BBADIS-63954; No. of pages: 9; 4C: 6, 8 Biochimica et Biophysica Acta xxx (2014) xxx–xxx
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Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion☆ Jie Xu a,1, Xinghua Qin a,1, Xiaoqing Cai a, Lu Yang a, Yuan Xing a, Jun Li a, Lihua Zhang b, Ying Tang c, Jiankang Liu c, Xing Zhang a,⁎⁎, Feng Gao a,⁎ a b c
Department of Physiology, School of Basic Medical Sciences, The Fourth Military Medical University, Xi'an, China Department of Geriatrics, Tangdu Hospital, The Fourth Military Medical University, Xi'an, China Institute of Mitochondrial Biology and Medicine, Xi'an Jiaotong University School of Life Science, Xi'an, China
a r t i c l e
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Article history: Received 15 February 2014 Received in revised form 10 May 2014 Accepted 13 May 2014 Available online xxxx Keywords: Myocardial ischemia/reperfusion JNK Mitochondria Autophagy Apoptosis
a b s t r a c t c-Jun N-terminal kinase (JNK) is a stress-activated mitogen-activated protein kinase that plays a central role in initiating apoptosis in disease conditions. Recent studies have shown that mitochondrial JNK signaling is partly responsible for ischemic myocardial dysfunction; however, the underlying mechanism remains unclear. Here we report for the first time that activation of mitochondrial JNK, rather than JNK localization on mitochondria, induces autophagy and apoptosis and aggravates myocardial ischemia/reperfusion injury. Myocardial ischemia/ reperfusion induced a dominant increase of mitochondrial JNK phosphorylation, while JNK mitochondrial localization was reduced. Treatment with Tat-SabKIM1, a retro-inverso peptide which blocks JNK interaction with mitochondria, decreased mitochondrial JNK activation without affecting JNK mitochondrial localization following reperfusion. Tat-SabKIM1 treatment reduced Bcl2-regulated autophagy, cytochrome c-mediated apoptosis and myocardial infarct size. Notably, selective inhibition of mitochondrial JNK activation using Tat-SabKIM1 produced a similar infarct size-reducing effect as inhibiting universal JNK activation with JNK inhibitor SP600125. Moreover, insulin-treated animals exhibited significantly dampened mitochondrial JNK activation accompanied by reduced infarct size and diminished autophagy and apoptosis following reperfusion. Taken together, these findings demonstrate that mitochondrial JNK activation, rather than JNK mitochondrial localization, induces autophagy and apoptosis and exacerbates myocardial ischemia/reperfusion injury. Insulin selectively inhibits mitochondrial JNK activation, contributing to insulin cardioprotection against myocardial ischemic/reperfusion injury. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. © 2014 Published by Elsevier B.V.
1. Introduction c-Jun N-terminal kinase (JNK) is a member of an evolutionarily conserved subfamily of mitogen-activated protein kinases (MAP kinases), which is critical for cellular responses to a variety of environmental and cellular stimuli [1,2]. Analysis of JNK-regulated pathways has
Abbreviations: AI, apoptosis index; COX, cytochrome c oxidase; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; LVSP, left ventricular systolic pressure; MABP, mean arterial blood pressure; mitoJNK, mitochondrial JNK; mTOR, mammalian target of rapamycin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling ☆ This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. ⁎ Correspondence to: F. Gao, Department of Physiology and Department of Cardiology, The Fourth Military Medical University, Xi'an 710032, China. Tel.: + 86 29 84776423; fax: + 86 29 83246270. ⁎⁎ Correspondence to: X. Zhang, Department of Physiology, The Fourth Military Medical University, Xi'an 710032, China. Tel.: +86 29 84772779; fax: +86 29 84772779. E-mail addresses: [email protected] (X. Zhang), [email protected] (F. Gao). 1 These authors contributed equally to this study.
shown that JNK is indispensable for both cell proliferation and apoptosis, dependent on the stimuli and the cell type involved [3,4]. In a variety of pathological conditions, JNK activation has been observed to have a central role in both extrinsic and intrinsic pathways that initiate apoptosis [5]. Inhibition of JNK signaling has been shown to attenuate apoptosis in several cell types, including hepatocytes, endothelial cells and cardiomyocytes, under different conditions [6–8]. Upon activation, the phosphorylated JNK translocates to the nucleus resulting in apoptosis by increasing the expression of pro-apoptotic genes through the transactivation of c-Jun/AP1-dependent or p53/73 protein-dependent mechanisms [5]. Recently, a new subcellular location of JNK has been identified in mitochondria [9]. At the mitochondrial outer membrane, JNK can interact with and phosphorylate a scaffold protein, Sab, facilitating JNK's translocation to mitochondria for signaling events [9,10]. Mitochondrial translocation of JNK has been presented in cellular and animal levels in the studies of DNA damage, liver injury, ischemia and oxidative stress [11–13], which are responsive to decreased mitochondrial function, such as blunted mitochondrial respiration, inhibited mitochondrial substrate flux, enhanced production
http://dx.doi.org/10.1016/j.bbadis.2014.05.012 0925-4439/© 2014 Published by Elsevier B.V.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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of reactive oxygen species and, importantly, mitochondria-mediated apoptosis [14]. These studies establish that mitochondria are a primary target of pro-apoptotic signaling by JNK. It has been well documented that JNK signaling is involved in the induction of cardiac ischemia/reperfusion (I/R) injury [15–17], and inhibition of JNK activation using either genetic modulation or chemical inhibitors has been shown to protect cardiomyocytes against I/R injury both in vivo and in vitro [7,18]. A recent study by LoGrasso and colleagues showed that inhibition of mitoJNK signaling alone, via disrupting the JNK/sab interaction at the outer mitochondrial membrane using a newly developed biochemical probe, improved mitochondrial function and exerted cardioprotective effect similar to that afforded by universal JNK inhibition, indicating a pivot role of mitoJNK in the induction of cardiac injury following I/R [19]. Most studies on mechanisms responsible for I/R-induced cardiac abnormalities have been focused on necrosis and apoptosis. Until recently dysregulated autophagy has been shown to play a unique role in I/R injury and post-infarction cardiac remodeling [20]. It has been reported that autophagic flux triggered by ischemia at the early stage of I/R may be beneficial, whereas excessive autophagy during reperfusion at later stage of I/R is harmful [21,22]. Here, we report our findings that mitoJNK activation rather than JNK mitochondrial localization induces autophagy and apoptosis, consequently aggravating myocardial I/R injury.
2. Materials and methods
2.3. Preparation of mitochondrial and cytosolic/nuclear fractions Cardiac mitochondria were isolated from either sham-operated or I/R rats as described previously [19]. Briefly, rat hearts were washed with ice-cold isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA and 0.5 mg/ml BSA, pH 7.4), minced into small pieces, and then homogenized. The homogenate was centrifuged at 4 °C for 10 min at 1000 g. The supernatant was collected and further centrifuged at 4 °C for 10 min at 10,000 g. The pellet was re-suspended in lysis buffer for Western blot analysis of mitochondrial proteins, and the resulting supernatant was used as the soluble cytosolic/nuclear fraction. Cytochrome c oxidase IV (COX IV) was used as an internal control for mitochondria and GAPDH as an internal control for cytosol. 2.4. Determination of myocardial infarct size and apoptosis At the end of the 2 h reperfusion, myocardial infarct size was determined by means of a double-staining technique and was analyzed by a digital imaging system described previously [23]. Myocardial apoptotic index was analyzed by TUNEL (terminal deoxynucleotidyl nick end labeling) assay using an In Situ Cell Death Detection Kit (Roche Molecular Biochemical, Indianapolis, IN) according to the protocol provided by the manufacturer; TUNEL staining for apoptotic cell nuclei and DAPI staining for all myocardial cell nuclei. Cardiomyocytes from four slides per block which were randomly selected were evaluated to determine the number and percentage of cells exhibiting positive staining for apoptosis. The apoptosis index (AI) was determined [(no. of apoptotic myocytes / total no. of myocytes counted) × 100%].
2.1. Experimental protocol 2.5. Assessment of cardiac function The experiments were performed in adherence with the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Fourth Military Medical University Committee on Animal Care. Male Sprague–Dawley rats weighting 180–220 g were fasted overnight and anesthetized through intraperitoneal injection of 60 mg/kg pentobarbital sodium. Myocardial ischemia was produced by exteriorizing the heart with a left thoracic incision followed by making a slipknot (6–0 silk) around left anterior descending coronary artery, as described previously [23]. The success in coronary occlusion was confirmed by immediate ST elevation on electrocardiogram. The animals were subjected to 30 min of coronary occlusion followed by 2 h of reperfusion. The effects of insulin (60 U/l, intravenous infusion at 4 ml/kg/h for 2 h, beginning at 5 min before reperfusion) on myocardial I/R injury were examined in I/R + insulin rats. SP600125 (1 mg/kg, Sigma-Aldrich Co.) and anisomycin (0.1 mg/kg, Sigma-Aldrich Co.) were administered through femoral vein at 10 and 15 min respectively before reperfusion. Hearts were excised at 2 h after reperfusion and the tissue from the area at risk was separated, washed by ice-cold saline and then rapidly frozen in liquid nitrogen. The frozen hearts were stored in a refrigerator at − 80 °C while waiting for Western blot analysis.
2.2. Tat-Sab treatment Tat-scramble (LPSVFGDVGAPSRLPEVSLSPPRRRQRRKKRG-NH 2 ) and Tat-Sab KIM1 (GFESLSVPSPLDLSGPRVVAPPRRRQRRKKRG-NH2 ) were purchased from NeoPeptide. Direct injections (five 5-μl injections, 2 mg/kg) were delivered 5 min before reperfusion into the left ventricle wall in areas below the occlusion site of the ligation (four injections) and in the apex of the heart (one injection), as described previously [19]. Injections were made with a 30-gauge needle and a 10-μl syringe. Control groups subjected to the same I/R procedure included I/R + saline, I/R + Tat-scramble and a sham group without left anterior descending coronary artery occlusion.
Cardiac function was monitored at the end of the 2 h reperfusion. An artery microcatheter was intubated into the left ventricle to determine the left ventricular pressure (LVP). Electrocardiogram, heart rate and LVP were recorded on a hemodynamic analyzing system (Chengdu Instrument Co., China). Left ventricular systolic pressure (LVSP), mean arterial blood pressure (MABP) and the instantaneous first derivation of LVP (+dP/dtmax and −dP/dtmax) were obtained by computer algorithms. 2.6. Determination of plasma lactate dehydrogenase levels Blood samples were drawn at the end of the 2 h reperfusion. Plasma lactate dehydrogenase (LDH) levels were measured using commercial detection kits (DU 640; Beckman Coulter, Brea, CA) according to the manufacturer's instruction. All measurements were assayed in duplicates. 2.7. Western blot analysis The protein expression and phosphorylation levels were determined using Western blot as described previously [23]. The immunoblots were probed with anti-JNK, anti-p-JNK (Thr183/Tyr185 Cell Signaling Technology, USA), anti-COX IV, anti-cytochrome c (Cell Signaling Technology, USA), anti-LC3, anti-p62, anti-Beclin1, anti-Bcl2 (Santacruz Biotechnology, Inc, USA) and anti-GAPDH (Abcam, USA) antibodies overnight at 4 °C and subsequently incubated with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA). GAPDH was used as the internal loading control. 2.8. Statistical analysis All values are presented as mean ± SEM. Differences were compared with ANOVA followed by Bonferroni post hoc test. Probabilities of b0.05 were considered to be statistically significant. All of the statistical tests were performed with the GraphPad Prism software, version 5.0 (GraphPad Software Inc., San Diego, CA).
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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3. Results 3.1. I/R induced mitoJNK activation To elucidate the role of mitoJNK signaling in I/R injury, the localization and activation of JNK were determined after reperfusion. Western blotting of JNK and p-JNK showed two immunoreactive bands with
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approximate molecular weights of 46- and 55-kDa (Fig. 1A, E, I) in cytosol/nucleus, while the 55-kDa splice of JNK was dominant in mitochondria, which is consistent with previous studies [19,24]. The phosphorylation level of JNK was increased in both mitochondria (Fig. 1A, B) and cytosol/nucleus (Fig. 1E, F) after 2 h of reperfusion. However, it should be noted that total JNK in mitochondria was decreased (Fig. 1C) and total JNK in cytosol/nucleus was increased (Fig. 1G), indicating JNK
Fig. 1. Mitochondrial JNK activation is associated with I/R injury. A, representative Western blots of p-JNK and JNK in mitochondria following I/R. B–D, the normalized ratio of p-JNK/COX IV (B), JNK/COX IV (C) and p-JNK/JNK (D) in mitochondria following I/R. E, as in A, for cytosol/nucleus. F–H, the normalized ratio of p-JNK/GAPDH (F), JNK/GAPDH (G) and p-JNK/JNK (H) in cytosol/nucleus following I/R. I, as in A, for total extract. J–K, as in G–H, for total extract. A–K, treatment with insulin (60 U/l, 4 ml/kg/h for 2 h through femoral vein 5 min before reperfusion) restored JNK activation and localization in both mitochondria and cytosol/nucleus. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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translocation from mitochondria to cytosol/nucleus during reperfusion. A dominant JNK activation occurred in mitochondria as evidenced by the fact that p-JNK/JNK displayed an approximately 4-fold increase in mitochondria (Fig. 1D) and 2-fold increase in cytosol/nucleus (Fig. 1H). Consistent with JNK activation in mitochondria and cytosol/nucleus, universal JNK activation displayed an approximately 4-fold increase (Fig. 1K), and I/R had little effect on universal JNK expression (Fig. 1J). To further examine the role of mitoJNK signaling in I/R injury, insulin treatment which has been demonstrated by our laboratory and others as one of the most promising strategies in cardioprotection against I/R injury was employed [25–29]. As shown previously, insulin treatment (60 U/l, intravenous infusion at 4 ml/kg/h for 2 h, beginning at 5 min before reperfusion) exerted protective effect against I/R injury as evidenced by reduced lesion area (Fig. 3A) and plasma LDH concentration (Fig. 3B). Meanwhile, insulin treatment decreased both mitochondrial and cytosolic/nuclear JNK activation (Fig. 1B, F). Importantly, treatment with insulin decreased the ratio of p-JNK/JNK in mitochondria, but had little effect on the ratio of p-JNK/JNK in cytosol/ nucleus (Fig. 1D), indicating that insulin could selectively inhibit mitoJNK activation during reperfusion. In addition, treatment with insulin restored the JNK localization after reperfusion (Fig. 1C, G). These results suggested that I/R-induced JNK activation, especially mitoJNK activation, is associated with I/R injury. 3.2. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide exerted protective effect against I/R injury To examine the role of mitoJNK activation in I/R injury, we used a peptide containing 20 residues of the SabKIM1 domain along with a HIV-Tat sequence (Tat-SabKIM1) as described previously [19], which can be used to block JNK translocation to mitochondria in vivo. It has been reported that Tat-SabKIM1 does not affect JNK-mediated nuclear events and JNK translocation to the nucleus [14]. As shown in Fig. 2A,
Tat-SabKIM1 injection (2 mg/kg, delivered 5 min before reperfusion) decreased the phosphorylation level of JNK in mitochondria (Fig. 2B, D), and had no evident effect on cytosolic/nuclear JNK activation (Fig. 2E, F, H) following I/R. Unexpectedly, Tat-SabKIM1 injection had no evident effect on JNK localization following reperfusion (Fig. 2C, G). These results indicated that injection with Tat-SabKIM1 can specifically inhibit mitoJNK activation without impact on cytosolic/nuclear JNK activation and JNK localization. Importantly, Tat-SabKIM1 injection markedly reduced lesion area (Fig. 3A) and plasma LDH level (Fig. 3B) following I/R, exerting similar cardioprotective effect as treatment with insulin (Fig. 3A, B). In addition, assessment of cardiac function after 2 h of reperfusion revealed that injection with Tat-SabKIM1 increased LVSP (Fig. 3C) and ±LV dP/dtmax in rats (Fig. 3D, E), while no significant differences in MABP and heart rate have been detected (data not shown). These results suggested that inhibition of mitoJNK activation alone exerts cardioprotective effect against I/R injury. 3.3. mitoJNK activation contributed to Bcl2-regulated autophagy Next, we examined the effect of mitoJNK activation on autophagy after reperfusion. I/R resulted in biochemical evidence of cardiac muscle autophagy, including conversion of the non-lipidated form of LC3, LC3-I, to the autophagosome membrane-associated lipidated form, LC3-II, and degradation of the autophagy substrate protein p62 (Fig. 4A, B, C). Bcl2 inhibits autophagy through a direct interaction with the autophagy protein Beclin1 [30]. I/R-induced autophagy was further confirmed by increased Beclin1 and decreased Bcl2 after reperfusion (Fig. 4A, D, E). Inhibition of mitoJNK activation through either Tat-SabKIM1 injection or insulin treatment alleviated I/R-induced autophagy, as evidenced by the restored levels of LC3-II/LC3-I, p62, Bcl2 and Beclin1 (Fig. 4A, B, C, D, E). These results indicated that mitoJNK activation triggers the Bcl2-regulated autophagy following reperfusion.
Fig. 2. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide during I/R in vivo. A, representative Western blots of p-JNK and JNK in mitochondria following I/R with Tat-SabKIM1 (TS) injection (2 mg/kg, delivered 5 min before reperfusion). B–D, the normalized ratio of p-JNK/COX IV (B), JNK/COX IV (C) and p-JNK/JNK (D) in mitochondria following I/R with TS injection. E, as in A, for cytosol/nucleus. F–H, the normalized ratio of p-JNK/GAPDH (F), JNK/GAPDH (G) and p-JNK/JNK (H) in cytosol/nucleus following I/R with TS injection. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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Fig. 3. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide and treatment with insulin exert protective effect against I/R injury in vivo. A–E, treatment with Tat-SabKIM1 (TS) or insulin decreased myocardial infarct size (INF) expressed as percentage of area at risk (AAR) (A) and plasma lactate dehydrogenase (LDH) concentration (B), and increased left ventricular systolic pressure (LVSP) (C), and ±dP/dtmax (D, E) following I/R. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Fig. 4. Inhibition of mitoJNK activation through insulin or Tat-SabKIM1 treatment ameliorates Bcl2-regulated autophagy following I/R. A, representative Western blots of the following autophagy-related proteins in heart subjected to I/R with insulin or Tat-SabKIM1 (TS) treatment: LC3-I, LC3-II, p62, Beclin1, and Bcl2. B–E, The statistical results of LC3-II/LC3-I (B), p62 (C), Beclin1 (D) and Bcl2 (E). Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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3.4. mitoJNK activation contributed to mitochondria-mediated apoptosis As shown in Fig. 5A, the amount of cytochrome c release from mitochondria into the cytosol following I/R displayed an approximately 3-fold increase compared with that in sham group. Treatment with insulin reduced cytochrome c release into the cytosol by 48.3% (Fig. 5A), and Tat-SabKIM1 injection alleviated cytochrome c release by 68.9% (Fig. 5A). Inhibition of mitoJNK signaling through either Tat-SabKIM1 injection or insulin treatment decreases I/R-induced cytochrome c release from mitochondria. Next, we examined cardiac apoptosis using TUNEL staining to further examine the role of mitoJNK signaling in the induction of apoptosis. TUNEL-positive cells were increased in I/R rats (Fig. 5B). Treatment with insulin exerted a significant anti-apoptotic effect as evidenced by reduced TUNEL-positive staining (Fig. 5B). Meanwhile Tat-SabKIM1 injection also decreased TUNEL-positive staining, exerting cardioprotective effect similar to insulin treatment (Fig. 5B). These results indicated that mitoJNK signaling triggers the mitochondria-mediated apoptosis following I/R.
3.5. Inhibiting mitoJNK activation alone exerted similar cardioprotective effect as universal JNK inhibition It's reported that inhibition of universal JNK activation also exerts cardioprotective effect against I/R injury [7]. Next, we inhibited universal JNK activation to test the contribution of mitoJNK activation in the induction of I/R injury. The phosphorylation level of JNK displayed an approximately 3.5-fold increase after 2 h of reperfusion (Fig. 6A). To further elucidate the involvement of JNK in the induction of I/R-induced myocardium injury, SP600125, a selective JNK inhibitor, was used. Treatment with SP600125 (1 mg/kg, administered through femoral vein 10 min before reperfusion) decreased phosphorylation level of universal JNK by 71.4%, and reduced lesion area by 40.3% and plasma LDH
concentration by 46.7% (Fig. 6B, C). Similarly, inhibition of mitoJNK activation by 66.8% (Fig. 2B) could reduce lesion area by 32.6% and plasma LDH concentration by 39.7% (Fig. 3A, B). These results indicated that inhibiting mitoJNK activation alone could exert similar cardioprotective effect as universal JNK inhibition. Furthermore, treatment with anisomycin (0.1 mg/kg, administered through contralateral femoral vein 15 min before reperfusion) which activates JNK signaling abolished the cardioprotection of SP600125 in I/R rats (Fig. 5B, C), further indicating that the cardioprotective effect of SP600125 is through the inhibition of JNK activation.
4. Discussion The major findings of the present study are as follows. First, I/R induces a dominant increase of mitoJNK activation which triggers Bcl2regulated autophagy and cytochrome c-mediated apoptosis. Second, it is mitoJNK activation rather than JNK localization on mitochondria that mediates the induction of I/R injury. Third, insulin selectively inhibits mitoJNK activation during reperfusion, which constitutes a novel mechanism of insulin cardioprotection against I/R injury. JNK has been shown to have an essential role in modulating the functions of pro- and anti-apoptotic proteins located in the mitochondria, thus regulating mitochondrial function and mitochondria-mediated apoptosis [5,13,31]. However, previous studies lack evidence showing the role of mitoJNK signaling in the regulation of mitochondrial function. Until recently, with the aid of a new method to selectively inhibit JNK translocation to mitochondria using Tat-SabKIM1 peptide through inhibiting JNK interacting with Sab, a binding partner for JNK mitochondrial association [9], LoGrasso and Chambers showed that mitoJNK signaling mediated mitochondrial reactive oxygen species generation and mitochondrial cytochrome c release [24]. Our in vivo study further confirmed that inhibition of mitoJNK activation with Tat-SabKIM1 reduced mitochondrial cytochrome c release and thus reduced apoptosis and
Fig. 5. Inhibition of mitoJNK activation through insulin or Tat-SabKIM1 treatment ameliorates mitochondria-mediated apoptosis following I/R. A, levels of cytochrome c release (fold change normalized to sham group) in rats subjected to I/R with insulin or Tat-SabKIM1 (TS) treatment. B, representative photomicrographs of in situ detection of apoptotic myocytes by TUNEL staining in heart slices from rats subjected to I/R. TUNEL positive cells (green) and total number of nuclei (blue) are shown for each representative section of the heart. Scale bar: 50 μm. Percentage of TUNEL-positive nuclei was analyzed below. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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Fig. 6. Universal JNK inhibition exerts cardioprotective effect against I/R injury similarly as inhibiting mitoJNK activation. A, myocardial phosphorylation level of JNK following I/R evaluated by Western blot. B, myocardial infarct size (INF) expressed as percentage of area at risk (AAR) following I/R. C, plasma lactate dehydrogenase (LDH) concentration following I/R. SP (SP600125, 1 mg/kg), a selective JNK inhibitor, was administered through femoral vein 10 min before reperfusion. A (anisomycin, 0.1 mg/kg), a JNK activator, was administered through contralateral femoral vein 15 min before reperfusion. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
attenuated I/R injury. Importantly, in the present study, we observed that JNK mitochondrial localization was decreased, though activated JNK on mitochondria was increased following I/R, which is contrary to LoGrasso's results that I/R increased JNK localization on mitochondria [19]. One possible reason for the discrepancy is that they only determined the total JNK in both mitochondria and cytosol/nucleus in in vitro study. Furthermore, our results showed that I/R induced a dominant increase of mitoJNK activation rather than JNK activation in cytosol/nucleus. mitoJNK activation caused cell death through not only cytochrome c-mediated apoptosis but also Bcl2-regulated autophagy. Although the precise role of autophagy regulation contributing to cell survival and death in ischemic hearts remains controversial, a number of studies have shown that despite the seemingly cardioprotective role of autophagy during ischemia, autophagy can induce cell death during reperfusion [20–22]. This form of cell death is referred to as type 2 programmed cell death but may simply be caused by excessive activation of autophagy. Mechanistically, mitoJNK phosphorylates Bcl2, antagonizing Bcl2 antiapoptotic and anti-autophagic activities [32,33], which may contribute to mitoJNK's deleterious role in myocardial I/R injury. Other possible mechanisms that maintain autophagy at a higher level during reperfusion include oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress and calcium overload [34]. Moreover, selective inhibition of mitoJNK activation produced similar infarct size-reducing effect as universally inhibiting JNK with JNK inhibitor SP600125. This suggests that inhibition of mitoJNK activation could possibly be at least partially responsible for the protective effects of universal JNK inhibition against I/R. These results are potentially of clinical significance, and may suggest a new therapeutic strategy for treating ischemic heart disease. Compounds that are either competitive
versus JNK-interacting protein or that have some bidentate character might be achieved with therapeutic benefit [35–37]. Studies from our laboratory and others demonstrate that the possible mechanisms through which insulin attenuates cardiac ischemia injury include metabolic modulation, positive inotropic effect and directly activating survival signaling which exerts anti-apoptosis, vasodilatation, anti-inflammation and anti-oxidative stress effects [38]. A lot of studies have established that central to these effects is the mitochondria whose proper functioning is essential to myocardial health due to its vital role in energy production and cell survival [28]. The present study showed that treatment with insulin selectively inhibits mitoJNK activation, although the exact mechanism is still elusive, consequently reduced apoptosis and infarct size. This extends our understanding of insulin cardioprotection against I/R injury. Based on the findings from previous studies and the present study, we proposed a model illustrating the possible mechanism of mitoJNK activation in the induction of myocardial I/R injury (Fig. 7). JNK is activated at a relatively low level and localized in either mitochondria or cytosol/nucleus under unstressed condition. During I/R, JNK is phosphorylated and activated JNK translocates to mitochondria, resulting in increased mitoJNK activation. Increased mitoJNK activation results in enhanced autophagy and cytochrome c release from mitochondria. Either insulin or Tat-SabKIM1 intervention inhibits mitoJNK activation and exerts cardioprotective effect against I/R injury. In conclusion, this is the first demonstration on the molecular level that mitoJNK activation, rather than JNK mitochondrial localization, triggers autophagy and apoptosis following I/R. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide is as effective as inhibiting the catalytic activity of universal JNK in improving post-ischemic cardiac function and
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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Fig. 7. Schematic figure illustrating that mitoJNK activation mediates myocardial I/R injury. TS: Tat-SabKIM1. The JNK locates in mitochondria and cytosol/nucleus in the absence of stress. Once it confronted I/R, JNK is phosphorylated in cytosol/nucleus and activated JNK translocates to mitochondrial outer membrane which triggers Bcl2-regulated autophagy and mitochondria-mediated apoptosis. Treatment with insulin or Tat-SabKIM1 inhibits mitoJNK activation and protects myocardium against I/R injury.
reducing myocardial infarction. Moreover, insulin inhibits mitoJNK activation and thus reduces myocardial infarction, which constitutes a novel mechanism of insulin cardioprotection against I/R injury. These results suggest that strategies directed towards inhibiting mitochondriaspecific JNK activation may have therapeutic potential for ischemic heart disease. Disclosures None declared. Acknowledgements This work was supported by grants from the National Basic Research Program of China (No. 2013CB531204), the State Key Program of National Natural Science Foundation of China (No. 81030005) and the National Natural Science Foundation of China (Nos. 81070184, 81270301). References [1] C.R. Weston, R.J. Davis, The JNK signal transduction pathway, Curr. Opin. Cell Biol. 19 (2007) 142–149. [2] Y. Zhao, T. Herdegen, Cerebral ischemia provokes a profound exchange of activated JNK isoforms in brain mitochondria, Mol. Cell. Neurosci. 41 (2009) 186–195. [3] A. Lin, B. Dibling, The true face of JNK activation in apoptosis, Aging Cell 1 (2002) 112–116. [4] W. Chen, M.A. White, M.H. Cobb, Stimulus-specific requirements for MAP3 kinases in activating the JNK pathway, J. Biol. Chem. 277 (2002) 49105–49110. [5] D.N. Dhanasekaran, E.P. Reddy, JNK signaling in apoptosis, Oncogene 27 (2008) 6245–6251. [6] S.K. Venugopal, J. Chen, Y. Zhang, D. Clemens, A. Follenzi, M.A. Zern, Role of MAPK phosphatase-1 in sustained activation of JNK during ethanol-induced apoptosis in hepatocyte-like VL-17A cells, J. Biol. Chem. 282 (2007) 31900–31908. [7] C. Ferrandi, R. Ballerio, P. Gaillard, C. Giachetti, S. Carboni, P.A. Vitte, J.P. Gotteland, R. Cirillo, Inhibition of c-Jun N-terminal kinase decreases cardiomyocyte apoptosis and infarct size after myocardial ischemia and reperfusion in anaesthetized rats, Br. J. Pharmacol. 142 (2004) 953–960. [8] E.W. Son, S.J. Mo, D.K. Rhee, S. Pyo, Inhibition of ICAM-1 expression by garlic component, allicin, in gamma-irradiated human vascular endothelial cells via downregulation of the JNK signaling pathway, Int. Immunopharmacol. 6 (2006) 1788–1795. [9] C. Wiltshire, D.A. Gillespie, G.H. May, Sab (SH3BP5), a novel mitochondria-localized JNK-interacting protein, Biochem. Soc. Trans. 32 (2004) 1075–1077.
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Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion☆ Jie Xu a,1, Xinghua Qin a,1, Xiaoqing Cai a, Lu Yang a, Yuan Xing a, Jun Li a, Lihua Zhang b, Ying Tang c, Jiankang Liu c, Xing Zhang a,⁎⁎, Feng Gao a,⁎ a b c
Department of Physiology, School of Basic Medical Sciences, The Fourth Military Medical University, Xi'an, China Department of Geriatrics, Tangdu Hospital, The Fourth Military Medical University, Xi'an, China Institute of Mitochondrial Biology and Medicine, Xi'an Jiaotong University School of Life Science, Xi'an, China
a r t i c l e
i n f o
Article history: Received 15 February 2014 Received in revised form 10 May 2014 Accepted 13 May 2014 Available online xxxx Keywords: Myocardial ischemia/reperfusion JNK Mitochondria Autophagy Apoptosis
a b s t r a c t c-Jun N-terminal kinase (JNK) is a stress-activated mitogen-activated protein kinase that plays a central role in initiating apoptosis in disease conditions. Recent studies have shown that mitochondrial JNK signaling is partly responsible for ischemic myocardial dysfunction; however, the underlying mechanism remains unclear. Here we report for the first time that activation of mitochondrial JNK, rather than JNK localization on mitochondria, induces autophagy and apoptosis and aggravates myocardial ischemia/reperfusion injury. Myocardial ischemia/ reperfusion induced a dominant increase of mitochondrial JNK phosphorylation, while JNK mitochondrial localization was reduced. Treatment with Tat-SabKIM1, a retro-inverso peptide which blocks JNK interaction with mitochondria, decreased mitochondrial JNK activation without affecting JNK mitochondrial localization following reperfusion. Tat-SabKIM1 treatment reduced Bcl2-regulated autophagy, cytochrome c-mediated apoptosis and myocardial infarct size. Notably, selective inhibition of mitochondrial JNK activation using Tat-SabKIM1 produced a similar infarct size-reducing effect as inhibiting universal JNK activation with JNK inhibitor SP600125. Moreover, insulin-treated animals exhibited significantly dampened mitochondrial JNK activation accompanied by reduced infarct size and diminished autophagy and apoptosis following reperfusion. Taken together, these findings demonstrate that mitochondrial JNK activation, rather than JNK mitochondrial localization, induces autophagy and apoptosis and exacerbates myocardial ischemia/reperfusion injury. Insulin selectively inhibits mitochondrial JNK activation, contributing to insulin cardioprotection against myocardial ischemic/reperfusion injury. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. © 2014 Published by Elsevier B.V.
1. Introduction c-Jun N-terminal kinase (JNK) is a member of an evolutionarily conserved subfamily of mitogen-activated protein kinases (MAP kinases), which is critical for cellular responses to a variety of environmental and cellular stimuli [1,2]. Analysis of JNK-regulated pathways has
Abbreviations: AI, apoptosis index; COX, cytochrome c oxidase; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; LVSP, left ventricular systolic pressure; MABP, mean arterial blood pressure; mitoJNK, mitochondrial JNK; mTOR, mammalian target of rapamycin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling ☆ This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases. ⁎ Correspondence to: F. Gao, Department of Physiology and Department of Cardiology, The Fourth Military Medical University, Xi'an 710032, China. Tel.: + 86 29 84776423; fax: + 86 29 83246270. ⁎⁎ Correspondence to: X. Zhang, Department of Physiology, The Fourth Military Medical University, Xi'an 710032, China. Tel.: +86 29 84772779; fax: +86 29 84772779. E-mail addresses: [email protected] (X. Zhang), [email protected] (F. Gao). 1 These authors contributed equally to this study.
shown that JNK is indispensable for both cell proliferation and apoptosis, dependent on the stimuli and the cell type involved [3,4]. In a variety of pathological conditions, JNK activation has been observed to have a central role in both extrinsic and intrinsic pathways that initiate apoptosis [5]. Inhibition of JNK signaling has been shown to attenuate apoptosis in several cell types, including hepatocytes, endothelial cells and cardiomyocytes, under different conditions [6–8]. Upon activation, the phosphorylated JNK translocates to the nucleus resulting in apoptosis by increasing the expression of pro-apoptotic genes through the transactivation of c-Jun/AP1-dependent or p53/73 protein-dependent mechanisms [5]. Recently, a new subcellular location of JNK has been identified in mitochondria [9]. At the mitochondrial outer membrane, JNK can interact with and phosphorylate a scaffold protein, Sab, facilitating JNK's translocation to mitochondria for signaling events [9,10]. Mitochondrial translocation of JNK has been presented in cellular and animal levels in the studies of DNA damage, liver injury, ischemia and oxidative stress [11–13], which are responsive to decreased mitochondrial function, such as blunted mitochondrial respiration, inhibited mitochondrial substrate flux, enhanced production
http://dx.doi.org/10.1016/j.bbadis.2014.05.012 0925-4439/© 2014 Published by Elsevier B.V.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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of reactive oxygen species and, importantly, mitochondria-mediated apoptosis [14]. These studies establish that mitochondria are a primary target of pro-apoptotic signaling by JNK. It has been well documented that JNK signaling is involved in the induction of cardiac ischemia/reperfusion (I/R) injury [15–17], and inhibition of JNK activation using either genetic modulation or chemical inhibitors has been shown to protect cardiomyocytes against I/R injury both in vivo and in vitro [7,18]. A recent study by LoGrasso and colleagues showed that inhibition of mitoJNK signaling alone, via disrupting the JNK/sab interaction at the outer mitochondrial membrane using a newly developed biochemical probe, improved mitochondrial function and exerted cardioprotective effect similar to that afforded by universal JNK inhibition, indicating a pivot role of mitoJNK in the induction of cardiac injury following I/R [19]. Most studies on mechanisms responsible for I/R-induced cardiac abnormalities have been focused on necrosis and apoptosis. Until recently dysregulated autophagy has been shown to play a unique role in I/R injury and post-infarction cardiac remodeling [20]. It has been reported that autophagic flux triggered by ischemia at the early stage of I/R may be beneficial, whereas excessive autophagy during reperfusion at later stage of I/R is harmful [21,22]. Here, we report our findings that mitoJNK activation rather than JNK mitochondrial localization induces autophagy and apoptosis, consequently aggravating myocardial I/R injury.
2. Materials and methods
2.3. Preparation of mitochondrial and cytosolic/nuclear fractions Cardiac mitochondria were isolated from either sham-operated or I/R rats as described previously [19]. Briefly, rat hearts were washed with ice-cold isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, 1 mM EGTA and 0.5 mg/ml BSA, pH 7.4), minced into small pieces, and then homogenized. The homogenate was centrifuged at 4 °C for 10 min at 1000 g. The supernatant was collected and further centrifuged at 4 °C for 10 min at 10,000 g. The pellet was re-suspended in lysis buffer for Western blot analysis of mitochondrial proteins, and the resulting supernatant was used as the soluble cytosolic/nuclear fraction. Cytochrome c oxidase IV (COX IV) was used as an internal control for mitochondria and GAPDH as an internal control for cytosol. 2.4. Determination of myocardial infarct size and apoptosis At the end of the 2 h reperfusion, myocardial infarct size was determined by means of a double-staining technique and was analyzed by a digital imaging system described previously [23]. Myocardial apoptotic index was analyzed by TUNEL (terminal deoxynucleotidyl nick end labeling) assay using an In Situ Cell Death Detection Kit (Roche Molecular Biochemical, Indianapolis, IN) according to the protocol provided by the manufacturer; TUNEL staining for apoptotic cell nuclei and DAPI staining for all myocardial cell nuclei. Cardiomyocytes from four slides per block which were randomly selected were evaluated to determine the number and percentage of cells exhibiting positive staining for apoptosis. The apoptosis index (AI) was determined [(no. of apoptotic myocytes / total no. of myocytes counted) × 100%].
2.1. Experimental protocol 2.5. Assessment of cardiac function The experiments were performed in adherence with the National Institutes of Health Guidelines for the Use of Laboratory Animals and were approved by the Fourth Military Medical University Committee on Animal Care. Male Sprague–Dawley rats weighting 180–220 g were fasted overnight and anesthetized through intraperitoneal injection of 60 mg/kg pentobarbital sodium. Myocardial ischemia was produced by exteriorizing the heart with a left thoracic incision followed by making a slipknot (6–0 silk) around left anterior descending coronary artery, as described previously [23]. The success in coronary occlusion was confirmed by immediate ST elevation on electrocardiogram. The animals were subjected to 30 min of coronary occlusion followed by 2 h of reperfusion. The effects of insulin (60 U/l, intravenous infusion at 4 ml/kg/h for 2 h, beginning at 5 min before reperfusion) on myocardial I/R injury were examined in I/R + insulin rats. SP600125 (1 mg/kg, Sigma-Aldrich Co.) and anisomycin (0.1 mg/kg, Sigma-Aldrich Co.) were administered through femoral vein at 10 and 15 min respectively before reperfusion. Hearts were excised at 2 h after reperfusion and the tissue from the area at risk was separated, washed by ice-cold saline and then rapidly frozen in liquid nitrogen. The frozen hearts were stored in a refrigerator at − 80 °C while waiting for Western blot analysis.
2.2. Tat-Sab treatment Tat-scramble (LPSVFGDVGAPSRLPEVSLSPPRRRQRRKKRG-NH 2 ) and Tat-Sab KIM1 (GFESLSVPSPLDLSGPRVVAPPRRRQRRKKRG-NH2 ) were purchased from NeoPeptide. Direct injections (five 5-μl injections, 2 mg/kg) were delivered 5 min before reperfusion into the left ventricle wall in areas below the occlusion site of the ligation (four injections) and in the apex of the heart (one injection), as described previously [19]. Injections were made with a 30-gauge needle and a 10-μl syringe. Control groups subjected to the same I/R procedure included I/R + saline, I/R + Tat-scramble and a sham group without left anterior descending coronary artery occlusion.
Cardiac function was monitored at the end of the 2 h reperfusion. An artery microcatheter was intubated into the left ventricle to determine the left ventricular pressure (LVP). Electrocardiogram, heart rate and LVP were recorded on a hemodynamic analyzing system (Chengdu Instrument Co., China). Left ventricular systolic pressure (LVSP), mean arterial blood pressure (MABP) and the instantaneous first derivation of LVP (+dP/dtmax and −dP/dtmax) were obtained by computer algorithms. 2.6. Determination of plasma lactate dehydrogenase levels Blood samples were drawn at the end of the 2 h reperfusion. Plasma lactate dehydrogenase (LDH) levels were measured using commercial detection kits (DU 640; Beckman Coulter, Brea, CA) according to the manufacturer's instruction. All measurements were assayed in duplicates. 2.7. Western blot analysis The protein expression and phosphorylation levels were determined using Western blot as described previously [23]. The immunoblots were probed with anti-JNK, anti-p-JNK (Thr183/Tyr185 Cell Signaling Technology, USA), anti-COX IV, anti-cytochrome c (Cell Signaling Technology, USA), anti-LC3, anti-p62, anti-Beclin1, anti-Bcl2 (Santacruz Biotechnology, Inc, USA) and anti-GAPDH (Abcam, USA) antibodies overnight at 4 °C and subsequently incubated with the corresponding secondary antibodies at room temperature for 1 h. The blots were visualized with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA). GAPDH was used as the internal loading control. 2.8. Statistical analysis All values are presented as mean ± SEM. Differences were compared with ANOVA followed by Bonferroni post hoc test. Probabilities of b0.05 were considered to be statistically significant. All of the statistical tests were performed with the GraphPad Prism software, version 5.0 (GraphPad Software Inc., San Diego, CA).
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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3. Results 3.1. I/R induced mitoJNK activation To elucidate the role of mitoJNK signaling in I/R injury, the localization and activation of JNK were determined after reperfusion. Western blotting of JNK and p-JNK showed two immunoreactive bands with
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approximate molecular weights of 46- and 55-kDa (Fig. 1A, E, I) in cytosol/nucleus, while the 55-kDa splice of JNK was dominant in mitochondria, which is consistent with previous studies [19,24]. The phosphorylation level of JNK was increased in both mitochondria (Fig. 1A, B) and cytosol/nucleus (Fig. 1E, F) after 2 h of reperfusion. However, it should be noted that total JNK in mitochondria was decreased (Fig. 1C) and total JNK in cytosol/nucleus was increased (Fig. 1G), indicating JNK
Fig. 1. Mitochondrial JNK activation is associated with I/R injury. A, representative Western blots of p-JNK and JNK in mitochondria following I/R. B–D, the normalized ratio of p-JNK/COX IV (B), JNK/COX IV (C) and p-JNK/JNK (D) in mitochondria following I/R. E, as in A, for cytosol/nucleus. F–H, the normalized ratio of p-JNK/GAPDH (F), JNK/GAPDH (G) and p-JNK/JNK (H) in cytosol/nucleus following I/R. I, as in A, for total extract. J–K, as in G–H, for total extract. A–K, treatment with insulin (60 U/l, 4 ml/kg/h for 2 h through femoral vein 5 min before reperfusion) restored JNK activation and localization in both mitochondria and cytosol/nucleus. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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translocation from mitochondria to cytosol/nucleus during reperfusion. A dominant JNK activation occurred in mitochondria as evidenced by the fact that p-JNK/JNK displayed an approximately 4-fold increase in mitochondria (Fig. 1D) and 2-fold increase in cytosol/nucleus (Fig. 1H). Consistent with JNK activation in mitochondria and cytosol/nucleus, universal JNK activation displayed an approximately 4-fold increase (Fig. 1K), and I/R had little effect on universal JNK expression (Fig. 1J). To further examine the role of mitoJNK signaling in I/R injury, insulin treatment which has been demonstrated by our laboratory and others as one of the most promising strategies in cardioprotection against I/R injury was employed [25–29]. As shown previously, insulin treatment (60 U/l, intravenous infusion at 4 ml/kg/h for 2 h, beginning at 5 min before reperfusion) exerted protective effect against I/R injury as evidenced by reduced lesion area (Fig. 3A) and plasma LDH concentration (Fig. 3B). Meanwhile, insulin treatment decreased both mitochondrial and cytosolic/nuclear JNK activation (Fig. 1B, F). Importantly, treatment with insulin decreased the ratio of p-JNK/JNK in mitochondria, but had little effect on the ratio of p-JNK/JNK in cytosol/ nucleus (Fig. 1D), indicating that insulin could selectively inhibit mitoJNK activation during reperfusion. In addition, treatment with insulin restored the JNK localization after reperfusion (Fig. 1C, G). These results suggested that I/R-induced JNK activation, especially mitoJNK activation, is associated with I/R injury. 3.2. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide exerted protective effect against I/R injury To examine the role of mitoJNK activation in I/R injury, we used a peptide containing 20 residues of the SabKIM1 domain along with a HIV-Tat sequence (Tat-SabKIM1) as described previously [19], which can be used to block JNK translocation to mitochondria in vivo. It has been reported that Tat-SabKIM1 does not affect JNK-mediated nuclear events and JNK translocation to the nucleus [14]. As shown in Fig. 2A,
Tat-SabKIM1 injection (2 mg/kg, delivered 5 min before reperfusion) decreased the phosphorylation level of JNK in mitochondria (Fig. 2B, D), and had no evident effect on cytosolic/nuclear JNK activation (Fig. 2E, F, H) following I/R. Unexpectedly, Tat-SabKIM1 injection had no evident effect on JNK localization following reperfusion (Fig. 2C, G). These results indicated that injection with Tat-SabKIM1 can specifically inhibit mitoJNK activation without impact on cytosolic/nuclear JNK activation and JNK localization. Importantly, Tat-SabKIM1 injection markedly reduced lesion area (Fig. 3A) and plasma LDH level (Fig. 3B) following I/R, exerting similar cardioprotective effect as treatment with insulin (Fig. 3A, B). In addition, assessment of cardiac function after 2 h of reperfusion revealed that injection with Tat-SabKIM1 increased LVSP (Fig. 3C) and ±LV dP/dtmax in rats (Fig. 3D, E), while no significant differences in MABP and heart rate have been detected (data not shown). These results suggested that inhibition of mitoJNK activation alone exerts cardioprotective effect against I/R injury. 3.3. mitoJNK activation contributed to Bcl2-regulated autophagy Next, we examined the effect of mitoJNK activation on autophagy after reperfusion. I/R resulted in biochemical evidence of cardiac muscle autophagy, including conversion of the non-lipidated form of LC3, LC3-I, to the autophagosome membrane-associated lipidated form, LC3-II, and degradation of the autophagy substrate protein p62 (Fig. 4A, B, C). Bcl2 inhibits autophagy through a direct interaction with the autophagy protein Beclin1 [30]. I/R-induced autophagy was further confirmed by increased Beclin1 and decreased Bcl2 after reperfusion (Fig. 4A, D, E). Inhibition of mitoJNK activation through either Tat-SabKIM1 injection or insulin treatment alleviated I/R-induced autophagy, as evidenced by the restored levels of LC3-II/LC3-I, p62, Bcl2 and Beclin1 (Fig. 4A, B, C, D, E). These results indicated that mitoJNK activation triggers the Bcl2-regulated autophagy following reperfusion.
Fig. 2. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide during I/R in vivo. A, representative Western blots of p-JNK and JNK in mitochondria following I/R with Tat-SabKIM1 (TS) injection (2 mg/kg, delivered 5 min before reperfusion). B–D, the normalized ratio of p-JNK/COX IV (B), JNK/COX IV (C) and p-JNK/JNK (D) in mitochondria following I/R with TS injection. E, as in A, for cytosol/nucleus. F–H, the normalized ratio of p-JNK/GAPDH (F), JNK/GAPDH (G) and p-JNK/JNK (H) in cytosol/nucleus following I/R with TS injection. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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Fig. 3. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide and treatment with insulin exert protective effect against I/R injury in vivo. A–E, treatment with Tat-SabKIM1 (TS) or insulin decreased myocardial infarct size (INF) expressed as percentage of area at risk (AAR) (A) and plasma lactate dehydrogenase (LDH) concentration (B), and increased left ventricular systolic pressure (LVSP) (C), and ±dP/dtmax (D, E) following I/R. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Fig. 4. Inhibition of mitoJNK activation through insulin or Tat-SabKIM1 treatment ameliorates Bcl2-regulated autophagy following I/R. A, representative Western blots of the following autophagy-related proteins in heart subjected to I/R with insulin or Tat-SabKIM1 (TS) treatment: LC3-I, LC3-II, p62, Beclin1, and Bcl2. B–E, The statistical results of LC3-II/LC3-I (B), p62 (C), Beclin1 (D) and Bcl2 (E). Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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3.4. mitoJNK activation contributed to mitochondria-mediated apoptosis As shown in Fig. 5A, the amount of cytochrome c release from mitochondria into the cytosol following I/R displayed an approximately 3-fold increase compared with that in sham group. Treatment with insulin reduced cytochrome c release into the cytosol by 48.3% (Fig. 5A), and Tat-SabKIM1 injection alleviated cytochrome c release by 68.9% (Fig. 5A). Inhibition of mitoJNK signaling through either Tat-SabKIM1 injection or insulin treatment decreases I/R-induced cytochrome c release from mitochondria. Next, we examined cardiac apoptosis using TUNEL staining to further examine the role of mitoJNK signaling in the induction of apoptosis. TUNEL-positive cells were increased in I/R rats (Fig. 5B). Treatment with insulin exerted a significant anti-apoptotic effect as evidenced by reduced TUNEL-positive staining (Fig. 5B). Meanwhile Tat-SabKIM1 injection also decreased TUNEL-positive staining, exerting cardioprotective effect similar to insulin treatment (Fig. 5B). These results indicated that mitoJNK signaling triggers the mitochondria-mediated apoptosis following I/R.
3.5. Inhibiting mitoJNK activation alone exerted similar cardioprotective effect as universal JNK inhibition It's reported that inhibition of universal JNK activation also exerts cardioprotective effect against I/R injury [7]. Next, we inhibited universal JNK activation to test the contribution of mitoJNK activation in the induction of I/R injury. The phosphorylation level of JNK displayed an approximately 3.5-fold increase after 2 h of reperfusion (Fig. 6A). To further elucidate the involvement of JNK in the induction of I/R-induced myocardium injury, SP600125, a selective JNK inhibitor, was used. Treatment with SP600125 (1 mg/kg, administered through femoral vein 10 min before reperfusion) decreased phosphorylation level of universal JNK by 71.4%, and reduced lesion area by 40.3% and plasma LDH
concentration by 46.7% (Fig. 6B, C). Similarly, inhibition of mitoJNK activation by 66.8% (Fig. 2B) could reduce lesion area by 32.6% and plasma LDH concentration by 39.7% (Fig. 3A, B). These results indicated that inhibiting mitoJNK activation alone could exert similar cardioprotective effect as universal JNK inhibition. Furthermore, treatment with anisomycin (0.1 mg/kg, administered through contralateral femoral vein 15 min before reperfusion) which activates JNK signaling abolished the cardioprotection of SP600125 in I/R rats (Fig. 5B, C), further indicating that the cardioprotective effect of SP600125 is through the inhibition of JNK activation.
4. Discussion The major findings of the present study are as follows. First, I/R induces a dominant increase of mitoJNK activation which triggers Bcl2regulated autophagy and cytochrome c-mediated apoptosis. Second, it is mitoJNK activation rather than JNK localization on mitochondria that mediates the induction of I/R injury. Third, insulin selectively inhibits mitoJNK activation during reperfusion, which constitutes a novel mechanism of insulin cardioprotection against I/R injury. JNK has been shown to have an essential role in modulating the functions of pro- and anti-apoptotic proteins located in the mitochondria, thus regulating mitochondrial function and mitochondria-mediated apoptosis [5,13,31]. However, previous studies lack evidence showing the role of mitoJNK signaling in the regulation of mitochondrial function. Until recently, with the aid of a new method to selectively inhibit JNK translocation to mitochondria using Tat-SabKIM1 peptide through inhibiting JNK interacting with Sab, a binding partner for JNK mitochondrial association [9], LoGrasso and Chambers showed that mitoJNK signaling mediated mitochondrial reactive oxygen species generation and mitochondrial cytochrome c release [24]. Our in vivo study further confirmed that inhibition of mitoJNK activation with Tat-SabKIM1 reduced mitochondrial cytochrome c release and thus reduced apoptosis and
Fig. 5. Inhibition of mitoJNK activation through insulin or Tat-SabKIM1 treatment ameliorates mitochondria-mediated apoptosis following I/R. A, levels of cytochrome c release (fold change normalized to sham group) in rats subjected to I/R with insulin or Tat-SabKIM1 (TS) treatment. B, representative photomicrographs of in situ detection of apoptotic myocytes by TUNEL staining in heart slices from rats subjected to I/R. TUNEL positive cells (green) and total number of nuclei (blue) are shown for each representative section of the heart. Scale bar: 50 μm. Percentage of TUNEL-positive nuclei was analyzed below. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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Fig. 6. Universal JNK inhibition exerts cardioprotective effect against I/R injury similarly as inhibiting mitoJNK activation. A, myocardial phosphorylation level of JNK following I/R evaluated by Western blot. B, myocardial infarct size (INF) expressed as percentage of area at risk (AAR) following I/R. C, plasma lactate dehydrogenase (LDH) concentration following I/R. SP (SP600125, 1 mg/kg), a selective JNK inhibitor, was administered through femoral vein 10 min before reperfusion. A (anisomycin, 0.1 mg/kg), a JNK activator, was administered through contralateral femoral vein 15 min before reperfusion. Data are expressed as mean ± SEM. n = 6/group. *P b 0.05, **P b 0.01.
attenuated I/R injury. Importantly, in the present study, we observed that JNK mitochondrial localization was decreased, though activated JNK on mitochondria was increased following I/R, which is contrary to LoGrasso's results that I/R increased JNK localization on mitochondria [19]. One possible reason for the discrepancy is that they only determined the total JNK in both mitochondria and cytosol/nucleus in in vitro study. Furthermore, our results showed that I/R induced a dominant increase of mitoJNK activation rather than JNK activation in cytosol/nucleus. mitoJNK activation caused cell death through not only cytochrome c-mediated apoptosis but also Bcl2-regulated autophagy. Although the precise role of autophagy regulation contributing to cell survival and death in ischemic hearts remains controversial, a number of studies have shown that despite the seemingly cardioprotective role of autophagy during ischemia, autophagy can induce cell death during reperfusion [20–22]. This form of cell death is referred to as type 2 programmed cell death but may simply be caused by excessive activation of autophagy. Mechanistically, mitoJNK phosphorylates Bcl2, antagonizing Bcl2 antiapoptotic and anti-autophagic activities [32,33], which may contribute to mitoJNK's deleterious role in myocardial I/R injury. Other possible mechanisms that maintain autophagy at a higher level during reperfusion include oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress and calcium overload [34]. Moreover, selective inhibition of mitoJNK activation produced similar infarct size-reducing effect as universally inhibiting JNK with JNK inhibitor SP600125. This suggests that inhibition of mitoJNK activation could possibly be at least partially responsible for the protective effects of universal JNK inhibition against I/R. These results are potentially of clinical significance, and may suggest a new therapeutic strategy for treating ischemic heart disease. Compounds that are either competitive
versus JNK-interacting protein or that have some bidentate character might be achieved with therapeutic benefit [35–37]. Studies from our laboratory and others demonstrate that the possible mechanisms through which insulin attenuates cardiac ischemia injury include metabolic modulation, positive inotropic effect and directly activating survival signaling which exerts anti-apoptosis, vasodilatation, anti-inflammation and anti-oxidative stress effects [38]. A lot of studies have established that central to these effects is the mitochondria whose proper functioning is essential to myocardial health due to its vital role in energy production and cell survival [28]. The present study showed that treatment with insulin selectively inhibits mitoJNK activation, although the exact mechanism is still elusive, consequently reduced apoptosis and infarct size. This extends our understanding of insulin cardioprotection against I/R injury. Based on the findings from previous studies and the present study, we proposed a model illustrating the possible mechanism of mitoJNK activation in the induction of myocardial I/R injury (Fig. 7). JNK is activated at a relatively low level and localized in either mitochondria or cytosol/nucleus under unstressed condition. During I/R, JNK is phosphorylated and activated JNK translocates to mitochondria, resulting in increased mitoJNK activation. Increased mitoJNK activation results in enhanced autophagy and cytochrome c release from mitochondria. Either insulin or Tat-SabKIM1 intervention inhibits mitoJNK activation and exerts cardioprotective effect against I/R injury. In conclusion, this is the first demonstration on the molecular level that mitoJNK activation, rather than JNK mitochondrial localization, triggers autophagy and apoptosis following I/R. Inhibiting mitoJNK activation with Tat-SabKIM1 peptide is as effective as inhibiting the catalytic activity of universal JNK in improving post-ischemic cardiac function and
Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
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Fig. 7. Schematic figure illustrating that mitoJNK activation mediates myocardial I/R injury. TS: Tat-SabKIM1. The JNK locates in mitochondria and cytosol/nucleus in the absence of stress. Once it confronted I/R, JNK is phosphorylated in cytosol/nucleus and activated JNK translocates to mitochondrial outer membrane which triggers Bcl2-regulated autophagy and mitochondria-mediated apoptosis. Treatment with insulin or Tat-SabKIM1 inhibits mitoJNK activation and protects myocardium against I/R injury.
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Please cite this article as: J. Xu, et al., Mitochondrial JNK activation triggers autophagy and apoptosis and aggravates myocardial injury following ischemia/reperfusion, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/j.bbadis.2014.05.012
