations to update and disseminate resuscitation guidelines, the in-hospital mortality among successfully resuscitated patients remains up to 70% (Neumar et al., 2008; Nichol et al., 2010). Moreover, nearly 60% of survival patients had moderate to severe cognitive deficits due to cerebral ischemia–reperfusion injury at three months after cardiac arrest (Roine et al., 1993). Neurological injury after cardiac arrest is a major contributor to morbidity and mortality in survivors of resuscitation (Neumar et al., 2008). Although therapeutic hypothermia was the first recommended protection demonstrated to improve neurologic outcomes in comatose survivors after cardiac arrest (Bernard et al., 2002; Hypothermia after Cardiac Arrest Study Group, 2002), it is limited by practical difficulties in implementation. Alternative approaches should be thus developed to further improve neurologic outcomes in patients after cardiac arrest. Mitochondria are important organelles in all cell types, but they are particularly important in the nervous system. Mitochondria are essential to neuronal processes such as energy production, Ca2+ regulation, maintenance of plasma membrane potential, protein folding by chaperones (Chan, 2006). There is mounting evidence that the mitochondria-dependent apoptosis is involved in the neuron damage during the cerebral ischemia–reperfusion (Mattson et al., 2001; Chen et al., 2013). Mitochondria exist in dynamic networks that continuously undergo fusion and fission, which play an important role in maintaining their function in neurons (Knott et al., 2008). Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has recently been demonstrated to be an intrinsic component of multiple mitochondria-dependent apoptosis pathways (Martinou and Youle, 2011). Drp1 usually resides in an inactive form in the cytosol and on activation translocates to the mitochondria. Inhibition of Drp1 translocation onto the mitochondria by mitochondrial division inhibitor-1 (Mdivi-1) prevents mitochondrial fission (Tanaka and Youle, 2008). Mdivi-1 is a derivative of quinazolinone
c
Department of Emergency Medicine, People’s Hospital of Baoan District, Shenzhen, China d Department of Emergency Medicine, Zhongshan People’s Hospital, Zhongshan, China e
Department of Cardiovascular Medicine, The First Affiliated Hospital, Guangxi Medical University, Nanning, China
Abstract—Mitochondrial fission is predominantly controlled by the activity of dynamin-related protein1 (Drp1), which has been reported to be involved in mitochondria apoptosis pathways. However, the role of Drp1 in a rat model of cardiac arrest remains unknown. In this study, we found that activation of Drp1 in the mitochondria was increased after cardiac arrest and inhibition of Drp1 by 1.2 mg/kg of mitochondrial division inhibitor-1 (Mdivi-1) administration after the restoration of spontaneous circulation (ROSC) significantly protected against cerebral ischemic injury, shown by the increased 72-h survival rate and improved neurological function. Moreover, the increase of the vital neuron and the reduction of cytochrome c (CytC) release, apoptosis-inducing factor (AIF) translocation and caspase-3 activation in the brain indicate that this protection might result from the suppression of neuron apoptosis. Altogether, these results indicated that Drp1 is activated after cardiac arrest and the inhibition of Drp1 is protective against cerebral ischemic injury in a rat of cardiac arrest model via inhibition of the mitochondrial apoptosis pathway. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.
*Correspondence to: Z. Huang, Department of Emergency Medicine, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, 107# West Yanjiang Road, Guangzhou 510120, China. Tel: +86-20-81332084; fax: +86-20-81332410. E-mail address: [email protected] (Z. Huang). y These authors contributed equally. Abbreviations: AIF, apoptosis-inducing factor; CA-1, cornu ammonis 1; CPR, cardiopulmonary resuscitation; CytC, cytochrome c; DMSO, dimethyl sulfoxide; Drp1, dynamin-related protein1; HE, Hematoxylin– eosin; MAP, mean aortic pressure; Mdivi-1, mitochondrial division inhibitor-1; NDS, neurologic deficit score; OPC, overall performance category; ROSC, restoration of spontaneous circulation; TUNEL, TdTmediated dUTP nick-end labeling. http://dx.doi.org/10.1016/j.neuroscience.2015.10.020 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 67
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and serves as a selective inhibitor of mitochondrial fission protein Drp1 (Cassidy-Stone et al., 2008). Recent studies have shown that Mdivi-1 could protect the heart against ischemia/reperfusion injury (Ong et al., 2010) and block apoptotic cell death and increase retinal ganglion cell survival in ischemic mouse retina (Park et al., 2011). In addition, pretreatment with Mdivi-1 could provide neuroprotection against transient ischemic brain damage in vivo (Grohm et al., 2012). Therefore, we hypothesize that inhibition of Drp1 could represent a potential therapeutic strategy in a cardiac arrest model. Herein, we showed that cardiac arrest-induced global cerebral ischemia result in mitochondrial fission protein Drp1 activation in vivo. Importantly, we found that inhibition of Drp1 by Midvi-1 could protect against cerebral ischemic injury after cardiac arrest probably via the suppression of cytochrome c (CytC) and apoptosisinducing factor (AIF)-dependent mitochondrial apoptosis pathway.
EXPERIMENTAL PROCEDURES Animals and drug preparation Male Sprague–Dawley rats (weight, 350–450 g) were purchased from the Laboratory Animal Center of Sun Yat-Sen University. Animals were housed light and temperature controlled environment. Food and water were supplied ad libitum. Animal experiments were followed the Guide for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Sun Yat-Sen University. All drugs used were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise specified. Mdivi1 was dissolved in dimethyl sulfoxide (DMSO) and used at a concentration of 0.24 and 1.2 mg/kg individually. DMSO vehicle was used as a control for cardiac arrestinduced mice. Cardiac arrest model All rats were fasted on the night prior to the experiment. After the induction of anesthesia by an intraperitoneally injection of 45 mg/kg of pentobarbital sodium, rats were intubated with a 14-gauge cannula mounted on a blunt needle with a 145° angled tip. A 23-guage catheter (PE-50) was advanced through the left femoral artery into the aorta to measure mean aortic pressure (MAP). Another 23-guage catheter (PE-50) was inserted into the left femoral vein for intravenous infusion. End-tidal PaCO2 (PETCO2) was measured with a side-stream infrared CO2 analyzer (CAPSTAR-100, CWE Inc., Ardmore, PA, USA) interposed between the tracheal cannula and the respirator. All hemodynamic data include MAP and electrocardiogram (ECG) lead II was continuously monitored via a WinDaq data-acquisition system (DataQ, Akron, OH, USA). Rectal core temperature was monitored and maintained at 36.5 ± 0.5 °C with a heat lamp. Anesthetized rats were paralyzed with 2 mg/kg vecuronium bromide and baseline measurements were accomplished. Asphyxia was induced by clamping the
endotracheal tube. After a short induction of apnea, cardiac arrest was determined by absent pulsation of aortic artery, defined as MAP <20 mmHg. Precordial compression was begun and mechanical ventilation with 100% FiO2 was performed 6 min after the onset of cardiac arrest. Precordial compression at a rate of 250/min was synchronized to conduct a compression/ ventilation ratio of 5:1. Adrenaline (20 lg/kg) was injected after 2 min of cardiopulmonary resuscitation (CPR). In unsuccessfully resuscitated animals, the 30 s of CPR and adrenaline administrations were repeated and maintained until restoration of spontaneous circulation (ROSC) or 10 min after cardiac arrest. ROSC was defined as the return of supraventricular cardiac rhythm with mean aortic pressure (MAP) over 60 mmHg for P5 min. Animals were observed for 1 h after successful resuscitation. Experimental design After ROSC, the animals were randomized into the following three groups: (1) vehicle group; (2) Mdivi-1 low-dose group (Mdivi-1 dose1 group); (3) Mdivi-1 highdose group (Mdivi-1 dose2 group). Animals in the vehicle group were given an intravenous injection of 0.1% DMSO after 1 min of ROSC. Rats in Mdivi-1 dose1 group and Mdivi-1 dose2 group were intravenously infused with Mdivi-1 at doses of 0.24 and 1.2 mg/kg, respectively. Animals underwent identical anesthetic and surgical procedures except cardiac arrest were used as the sham group. Survival study and neurological outcome evaluation The survival rate after cardiac arrest was observed until 72 h. At 24, 48 and 72 h after ROSC, the neurologic function scores were performed blindly, using an overall performance category (OPC) scoring system (OPC: 1 = normal; 2 = mild disability; 3 = moderate disability; 4 = severe disability/coma; 5 = death/brain death) and neurologic deficit score (NDS, 0–10%, normal; 100%, brain death) (Neumar et al., 1995). All survived animals were sacrificed at 72 h after surgery and the brain tissues were removed for the following examinations. HE staining and TUNEL (TdT-mediated dUTP nick-end labeling) assay Separated left hemisphere of rats fixed in 4% paraformaldehyde and the paraffin-embedded brain tissues were cut into sections of 4-lm. Hematoxylin– eosin (HE) staining (Beyotime, Hangzhou, China) and TUNEL assay (Roche, Boston, MA, USA) were performed for in situ detection of neuronal injury, according to the manufacture’s instruction. Six visual fields from each brain tissue sample, in the hippocampal cornu ammonis 1 (CA-1) region were randomly selected and the HE-stained neuronal cells were counted. The immunohistochemical score (IHS) is determined by the percentage of positive-stained cells and the intensity of staining for TUNEL staining. All analyses were performed blinded.
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Western blot analysis To obtain the mitochondrial, cytosolic and nuclear protein fractions, separated right hemisphere were homogenized in lysis buffer. Thirty micrograms of protein were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Thirty micrograms of protein were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunolabelling was performed using mouse monoclonal Drp1 antibody (diluted 1:200; Abcam, Boston, MA, USA), rabbit polyclonal cleaved caspase-3 antibody (diluted 1:1000; CST, Beverly, MA, USA), rabbit monoclonal CytC antibody (diluted 1:1,000; CST, USA), rabbit monoclonal AIF antibody (diluted 1:1,000; CST, USA), rabbit monoclonal Histone H2A.X antibody (diluted 1:1,000; CST, USA), rabbit monoclonal Cyclooxygenase (COX)-IV antibody (diluted 1:1,000; CST, USA), rabbit monoclonal b-actin antibody (diluted 1:1,000; CST, USA). Immunoreactivity was developed by chemiluminescence kit and exposed to film, and quantitative analysis of band intensity was carried out by Quantity One Software (BIO-RAD, Hercules, CA, USA).
Statistical analysis Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using SPSS version 20.0 (SPSS, Chicago, IL). Comparison of the same parameters among groups was done using one-way analysis of variance (ANOVA), and the difference between pairs of means was tested post hoc with Fisher’s Least Significant Difference (LSD) test. A two-tailed Fisher’s exact test was employed to compare proportions of OPC values between groups (favorable vs. unfavorable outcome, OPC 1–2 vs. OPC 3–5). The difference in survival rates among the various groups was compared using log-rank test. A P-value <0.05 was considered statistically significant.
RESULTS Mdivi-1 inhibited Drp1 activation induced by cardiac arrest Western blot analysis demonstrated that Drp1 protein level increased in the mitochondria and decreased in the cytosol of brain after cardiac arrest (Fig. 1), indicating that ischemic injury induced by cardiac arrest lead to the trigger of Drp1 translocation onto mitochondria. As expected, treatment with Mdivi-1 at the dose of 1.2 mg/kg significantly attenuated cardiac arrestinduced translocation of cytosolic Drp1 (P < 0.05). However, lower dose of Mdivi-1 did not show a similar effect. Taken together, these results suggest that Mdivi-1 of 1.2 mg/kg could inhibit Drp1 activation induced by cardiac arrest.
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Inhibition of Drp1 by Mdivi-1 improved neurological outcome after ROSC To determine the effect of Drp1 inhibition by Mdivi-1 on the mortality and morbidity after cardiac arrest, total of 50 rats were subjected to asphyxial cardiac arrest. Of those, 42 rats achieved ROSC and were randomly allocated into vehicle (n = 14), Mdivi-1 dose1 (n = 14) and Mdivi-1 dose2 (n = 14). Rats were intravenously infused with either 0.1% DMSO or Mdivi-1 (0.24 or 1.2 mg/kg) after 1 min of ROSC. All survived animals showed no significant differences in body weight, basal body temperature, arterial blood gas, end-tidal PaCO2, heart rate and mean arterial pressure of rats as compared with animals in the sham group (n = 8) before inducing cardiac arrest (Table 1). At 72 h after ROSC, the survival rates of vehicle group, Mdivi-1 dose1 group and Mdivi-1 dose2 group were 21.4% (3/14), 28.6% (4/14) and 64.3% (9/14), respectively. The survival rates of rats of vehicle and Mdivi-1 dose1 group were significantly lower than those of the sham group (100%, P < 0.05). Compared with the vehicle-treated group, treatment with 1.2 mg/kg of Mdivi-1 significantly improved the survival rate after ROSC (threefold, P < 0.05), although the lower dose (0.24 mg/kg) did not show any effect (Fig. 2). OPC were not significantly different between Mdivi-1 dose2 group and vehicle group at 24 and 48 h and similar results were observed according to NDS. In the Mdivi-1 dose2 group, six of 14 animals achieved the favorable outcome (OPC 1 or 2) at 72 h after ROSC, compared to one of 14 in Mdivi-1 dose1 group and 0 of 14 in the vehicle group (Fig. 3). Treatment with 1.2 mg/kg of Mdivi-1 significantly improved the OPC score compared to the vehicle-treated group (P < 0.05). At 72 h after ROSC, survived animals treated with Mdivi-1 at the dose of 1.2 mg/kg showed better neurological function by NDS scores than those of vehicle-treated animals (P < 0.05) (Fig. 4). However, no significant differences were found between vehicle and Mdivi-1 dose1 group at the different time points (P > 0.05). Taken together, these data suggest that 1.2 mg/kg of Mdivi-1 treatment improves neurological outcomes after cardiac arrest. Inhibition of Drp1 by Mdivi-1 attenuated neuronal ischemic injury after ROSC To evaluate the effects on brain neurons after inhibition of Drp1, HE staining and TUNEL assay were performed to detect neuronal ischemic injury at 72 h after ROSC. The number of vital neurons in the hippocampal CA-1 region was significantly reduced, and the nucleus was compressed in animals of the vehicle-treated group. As compared, treatment with 1.2 mg/kg of Mdivi-1 significantly increased the number of vital neurons with normal nucleus, although the number of ischemic neurons in Mdivi-1 dose1 group was not significantly reduced (Fig. 5A, C). Apoptotic neurons were mainly found around the ischemic hippocampal CA-1 region as illustrated by TUNEL staining. Few TUNEL-positive neurons were found in the sham group, where
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Fig. 1. The protein levels of Drp1 in the brain after cardiac arrest. (A) Western blot analysis and (B) quantitative analysis of Drp1 expression at 72 h after ROSC. Cyclooxygenase (COX)-IV and b-actin were used as internal controls of mitochondrial and cytosolic subfractions, respectively. * P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. Mito, mitochondria; Cyto, cytoplasm.
Table 1. Basic characteristics
Body weight (g) HR (bpm) MAP (mmHg) PETCO2 (mmHg) Temperature (°C) PH PaCO2 (mmHg) PaO2 (mmHg) Lactate (mmol/L)
Sham (n = 8)
CPR (n = 14)
Mdivi-1 dose1 (n = 14)
Mdivi-1 dose2 (n = 14)
403.38 ± 12.97 361.88 ± 34.01 131.50 ± 18.09 39.25 ± 3.49 36.65 ± 0.26 7.40 ± 0.01 39.38 ± 3.16 95.13 ± 2.59 0.89 ± 0.08
400.50 ± 15.97 376.09 ± 31.17 131.93 ± 14.44 39.21 ± 3.64 36.54 ± 0.28 7.41 ± 0.02 39.28 ± 3.47 94.50 ± 2.24 0.90 ± 0.09
402.29 ± 14.86 374.50 ± 30.89 132.71 ± 14.25 38.57 ± 3.30 36.60 ± 0.22 7.42 ± 0.02 38.64 ± 3.13 94.07 ± 2.67 0.94 ± 0.13
400.14 ± 17.69 373.64 ± 35.03 133.57 ± 16.25 38.85 ± 2.82 36.52 ± 0.25 7.41 ± 0.01 38.93 ± 2.62 94.00 ± 2.94 0.93 ± 0.11
HR, heart rate; MAP, mean aortic pressure; PETCO2, end-tidal PaCO2.
Inhibition of Drp1 by Mdivi-1 inhibited mitochondriadependent apoptosis in the brain
Fig. 2. Inhibition of Drp1 by Mdivi-1 increased survival after cardiac arrest. *P < 0.05 vs. Sham group revealed by log-rank test; # P < 0.05 vs. Vehicle group.
numerous apoptotic neurons were evident in the vehicletreated group. As compared, less apoptotic neurons were observed (P < 0.05) in Mdivi-1 dose2 group. However, no difference in the scores was seen between vehicle and Mdivi-1 dose1 group (P > 0.05) (Fig. 5B, D).
To further find out the underlying mechanism of attenuating cerebral ischemia induced by cardiac arrest after inhibition of Drp1, we examine the molecules involved in apoptosis. As shown in Figs. 6 and 7. Caspases regulate cell apoptosis. CytC release activates caspase-3 and AIF is a caspase-independent mitochondrial death effector. Consistent with prominent increases in activated cleavage product of caspase-3, mitochondrial CytC and AIF were released into the cytosol and nucleus in the vehicle-treated group, respectively. As compared, treatment with 1.2 mg/kg of Mdivi-1 significantly inhibited release of CytC, AIF translocation and caspase-3 activation (P < 0.05). In contrast, there were no significant differences in the protein expression of cleaved caspase-3, CytC and AIF between the vehicle group and Mdivi-1 dose1 group
Fig. 3. Inhibition of Drp1 by Mdivi-1 improved OPC score after cardiac arrest. (A) 24 h, (B) 48 h, (C) 72 h. Score 1 and 2 were defined as a good outcome of neurological function, whereas score 3, 4, and 5 were defined as a poor outcome. Each dot represents one rat. *P < 0.05 vs. Sham group revealed by Fisher’s exact test; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. OPC, overall performance categories.
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Fig. 4. Inhibition of Drp1 by Mdivi-1 improved NDS of survival rats after cardiac arrest. Data are expressed as mean ± SD. #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. NDS, neurological deficit scores.
(P > 0.05). Taken together, these data suggest that Mdivi-1 (1.2 mg/kg) treatment reduced cardiac arrestinduced apoptosis by inhibiting release of CytC, translocation of AIF, caspase-3 activation.
DISCUSSION In this study, cerebral ischemia–reperfusion injury after cardiac arrest led to a prominent increase of Drp1 activation in the mitochondria. Most importantly, we provide the first direct evidence that the inhibition of
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Drp1 by Mdivi-1 improves neurological outcome and attenuated neuronal damage after cardiac arrest. The underlying mechanism maybe through the inhibition of mitochondria-dependent apoptosis pathway. These results suggested that Drp1 activation contribute to cerebral ischemia after cardiac arrest. Given the fact that Mdivi-1, the selective Drp1 inhibitor was delivered after ROSC, a clinically relevant intervention time, inhibition of Drp1 may be a promising therapeutic target for improving neurological outcome after cardiac arrest. We found cardiac arrest induced Drp1 activation in this study. The activity of Drp1 can be regulated by several post-modifications of Drp1, such as phosphorylation, sumoylation, ubiquitination and S-nitrosylation (Chang and Blackstone, 2010). Studies suggests that dephosphorylation of Drp1 at Serine 637 by calcineurin accelerates Drp1 activity (Cribbs and Strack, 2007). Cerebral ischemia–reperfusion could result in activation of the phosphatase calcineurin (Phillis et al., 2002), thus calcineurin may contribute to Drp1 activation after cardiac arrest. Drp1 accumulate on mitochondria and result in mitochondria-dependent apoptosis (Frank et al., 2001; Karbowski and Youle, 2003; Arnoult, 2007). Evidence suggests that inhibition of Drp1 by overexpression of a
Fig. 5. Inhibition of Drp1 by Mdivi-1 attenuated neuronal injury in the hippocampal CA-1 region after cardiac arrest. (A) Representative pictures of HE staining with a magnifying power of 400; (B) Representative pictures of TUNEL staining with a magnifying power of 400; (C) Proportions of ischemic neurons per total neurons; (D) IHS of TUNEL staining. Data are expressed as mean ± SD.*P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. IHS, immunohistochemical score.
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Fig. 6. Effect of inhibition of Drp1 by Mdivi-1 on protein levels of CytC and AIF after cardiac arrest. (A) Western blot analysis and (B) quantitative analysis of CytC expression at 72 h after ROSC (C) quantitative analysis of AIF expression at 72 h after ROSC. Histone, Cyclooxygenase (COX)-IV and b-actin were used as internal controls of nuclear, mitochondrial and cytosolic subfractions, respectively. Data are expressed as mean ± SD. * P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group. Mito, mitochondria; Cyto, cytoplasm; Nu, nucleus; CytC, cytochrome c; AIF, apoptosis-inducing factor.
Fig. 7. Effect of inhibition of Drp1 by Mdivi-1 on protein levels of caspase-3 after cardiac arrest. (A) Western blot analysis and (B) quantitative analysis of caspase-3 expression at 72 h after ROSC. b-actin were used as internal controls of cytosolic subfractions. Data are expressed as mean ± SD. *P < 0.05 vs. Sham group; #P < 0.05 vs. Vehicle group; 4P > 0.05 vs. Vehicle group.
dominant negative mutant of Drp1 or knockdown of Drp1 could prevent the release of CytC and AIF from mitochondria and attenuate apoptosis (Brooks et al., 2011), which is further confirmed by our data. Drp1 is required for CytC release and subsequently apoptosis execution (Frank et al., 2001; Lee et al., 2004). However, opposite views support that Drp1 is not prerequisite for apoptosis activation (Breckenridge et al., 2008). Regardless, ischemicreperfusion injury after cardiac arrest in our animal model promotes the Drp-1 activity and ultimately results in apoptosis execution. CytC and AIF as pro-apoptotic proteins are all localized in the mitochondria of healthy neurons, and the regulated release of these proteins by death signals plays a vital role in the execution of the mitochondriadependent apoptotic pathway (Wang and Youle, 2009). AIF release results in caspase-independent apoptosis (Zhang et al., 2002), while CytC release activates caspase-3 and finally results in caspase-dependent apoptosis (Sheridan and Martin, 2010). In this study, we find
that inhibition of Drp1 by Mdivi-1 prevented cardiac arrest-induced CytC release, AIF translocation and caspase-3 activation. Thus, the mitochondria-dependent apoptotic pathway may be involved in the protective mechanism of Drp1 inhibition against global cerebral ischemia induced by cardiac arrest. Global cerebral ischemia mostly leads to hippocampal damage, which results in necrotic and apoptotic neuronal death usually detectable 3 days after ischemia (Sugawara et al., 2000). The hippocampus is the most sensitive region to cerebral ischemia, which is closely related to cognitive dysfunction after ischemia–reperfusion injury. In the present study, therefore, the hippocampal CA-1 region of rats surviving until 72 h was selected for pathological examination. HE staining and TUNEL assay revealed various degrees of neuronal necrosis and apoptosis in rats after cardiac arrest, and the degree of pathological damage was consistent with the general neurological function impairment. Additionally, inhibition
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of Drp1 by Mdivi-1 improved survival and neurological function in our model. These neurological benefits could be associated with the attenuation of the cardiac arrestinduced neuronal necrosis and apoptosis following Mdivi-1 administration after ROSC, adding more supportive evidences for the neuroprotective effects of inhibition of Drp1. Consistent with previous studies (Ong et al., 2010; Xie et al., 2013), we found that 1.2 mg/kg of Mdivi-1 inhibited Drp1 translocation to the mitochondria and thereby attenuated cerebral ischemic injury from cardiac arrest, although Mdivi-1 (0.24 mg/kg) had no benefits on neurological outcomes, indicating that Mdivi-1 may offer therapeutic effects in a dose-dependent manner. In our model of cardiac arrest, we found that Drp1 translocation to the mitochondria could be suppressed by Mdivi-1. Importantly, this suppression markedly improves the neurological outcome and survival after cardiac arrest. Since our interventions of Drp1 activity were based with pharmacologically use of Mdivi-1, our results still require a further confirmation by gene approach or other novel inhibitors of Drp1.
CONCLUSION In summary, we suggest that Drp1 plays an important role in cerebral ischemic injury after cardiac arrest and pharmacological inhibition of Drp1 might be a novel strategy for neuroprotection after cardiac arrest. The underlying mechanism may be via suppression of the mitochondrial apoptosis pathway. Acknowledgments—This work was supported by the National Natural Science Foundation of China (81501137) and the Science and Technology Foundation of Guangdong Province, China (2015A030310042)
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(Accepted 11 October 2015) (Available online 20 October 2015)